JP2013066463A - Multi-nucleic acid reaction tool and detection method using same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multi-nucleic reaction tool capable of subjecting a plurality of different target sequences to an amplification reaction simultaneously and independently using a plurality of different primer sets in one reaction field, and independently determining a presence/absence or the amount of the target sequences for each of amplified products of the primer sets, and to provide a method for detecting a target nucleic acid using the same.SOLUTION: A multi-nucleic acid reaction tool of an embodiment is equipped with a support, a plurality of different primer sets, and probe nucleic acids comprising a complementary sequence of a target sequence. The support is configured to support a liquid-phase reaction field. On at least one surface of the support which is in contact with the reaction field when the reaction field is formed using the liquid phase, the plurality of different primer sets are fixed in a releasable manner to mutually independent primer immobilization regions for each type of primer. Each of the plurality of different primer sets is configured to amplify the respective corresponding target sequence. The probe nucleic acids are fixed to the same positions as the primer immobilization regions or to probe immobilization regions in proximity thereto, and hybridized signals are independently detected.

Description

  Embodiments described herein relate generally to a multi-nucleic acid reaction device and a detection method using the same.

  Currently, with the advancement of genetic testing technology, genetic testing is being carried out in various situations such as clinical practice and criminal investigation. These genetic tests often become useful only when a plurality of target genes are detected and the results are combined. For example, pathogens are identified in clinical settings. In that case, a plurality of types of microorganisms suspected of being infected or the type of each microorganism is determined based on the patient's symptoms. Thereby, diagnosis is performed. At the crime investigation site, for example, individual identification is performed. In that case, the number of repetitions is specified for the repetitive sequences at a plurality of loci possessed by all persons on the genome. Individuals are comprehensively identified from the number of repetitions at a plurality of identified loci. Thereby, an individual can be specified with high probability. Thus, a technique for detecting a plurality of target genes has become very important.

  Conventionally, when detecting a plurality of target genes, sample nucleic acid is first amplified in a specific reaction vessel. Thereafter, detection of the obtained amplification product is performed in a reaction apparatus for further detection.

  Amplification takes place mainly in the next plurality or one reaction vessel. When amplification is performed in a plurality of reaction containers, reaction containers for amplifying each target gene are prepared. When amplification is performed in one reaction container, reagents for detecting all target genes are stored in one reaction container, and a multi-nucleic acid amplification reaction is performed. The detection of the nucleic acid to be detected is generally performed by subjecting the amplification product to a DNA chip or electrophoresis.

Japanese Patent No. 4127679 JP 2008-263959 A JP 2005-261298 A

  The problem to be solved by the present invention is that, in one reaction field, a plurality of types of target sequences are simultaneously and independently amplified using a plurality of types of primer sets, and each amplification product of the primer set is independently determined. It is an object to provide a multi-nucleic acid reaction device capable of determining the presence or absence or amount of a target nucleic acid and a target nucleic acid detection method using the same.

  The multi-nucleic acid reaction device of the embodiment includes a support, a plurality of types of primer sets, and a probe nucleic acid including a complementary sequence of a target sequence. The support is configured to support a liquid phase reaction field. When a reaction field is formed by a liquid phase, a plurality of types of primer sets are releasably immobilized for each kind in an immobilization region independent from each other on at least one surface of a support in contact with the reaction field. The plurality of types of primer sets are configured to amplify target sequences corresponding to the respective primer sets. The probe nucleic acid is immobilized at the probe immobilization region at the same position as or near the primer immobilization region. The hybridization signal for the probe nucleic acid is arranged so as to be independently detectable for each probe immobilization region.

FIG. 1 is a diagram showing a multi-nucleic acid reaction device of an embodiment. FIG. 2 is a perspective view showing a state in use of the embodiment. FIG. 3 is a plan view of the array-type primer probe chip of the embodiment. FIG. 4 is a plan view of the array-type primer probe chip of the embodiment. FIG. 5 is a diagram illustrating a part of the array-type primer probe chip according to the embodiment and a detection current. FIG. 6 is a diagram showing a part of the array-type primer probe chip of the embodiment and a detection current obtained thereby. FIG. 7 is a diagram showing a part of the array-type primer probe chip of the embodiment and a detection current obtained thereby. FIG. 8 is a diagram showing an array-type primer probe chip according to an embodiment. FIG. 9 is a diagram showing an array-type primer probe chip according to an embodiment. FIG. 10 is a diagram showing a part of the array type primer probe chip of the embodiment. FIG. 11 is a diagram showing a chip material for the array-type primer probe chip of the embodiment. FIG. 12 is a diagram illustrating an array-type primer probe chip according to an embodiment. FIG. 13 is a diagram showing the array-type primer probe chip of the embodiment in use. FIG. 14 is a diagram showing the array-type primer probe chip of the embodiment in use. FIG. 15 is a graph showing experimental results obtained with the array-type primer probe chip of the example. FIG. 16 is a plan view of the array-type primer probe chip of the embodiment. FIG. 17 is a diagram illustrating a multi-nucleic acid reaction device according to an embodiment. FIG. 18 is a diagram illustrating an array-type primer probe chip according to an embodiment. FIG. 19 is a diagram showing a chip material for the array-type primer probe chip of the embodiment. FIG. 20 is a diagram illustrating an array-type primer probe chip according to an embodiment. FIG. 21 is a diagram showing the array-type primer probe chip of the embodiment in use. FIG. 22 is a diagram showing the array-type primer probe chip of the embodiment in use. FIG. 23 is a diagram illustrating an array-type primer chip according to an embodiment. FIG. 24 is a diagram showing experimental results obtained with the array-type primer chip of the embodiment. FIG. 25 is a schematic view of a nucleic acid detection device according to an embodiment. FIG. 26 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 27 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 28 is a schematic view of a nucleic acid detection device according to an embodiment. FIG. 29 is a schematic view of a support according to the embodiment. FIG. 30 is a schematic view of a nucleic acid detection device according to an embodiment. FIG. 31 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 32 is a graph showing the results of the nucleic acid amplification reaction according to the embodiment. FIG. 33 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 34 is a schematic view of a nucleic acid detection device according to an embodiment. FIG. 35 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 36 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 37 is a schematic diagram of a nucleic acid detection device according to an embodiment. FIG. 38 is a schematic diagram of a nucleic acid detection device according to an embodiment and a graph showing the results of a nucleic acid amplification reaction. FIG. 39 is a diagram illustrating a procedure for creating the nucleic acid detection device according to the embodiment. FIG. 40 is a cross-sectional view illustrating a schematic configuration of the nucleic acid detection device according to the embodiment. FIG. 41 is a diagram illustrating a schematic configuration of the nucleic acid detection device according to the embodiment. FIG. 42 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device according to the embodiment. FIG. 43 is a diagram showing a schematic configuration of a cassette with a built-in nucleic acid detection device according to the embodiment. FIG. 44 is a diagram showing the opposing arrangement of the nucleic acid detection device and the reaction part defining member according to the embodiment. FIG. 45 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device according to the embodiment. FIG. 46 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device according to the embodiment. FIG. 47 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device according to the embodiment. FIG. 48 is a graph showing the results of the nucleic acid amplification reaction of the embodiment. FIG. 49 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device of the embodiment. FIG. 50 is a graph showing the results obtained from each electrode in the embodiment. FIG. 51 is an enlarged view of the vicinity of the reaction region on the surface of the nucleic acid detection device of the embodiment. FIG. 52 is a graph showing the results obtained from the electrodes in the embodiment. FIG. 53 is a plan view showing the array type probe chip of the embodiment. FIG. 54 is a perspective view showing the array-type primer chip of the embodiment. FIG. 55 is a perspective view showing the array-type primer chip of the embodiment. FIG. 56 is a perspective view showing the array-type primer probe chip of the embodiment. FIG. 57 is a perspective view showing the array-type primer probe chip of the embodiment. FIG. 58 is a diagram showing a chip material for the array-type primer probe chip of the embodiment. FIG. 59 is a diagram showing an array-type primer probe chip according to an embodiment. FIG. 60 is a diagram showing an array-type primer probe chip according to an embodiment. FIG. 61 is a diagram showing an array-type primer probe chip according to an embodiment. FIG. 62 is an exploded perspective view showing a schematic configuration of the nucleic acid detection cassette according to the embodiment. FIG. 63 is a perspective view showing a schematic configuration of the nucleic acid detection cassette according to the embodiment. FIG. 64 is a perspective view illustrating a schematic configuration of the nucleic acid detection device according to the embodiment.

  Various embodiments will be described below with reference to the drawings. In addition, the same code | symbol shall be attached | subjected to a common structure through embodiment, and the overlapping description is abbreviate | omitted. In addition, each drawing is a schematic diagram for promoting the embodiment and its understanding, and its shape, dimensions, ratio, etc. are different from the actual device, but these are considered in consideration of the following description and known techniques. The design can be changed as appropriate.

1. Definitions “Multinucleic acid amplification” refers to the simultaneous amplification of multiple types of target sequences to be amplified. “Amplification” refers to a step of continuously replicating a template nucleic acid using a primer set. The amplification method that can be used may be a method of amplifying a target nucleic acid using a primer set, and is not limited thereto. For example, PCR amplification, LAMP amplification, RT-LAMP amplification, SMAP amplification, ICAN amplification , SDA amplification, NASBA amplification, RCA amplification, LCA amplification, TMA amplification, primer extension (extension), Invader, bDNA method, PALSAR method and the like.

  “Target sequence” refers to a sequence to be amplified by a primer set, and includes a region to which a primer to be used binds.

  A “target nucleic acid” is a sequence that includes at least a target sequence, is a nucleic acid that is used as a template by a primer set to be used, and is also referred to as a “template nucleic acid”.

  A “primer set” is a collection of primers necessary for amplifying one target nucleic acid. For example, in the case of a primer set for PCR amplification, one primer set may include one kind of forward primer and one kind of reverse primer for amplifying one target nucleic acid. For example, in the case of a primer set for LAMP amplification, one primer set may include an FIP primer and a BIP primer for amplifying at least one target nucleic acid, and an F3 primer, a B3 primer, and an LP primer as necessary. That is, an LF primer and / or an LB primer may be included. For example, in the case of primer extension (extension), one kind of extension primer may be included.

  “Target sequence” refers to a sequence to be detected by the multi-nucleic acid reaction device. The target nucleic acid to be detected includes a “target sequence”. A nucleic acid comprising a target sequence or a nucleic acid containing a target sequence is referred to as a “target sequence strand”. The target sequence strand is hybridized with a probe nucleic acid containing the complementary sequence, and the presence / absence or amount of this hybridization is detected, and the presence / absence or amount of the target nucleic acid is detected or measured.

The “sample” may be a substance containing a nucleic acid to be amplified and / or detected by a multi-nucleic acid reaction device. The sample may be, but is not limited to, for example, blood, serum, leukocytes, urine, stool, semen, saliva, tissue, biopsy, oral mucosa, cultured cells, sputum, etc. The nucleic acid component may be extracted from any of the above or a mixture thereof by known means.

  “Hybridization signal” is a signal generated by hybridization of a probe nucleic acid and its complementary sequence, and is a generic term for detection signals detected as, for example, current value, fluorescence intensity, emission intensity, etc., by the detection method of the microarray. To do.

2. Multi-Nucleic Acid Reactor <First Embodiment>
One example of a multi-nucleic acid reaction tool will be described with reference to FIGS. 1 (a), (b) and (c). This multi-nucleic acid reaction tool is an example of a nucleic acid reaction tool for multi-amplifying a plurality of types of target nucleic acids and detecting amplification products obtained by multi-amplification.

  FIG. 1A is a perspective view of an example of a multi-nucleic acid reaction tool. A multi-nucleic acid reaction tool 1 shown in FIG. 1A has a container-shaped support 2. A plurality of primer immobilization regions 4 independent from each other are arranged on the inner bottom surface 3 of the support 2. A plurality of probe immobilization regions 5 are arranged adjacent to the plurality of primer immobilization regions 4 and corresponding to the respective primer immobilization regions 4.

  FIG. 1B is a schematic diagram in which the primer immobilization region 4 is enlarged. As shown therein, one kind of primer set 6 is fixed to one primer fixing region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are fixed for each type.

  Plural types of primer sets 6 are prepared in order to amplify a plurality of target nucleic acids. One primer set 6 for amplifying one specific target nucleic acid is fixed to one primer fixing region 4. For example, in the case of a PCR amplification reaction tool, one primer-immobilized region 4 includes a plurality of forward primers and reverse primers necessary for amplifying one type of specific target nucleic acid. In the case of a reaction tool for LAMP amplification, the FIP primer, BIP primer, and F3 primer, B3 as necessary, for amplifying one specific target nucleic acid in one primer immobilization region 4. A plurality of primers and LP primers are included.

  The primer set 6 is fixed to the primer immobilization region 4 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 6 to the primer immobilization region 4 can be achieved, for example, by dropping a solution containing a set of primer sets 6 onto one primer immobilization region 4 and then drying it. . Further, similarly, a solution containing a desired primer set 6 may be dropped and dried for each of the other primer immobilization regions 4 to fix a desired number of primer sets 6 to the support 2. As a result, the primer set 6 is fixed to all the primer fixing regions 4 arranged independently on one surface of the support 2. However, the primer set 6 may be fixed to the primer immobilization region 4 in such a manner that it can be released in contact with a liquid phase for providing a reaction field. Therefore, any fixing method known per se capable of such fixing may be used. In the case of dropping the solution containing the primer set 6, the solution containing the primer set may be, for example, water, a buffer solution or an organic solvent.

  The plurality of primer immobilization regions 4 arranged on the support 2 may be arranged independently of each other. The term “independently arranged” means that they are arranged at intervals that do not interfere with amplification initiated and / or advanced for each primer set 6 in the reaction field. For example, the adjacent primer immobilization regions 4 may be arranged in contact with each other, may be arranged in the vicinity of each other at a slight distance, or fixed in a commonly used detection device such as a so-called DNA chip. The probe nucleic acids may be arranged at the same distance from each other at the same distance.

  For example, the distance between adjacent primer immobilization regions 4 may be 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or more, preferably 100 μm to 10 mm There may be.

  The length of the primer is not limited to this, but about 5 bases or more, about 6 bases or more, about 7 bases or more, about 8 bases or more, about 9 bases or more, about 10 bases or more, about 15 bases More than about 20 bases, about 25 bases or more, about 30 bases or more, about 35 bases or more, about 40 bases or more, about 45 bases or more, about 55 bases or more, about 80 bases or less, about 75 bases or less About 70 bases or less, about 65 bases or less, about 60 bases or less, about 55 bases or less, about 50 bases or less, about 45 bases or less, about 40 bases or less, about 35 bases or less, about 30 bases or less, about 25 bases or less , About 20 bases or less, about 25 bases or less, or about 20 bases or less, or a combination of any of these lower and upper limits. For example, examples of preferable base length may be about 10 bases to about 60 bases, about 13 to 40 bases, about 10 to 30 bases, and the like. In addition, the length of the primer simultaneously fixed to one support may be the same for all primers, may be different for all primers, or may be the same length for some primers. Well, some primers may have different lengths. Moreover, you may differ for every primer set. In addition, the primer sets fixed in one region may have different lengths for each type, and all the primer sets fixed in one region may have the same length.

  The liquid phase for providing the reaction field only needs to be a liquid phase that allows the amplification reaction to proceed after the immobilized primer set 6 is released, for example, a reaction liquid necessary for the desired amplification. It may be.

  The container-shaped support 2 may be, for example, a tube, a well, a chamber, a flow path, a cup and a dish, and a plate including a plurality of them, such as a multiwell plate. The material of the support 2 may be any material that does not itself participate in the reaction, and may be any material that can perform the amplification reaction there. For example, it may be arbitrarily selected from silicon, glass, resin and metal. The support 2 in the form of a container may use any commercially available container.

  Although FIG. 1 shows an example in which the primer immobilization region 4 is disposed on the inner bottom surface 3 of the support 2, the present invention is not limited to this and may be disposed on at least a part of the inner side surface of the support 2. The inner bottom surface and the inner side surface, for example, the ceiling surface formed by the covering portion attached to the support 2 may be arranged on any or all of them.

  FIG. 1 (c) is an enlarged view of the probe immobilization region 5 arranged in the vicinity of the primer immobilization region 4. A plurality of probe nucleic acids 7 including a complementary sequence of a desired sequence to be detected are immobilized on the probe immobilization region 5.

  The desired sequence to be detected may be the target sequence. The probe immobilization region 5 is arranged so that a hybridization signal between the probe nucleic acid 7 and the target sequence chain can be detected independently between the plurality of probe immobilization regions 5.

  For fixing the probe nucleic acid 7 to the probe immobilization region 5, any general technique for fixing the probe nucleic acid to the substrate surface in a so-called DNA chip known per se may be used. The primer set 6 may be fixed after the probe nucleic acid 7 is fixed, the probe nucleic acid 7 may be fixed after the primer set 6 is fixed, or the primer set 6 and the probe nucleic acid 7 may be fixed simultaneously. Good.

  For example, the distance between adjacent probe immobilization regions 5 may be 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or more, preferably 100 μm to 10 mm It may be.

  For example, the distance between the probe immobilization region 5 and the primer immobilization region 4 is 0 μm to 0.1 μm, 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or The above may be sufficient, Preferably, it may be 100 micrometers-10 mm.

  For example, when the distance between the probe immobilization region 5 and the primer immobilization region 4 is 0 μm, it is understood that the probe immobilization region 5 and the primer immobilization region 4 are at the same position on the surface of the support 2. May be. Further, the probe immobilization region 5 may be included in the primer immobilization region 4, and the primer immobilization region 4 may be included in the probe immobilization region 5.

  The length of the probe nucleic acid 7 is, for example, 3 bases to 10 bases, 10 bases to 20 bases, 20 bases to 30 bases, 30 bases to 40 bases, 40 bases to 50 bases, 50 bases to 60 bases, preferably 10 bases It may be ~ 50 bases. The probe nucleic acid 7 includes a complementary sequence of the target sequence to be detected. The probe nucleic acid 7 may include a further sequence such as a spacer sequence in addition to the complementary sequence of the target sequence.

  The length of the target sequence may be, for example, 10 bases to 100 bases, 100 bases to 200 bases, bases 200 to 300 bases, 300 bases to 400 bases, preferably 100 bases to 300 bases.

  The length of the target sequence is, for example, 3 bases to 10 bases, 10 bases to 20 bases, 20 bases to 30 bases, 30 bases to 40 bases, 40 bases to 50 bases, 50 bases to 60 bases, preferably 10 bases to It may be 50 bases.

  The kind of primer set 6 immobilized on one primer immobilization region 4 may be one kind for amplifying one kind of target nucleic acid, or plural kinds for amplifying two or more kinds of target nucleic acids. It may be.

  The kind of the group of probe nucleic acids 7 fixed to one probe immobilization region 5 may be one kind for hybridizing with one kind of target sequence, in order to amplify two or more kinds of target nucleic acids, respectively. There may be multiple types. Alternatively, the probe nucleic acid 7 may be a probe nucleic acid 7 having a common target sequence portion and a sequence different from other target sequences.

  The lower limit of the number of primer immobilization regions 4 arranged in one multi-nucleic acid reaction tool 1 is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, and the upper limit is 10,000 or less It may be 5000 or less, 2500 or less, 2000 or less, 1500 or less, 1000 or less, 500 or less, 250 or less, 200 or less, 150 or less, and may be a range in which any of these upper and lower limits is combined.

  The number of primer immobilization regions 4 and probe immobilization regions 5 arranged in one multi-nucleic acid reaction device 1 may be the same or different. That is, the same number of probe immobilization regions 5 may be arranged so as to correspond to all the primer immobilization regions 4, and the number of primer immobilization regions 4 may be larger than the number of probe immobilization regions 5, The number of primer immobilization regions 4 may be smaller than the number of probe immobilization regions 5. Further, a positive control and / or a negative control for confirming the amplification reaction state or for confirming the state of the hybridization reaction may be included. Such positive control and / or negative control may be provided for the primer set 6 and / or the probe nucleic acid 7.

  In the above example, only the primer set 6 was fixed to the support 2 as a reagent for amplification. However, the present invention is not limited to this, and other components necessary for amplification, for example, enzymes such as polymerase and reverse transcriptase, and substrates, under the condition that the primer set 6 is fixed to each immobilization region 4 for each type. A substrate and / or a buffer may be fixed to the support 2 together with the primer set 6. In that case, the substance to be fixed may be contained in a desired liquid medium together with the primer set 6 and fixed by dropping, drying, or the like in the same manner as described above. In such a multi-nucleic acid reaction tool 1, when an amplification reaction is performed, the composition of the reaction solution added thereto may be selected according to the fixed component.

  The support body 2 is not limited to the container shape, and may be a plate shape, a spherical shape, a rod shape, or a part thereof, and the size and shape of the support body 2 may be arbitrarily selected by the practitioner. Good. Further, a substrate having a flow path may be used as the support 2. In that case, the primer set 6 and the probe nucleic acid 7 may be fixed inside the channel.

  In the conventional method, reaction containers for amplifying each target gene are prepared. In this case, as the number of target genes increases, the number of necessary reaction containers and the amount of work at the time of testing increase. In addition, when reagents for detecting all target genes are put in one reaction container, the number of target genes is limited due to factors such as bias in amplification reaction efficiency. This situation is the same in the amplification process. Furthermore, since it is necessary to perform the number of reaction containers and amplification and detection in different reaction containers, there is a possibility that sample may be mixed, and there is a risk that even if a mistake is made, it will not be noticed. Moreover, since it is necessary to perform at least the amplification process and the detection process, a long time is required for the inspection.

  According to the above-described embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

<Second Embodiment>
A method for amplifying and detecting nucleic acids using the above-described multi-nucleic acid reaction device of the first embodiment will be described with reference to FIG. FIG. 2 is a schematic diagram showing the state of a nucleic acid reaction performed in the multi-nucleic acid reaction tool 1 similar to that of the first embodiment over time.

  FIG. 2 (a-1) and (b-1) show the multi-nucleic acid reaction tool 1 before the reaction. A plurality of primer sets 6 are respectively fixed to a plurality of primer fixing regions 4 arranged on the inner bottom surface 3 of the support 2. Corresponding to each primer immobilization region 4, a probe immobilization region 5 is arranged in the vicinity of each primer immobilization region 4. In the probe immobilization region 5, a plurality of probe nucleic acids 7 are immobilized for each desired type.

  A state in which the reaction solution 8 is added to the multi-nucleic acid reaction tool 1 and accommodated therein is shown in FIGS. 2 (a-2) and (b-2).

  The reaction solution 8 may contain components necessary for a desired amplification reaction. Although not limited to these, for example, when an enzyme such as polymerase or a primer is used as a starting point to form a new polynucleotide chain, a substrate such as oxynucleoside triphosphate, or when reverse transcription is performed simultaneously. In addition, a buffer such as salts for maintaining a suitable amplification environment, such as reverse transcriptase and a substrate necessary for the reverse transcriptase, may be contained in the reaction solution 8.

  The sample may be added to the inside of the support 2 by adding the sample to the reaction solution 8 in advance before adding the reaction solution 8 to the multi-nucleic acid reaction tool 1. Alternatively, it may be performed by adding the reaction solution 8 to the multi-nucleic acid reaction tool 1 and then adding it to the reaction solution 8. Alternatively, it may be performed by adding a sample to the multi-nucleic acid reaction device 1 before adding the reaction solution 8 to the multi-nucleic acid reaction device 1.

  As shown in FIGS. 2 (a-2) and (b-2), in the multi-nucleic acid reaction tool 1 after the reaction solution 8 is added, the primer set 6 fixed to the inner bottom surface 3 is released and gradually. To spread. The free and diffused area is schematically indicated by area 9. The primer set 6 that is released and diffuses encounters other components necessary for amplification such as template nucleic acid, polymerase, and substrate existing in the vicinity thereof, and an amplification reaction is started. A plurality of primer sets 6 independently fixed for each type can start and proceed with an amplification reaction for the template nucleic acid independently for each type. Thereby, amplification of a plurality of template sequences using a plurality of types of primer sets 6 is achieved independently and simultaneously.

  FIGS. 2 (a-3) and (b-3) schematically show a state in which a template nucleic acid to be amplified is present in the free and diffused regions, an amplification reaction has occurred, and it has progressed. .

  In FIG. 2 (a-3), an amplification reaction is caused by the primer set 6 fixed to all the primer-immobilized regions 4, and a region where the amplification reaction proceeds is schematically shown as a reaction region 10. In FIG. 2 (b-3), amplification occurs only in a part of all the primer-immobilized regions 4 fixed to the inner bottom surface 3, that is, in FIG. 2 (b-3), only three regions. A progressing region is schematically shown as a reaction region 10.

  Here, the “reaction field” theoretically refers to a region defined by the reaction solution 8 in which the amplification reaction can proceed, that is, a region where the reaction solution exists. Also, a region of the reaction field where the amplification reaction actually starts and proceeds there is called a “reaction region”. If the amplification reaction actually proceeds only within the region 10, the region 10 is interpreted as a reaction region.

  The reaction solution 8 may be a liquid phase capable of performing an amplification reaction between the primer set 6 and the target nucleic acid after the fixed primer set is released. The reaction solution 8 may be injected into the reaction field (initially filled with air) to which the primer set 6 is fixed by some method mechanically or artificially before starting the amplification reaction. .

  When the nucleic acid containing the target sequence is present in the amplification product amplified in the region 10, the probe nucleic acid 7 immobilized on the probe-immobilized region 5 hybridizes to the nucleic acid. The probe nucleic acid 7 immobilized on the probe immobilization region 5 is arranged and immobilized so as to hybridize only with the amplification product in the corresponding primer immobilization region 4. That is, the probe immobilization region 5 and the primer immobilization region so that the probe nucleic acid 7 immobilized on one probe immobilization region 5 hybridizes only with the amplification product obtained in the corresponding one primer immobilization region 4. A plurality of pairs consisting of 4 are arranged. That is, a plurality of pairs are arranged while maintaining a distance that is configured such that amplification products amplified in one pair can be detected in the pair.

  Detection of hybridization between the probe nucleic acid 7 and the target sequence strand may be performed by a known hybridization signal detection means. For example, a fluorescent substance may be previously given to the primer set, or a fluorescent substance may be given to a substrate such as oxynucleoside triphosphate. The presence / absence and amount of hybridization may be determined using the fluorescence intensity from these fluorescent substances as an index. Alternatively, the hybridization signal may be detected by electrochemical means.

  The detection of hybridization may be performed after the surface of the multi-nucleic acid reaction tool 1 in contact with the reaction field is washed, or may be performed without washing. When detecting by electrochemical means, the hybridizing signal may be detected using an intercalator. In this case, for example, an intercalator may be included in the reaction solution 8 in advance, and the intercalator may be added before the hybridization reaction starts, during the hybridization reaction, or after the hybridization reaction. Even in these cases, the detection may be performed after the inside of the multi-nucleic acid reaction tool 1 is washed, or the detection may be performed without washing. Determination of the start of hybridization reaction, during reaction, and after reaction may be performed according to reaction conditions such as primer set, probe nucleic acid sequence and template nucleic acid sequence, reaction temperature, etc., and is based on criteria determined by preliminary experiments. It may be done.

  According to the above-described embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

<Third Embodiment>
A further example of a multi-nucleic acid reactor will be described with reference to FIG.

  FIG. 3 is a plan view of a chip-type multi-nucleic acid reaction device, that is, an array-type primer probe chip 1. The array-type primer probe chip 1 described in FIG. 3 is an example in which a substrate is used as the support 2. A plurality of primer immobilization regions 4 are arranged independently of each other on one surface 3 of the support 2. As in FIG. 1, one kind of primer set 4 is fixed to one primer fixing region 4 in the primer fixing region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are fixed for each type. Similarly to the first embodiment, the primer set 6 included in one primer immobilization region 4 may include different types of primers necessary for amplifying one specific target nucleic acid, for example.

  Corresponding to each primer immobilization region 4, a probe immobilization region 5 is arranged in the vicinity thereof. A plurality of probe nucleic acids 7 including a complementary sequence of one kind of target sequence are fixed to one probe fixing region 5. Different probe nucleic acids 7 for detecting different target sequences are immobilized on each of the plurality of arranged probe immobilization regions 5.

  The amplification reaction using this embodiment may be carried out by forming a reaction field by placing the reaction solution on at least the region or surface of the support 2 on which the primer set 6 is fixed.

  According to the above-described embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

<Fourth embodiment>
A fourth embodiment will be described with reference to FIG. The multi-nucleic acid reaction tool shown in FIG. 3 is a chip-type multi-nucleic acid reaction tool, that is, an array-type primer probe chip 1.

  The array type primer probe chip 1 uses a substrate as a support 2. A plurality of primer immobilization regions 4 are arranged independently of each other on one surface 3 of the support 2. As in FIG. 1, one kind of primer set 4 is fixed to one primer fixing region 4 in the primer fixing region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are fixed for each type. Similarly to the first embodiment, the primer set 6 included in one primer immobilization region 4 may include different types of primers necessary for amplifying one specific target nucleic acid, for example.

  Corresponding to each primer immobilization region 4, a probe immobilization region 5 is arranged in the vicinity thereof. A plurality of probe nucleic acids 7 including a complementary sequence of one kind of target sequence are fixed to one probe fixing region 5. Different probe nucleic acids 7 for detecting different target sequences are immobilized on each of the plurality of arranged probe immobilization regions 5.

  When the substrate is used as a support, the material may be any material that can perform an amplification reaction there, rather than a material that itself does not participate in the reaction. For example, it may be arbitrarily selected from silicon, glass, resin and metal. In addition, the primer and probe nucleic acid may be immobilized on the support in the same manner as in the first embodiment.

  When nucleic acid is amplified and detected using this array-type primer probe chip 1, it is placed in a container that can maintain the array-type primer probe chip 1. A reaction field is formed by adding a reaction solution into the container.

  In the case of this embodiment, the primer set 6 and the probe nucleic acid 7 may be fixed on both surfaces of the support 2. This makes it possible to fix more types of primer sets 6 and probe nucleic acids 7 to the support 2 of the array type primer probe chip 1. As a result, more target sequences can be amplified and detected.

  Hybridization detection may be performed by removing the array-type primer probe chip 1 from the container, or may be performed while being maintained in the container.

  Further, a label for recognizing the position of the primer set 6 and / or the probe nucleic acid 7 in the array type primer probe chip 1 may be further given to the support 2. Labeling may be performed by means known per se. In this embodiment, the array-type primer probe chip 1 corresponds to a support, a plurality of primer sets 6 that are independently and releasably fixed to at least one surface of the support, and a plurality of primer sets 6. And probe nucleic acid 7 immobilized so as to be independently detectable. Therefore, the form of the support 2 is not limited to a plate shape such as a substrate. The shape of the support may be any shape. The practitioner may arbitrarily select the size and shape of the support 2. For example, the form which consists of plate shape, spherical shape, rod shape, and those parts may be sufficient.

  The support material and the primer fixing method may be the same as those in the first embodiment. Such an array-type primer probe chip 31 includes a plurality of types of primer sets that are releasably fixed for each type to a substrate and at least one surface of the fixed region, and the plurality of primer-immobilized regions. At least one type of probe nucleic acid containing a complementary sequence of the target sequence, which is immobilized for each type so that a hybridization signal can be independently detected for each probe-immobilized region at the probe-immobilized region at or near the same position And may be used as a multinucleic acid amplification reaction carrier.

  According to the above-described embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

<Fifth Embodiment>
A further example of the array type primer probe chip will be described with reference to FIG.

  FIG. 4 is a plan view of the array-type primer probe chip 1. The array-type primer probe chip 1 illustrated in FIG. 4 is an example in which a substrate having a flow path is used as the support 2. On one surface 3 of the support 2, a plurality of flow paths 41 are formed that are formed by a plurality of grooves that extend linearly and are arranged in parallel to each other. A plurality of primer immobilization regions 4 are arranged independently of each other along the longitudinal direction of the flow channel 41 at the bottom 42 of each flow channel 41. As in FIG. 1, one kind of primer set 4 is fixed to one primer fixing region 4 in the primer fixing region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are fixed for each type. Similarly to the first embodiment, the primer set 6 included in one primer immobilization region 4 may include different types of primers necessary for amplifying one specific target nucleic acid, for example.

  A probe immobilization region 5 is arranged in the vicinity of the primer immobilization region 4 so as to correspond to each primer immobilization region 4. In one channel 41, the primer immobilization region 4 and the probe immobilization region 5 are alternately arranged along the longitudinal direction of the channel 41. A plurality of probe nucleic acids 7 including a complementary sequence of one kind of target sequence are fixed to one probe fixing region 5. Different probe nucleic acids 7 for detecting different target sequences are immobilized on each of the plurality of arranged probe immobilization regions 5.

  In the amplification reaction using this embodiment, a reaction field is formed by placing or including a reaction solution in at least one surface of the support 2 or a flow path formed on one surface of the support 2. Just do it. The support 2 supports the reaction field in a state where the reaction solution is placed on the support 2 or in a state where the reaction solution is included in the flow path.

  The array-type primer probe chip of the fifth embodiment described above has a channel formed on the surface of the support 2 and the primer set and the probe are immobilized on at least one wall surface in the formed channel. May be manufactured.

  The formation of the flow path 41 may be performed by forming a concave portion, a convex portion, and a concave portion and a convex portion on one surface of the support 2. Thereby, the shape of the flow path 41 should just be prescribed | regulated by a recessed part, a convex part, a recessed part, and a convex part. For example, the channel 41 may be formed by applying any means known per se for forming a groove in the substrate, such as etching, on the surface of the support 2. The number of the flow paths 41 included in the support 2 may be one or plural, and preferably plural.

  The arrangement of the primer set immobilization region 4 and the probe immobilization region 5 and the immobilization of the primer set 6 and the probe nucleic acid 7 may be performed in the same manner as in the first embodiment.

  The positions of the primer immobilization region 13 and the probe immobilization region 14 arranged with respect to the flow channel 41 are not limited to the bottom surface of the flow channel, and may be any surface in the flow channel. For example, such a surface may be the bottom surface, the side surface, and / or any ceiling surface of the flow path 41.

The ceiling surface of the channel is, for example, a channel provided by the covering by covering all the channels 41 or attaching a covering configured to cover each of the channels 41 to the support 2. The ceiling surface of 41 may be sufficient.

  The state of the amplification reaction and the detection result in the array type primer probe chip 1 in which the primer immobilization region 4 and the probe immobilization region 5 are arranged in the flow channel 41 will be described with reference to FIGS.

  FIG. 5 is a diagram showing one of the channels 41 included in the array-type primer probe chip 1 shown in FIG.

  FIG. 6 shows the array-type primer shown in FIG. 4 except that the position where the primer immobilization region 4 and the probe immobilization region 5 are arranged is not the bottom surface 42 of the flow channel 41 but the side surface of the flow channel 41. FIG. 3 is a view showing one flow path 41 provided in the probe tip 1.

  FIG. 7 shows one channel 41 provided in the array-type primer probe chip 1 shown in FIG. 4 except that the primer-immobilized region 4 and the probe-immobilized region 5 are arranged at substantially the same position. FIG.

  5A, 6A, and 7A are diagrams showing a state in which the primer set 6 and the probe set 7 are fixed to the channel 41. FIG. 5 (a), 6 (a), and 7 (a), regions A, B, C, D, E, F, and G are arranged on the inner surface of the flow path 41 along the longitudinal direction of the flow path 41. Has been. In each of the regions A, B, C, D, E, F and G, a primer set 6 is releasably immobilized, and a probe nucleic acid 7 to be arranged corresponding thereto is immobilized. The primer sets 6 immobilized on the regions A to G are designed to be different from each other in order to amplify different target sequences. The probe nucleic acid 7 has a sequence different from each other in order to detect a different target sequence for each region. That is, a primer set 6 and a probe nucleic acid 7 having different types of sequences as target sequences and target nucleic acids are immobilized on the primer-immobilized region 5 and the probe-immobilized region 5 arranged in the regions A to G, respectively. It has become.

  Specifically, the positions where the primer set 6 and the probe nucleic acid 7 are immobilized in the regions A to G in each channel are as follows. In FIG. 5A, the primer set 6 and the probe nucleic acid 7 corresponding to the primer set 6 are adjacent to each other on the bottom surface 42 of the channel 41 corresponding to the position of each region, and are arranged along the longitudinal direction of the channel 41. Yes. In FIG. 6A, the primer set 6 is releasably immobilized on one side surface corresponding to the position of each region of the flow path 41. The probe nucleic acid 7 is immobilized on the other side surface of the channel 41 facing the side surface on which the primer set 6 is immobilized. In FIG. 7A, the primer set 6 is releasably immobilized to the same position of the bottom surface 42 corresponding to the position of each region of the flow path 41, and the probe nucleic acid 7 is immobilized.

  FIGS. 5B, 6B, and 7B show the state after the reaction solution is added to the respective flow paths 41 in FIGS. 5A, 6A, and 7A. Shown in When the reaction solution is added to each channel 41, the primer set 6 is released into the reaction solution and diffuses. When the target template nucleic acid is present in the reaction solution, an amplification reaction occurs and an amplification product is produced. In FIG. 5B, FIG. 6B, and FIG. 7B, a region where an amplification reaction has occurred and is proceeding is schematically shown as an amplification region 10. When the target sequence is included in the amplification product produced in the amplification region 10, the target sequence strand and the corresponding probe nucleic acid 7 are hybridized to generate a hybrid signal. 5 (c), 6 (c), and 7 (c) show graphs representing the magnitudes of the hybridization signals detected in the flow path regions A to G, respectively. This graph shows that the sample contained in the added reaction solution contains a target sequence amplified by the primer set 6 immobilized in the regions A, C and F of each flow path, and obtained by the primer set 6. The obtained amplification product shows that the target sequence complementary to the sequence of the probe nucleic acid 7 immobilized in the regions A, C and F is included. That is, in the graphs of FIG. 5C, FIG. 6C, and FIG. 7C, the signals (referred to as “detection signals” in the figure) obtained for the regions A, C, and F are based on the background level. Is a large hybrid signal. Such a result indicates that the target sequence and the target sequence are present in the sample. In contrast, the signals obtained for regions B, D, E and G are below the background level. Based on these results, the target nucleic acid detection method determines that “the target nucleic acid is present in the sample”.

  According to such an embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

  In addition, since the reaction can be performed in the flow path, an array type primer probe chip with better operability is provided.

  Further, by providing the channel 41, it is possible to spread the reaction solution to all the primer sets 6 and the probe nucleic acids 7 more easily and in a short time. In addition, by providing a plurality of channels, it is possible to detect the target nucleic acid for each of a plurality of samples.

<Sixth Embodiment>
An array type primer probe chip according to a sixth embodiment will be described with reference to FIG. FIG. 8A is a plan view of the array-type primer probe chip 1, and FIG. 8B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 8A.

  The array-type primer probe chip 1 includes a support 2, a covering 71, a channel 41, a plurality of primer immobilization regions 4, a plurality of probe immobilization regions 5, a primer set 6, and a probe nucleic acid 7. .

  The flow path 41 is defined by one surface of the support body 2 and a recess formed on one surface of the covering body 71. The channel 41 has a wavy shape in which opposing portions are parallel to each other. The channel 41 has a flow direction from one end to the other end. The covering 71 is disposed in close contact with the support 2 so as to seal the liquid in the flow path 41.

  The plurality of primer immobilization regions 4 are arranged on the bottom surface 42 of the flow path 41 along the flow direction of the flow path 41. The plurality of probe immobilization regions 5 are arranged on the bottom surface 42 of the channel 41 in the vicinity of the corresponding primer immobilization region 4. In each of the plurality of primer immobilization regions 4, a plurality of types of primer sets 6 configured to amplify different target sequences are immobilized in a releasable manner. A plurality of types of probe nucleic acids 7 configured to hybridize to different target sequences are immobilized on each of the plurality of probe immobilization regions 5. Amplification reaction by the primer set 6 fixed to each of the plurality of primer-immobilized regions 4 is performed independently, and amplification products corresponding to the probe nucleic acids 7 respectively fixed to the plurality of probe-immobilized regions 5 The primer-immobilized region 4 and the probe-immobilized region 5 are arranged so that the hybridization with is performed independently.

  The array type primer probe chip 1 is produced as follows, for example. First, a plurality of types of primer sets 6 and corresponding probe nucleic acids 7 are immobilized in a region corresponding to the channel 41 on one surface of the substrate used as the support 2. Next, a covering 71 having a recess formed in a region corresponding to the flow path 41 is bonded to the support 2. At this time, the surface of the cover 71 on which the concave portion is formed is directed to the support 2 side.

  Adhesion of the covering 71 to the support 2 is performed by an adhesive means known per se, such as an adhesive or pressure bonding.

  The addition of the reaction solution to the flow path 41 is performed through the covering 71. Therefore, an opening that penetrates the outside and the inside of the covering 71 is formed as an injection portion 72 at one end of the channel 41 above the covering 71, or a puncturable material such as a resin. Thus, the covering 71 may be formed. When the covering body 71 is formed of a puncturable material, the reaction solution is added by, for example, using a syringe having a sharp tip to puncture the covering body 71 with the needle and reacting in the syringe. What is necessary is just to inject | pour a liquid with respect to a flow path. Further, only a part of the covering 71, for example, a part corresponding to one end part or both end parts of the channel 41 may be made of a puncturable material. Alternatively, there may be a plurality of openings in the covering 71.

  The material of the support 2 and the material of the covering 71 may be the same or different. The material of the support 2 and the covering 71 may be any material that does not itself participate in the reaction, and any material that can perform a nucleic acid reaction such as an amplification reaction there may be used. For example, it may be arbitrarily selected from silicon, glass, resin and metal.

  The support 2 and the covering 71 may be formed according to the material. For example, it may be performed using a molding method known per se, such as etching, extrusion molding, injection molding and / or stamping.

  In this embodiment, the example in which the primer set 6 and the probe nucleic acid 7 are immobilized on the bottom surface 42 of the channel 41 of the array type primer probe chip having one channel with a wavy line shape is shown. However, the shape, number and arrangement of the flow paths are not limited to this. The surface on which the primer set 6 and the probe nucleic acid 7 are immobilized may be any surface constituting the flow path. For example, such a surface may be a primer set 5 and / or a probe nucleic acid set that is circular with respect to the bottom surface of the channel, at least one side surface, a plurality of side surfaces and / or the ceiling surface, or all surfaces constituting the channel. May be fixed.

  In the above example, the array type primer probe chip is formed by using the covering body in which the flow path is formed and the substrate-like support body. However, the flow path may be defined by a recess formed on the support side and a substrate-like covering. For example, a first substrate in which a flow path is formed by forming a recess or a groove in advance may be used as a support, and a second substrate as a covering may be bonded to the support. In that case, a plurality of primer sets are independently and releasably immobilized on at least a part of the wall surface of the flow path, and a plurality of probe nucleic acids are independently immobilized corresponding to the primer sets. Is done. Thereafter, the array-type primer probe chip as shown in FIG. 8 may be manufactured by covering with silicon rubber as the second substrate.

  According to such an embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

  In addition, since the reaction can be performed in the flow path, an array type primer probe chip with better operability is provided.

  Further, by providing the channel 41, it is possible to spread the reaction solution to all the primer sets 6 and the probe nucleic acids 7 more easily and in a short time.

<Seventh Embodiment>
A further example of the array type primer probe chip will be described with reference to FIG. FIG. 9A is a plan view of the array-type primer probe chip 1, and FIG. 9B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 9A.

  The array-type primer probe chip 1 includes a support 2, a covering 71, a channel 41, a plurality of primer immobilization regions 4, a plurality of probe immobilization regions 5, a primer set 6, and a probe nucleic acid 7. .

  The flow path 41 is defined by one surface of the support body 2 and a recess formed on one surface of the covering body 71. The channel 41 has a wavy shape in which opposing portions are parallel to each other. The channel 41 has a flow direction from one end to the other end. The covering 71 is disposed in close contact with the support 2 so as to seal the liquid in the flow path 41.

  Five primer immobilization regions 4 are arranged on one side surface of the channel 41 along the flow direction of the channel 41. Six probe immobilization regions 5 corresponding to one primer immobilization region 4 are arranged in the flow direction of the flow channel 41 in the vicinity of the corresponding primer immobilization region 4 and on the side surface facing the side surface of the flow channel 41. Are arranged along.

  In each of the five primer immobilization regions 4, five types of primer sets 6 configured to amplify target sequences different from each other are releasably immobilized. In each of the five probe immobilization regions 5 corresponding to one primer immobilization region 4, six types of probe nucleic acids 7 configured to hybridize with different target sequences are immobilized. Amplification reaction using the primer set 6 fixed to each of the five primer fixing regions 4 is performed independently, and the amplification product corresponding to the probe nucleic acid 7 fixed to each probe fixing region 5 The primer-immobilized region 4 and the probe-immobilized region 5 are arranged so that the hybridizations are independently performed.

  The array type primer probe chip 1 is produced as follows, for example. First, a covering 71 having a recess formed in a region corresponding to the flow path 41 is produced. The primer set 6 is releasably fixed to the primer fixing region 4 on the side surface of the concave portion of the cover 71. The probe nucleic acid 7 is immobilized on the probe immobilization region 5 disposed on the other side surface facing the side surface of the flow path 41 in which the primer immobilization region 4 is disposed. Next, the obtained covering 71 is bonded to the support 2. At this time, the surface of the cover 71 on which the concave portion is formed is directed to the support 2 side.

  Adhesion of the covering 71 to the support 2 is performed by an adhesive means known per se, such as an adhesive or pressure bonding.

  The addition of the reaction solution to the flow path 41 is performed through the covering 71. Therefore, an opening that penetrates the outside and the inside of the covering 71 is formed as an injection portion 72 at one end of the channel 41 above the covering 71, or a puncturable material such as a resin. Thus, the covering 71 may be formed. When the covering body 71 is formed of a puncturable material, the reaction solution is added by, for example, using a syringe having a sharp tip to puncture the covering body 71 with the needle and reacting in the syringe. What is necessary is just to inject | pour a liquid with respect to a flow path. Further, only a part of the covering 71, for example, a part corresponding to one end part or both end parts of the channel 41 may be made of a puncturable material. Alternatively, there may be a plurality of openings in the covering 71.

  The material of the support 2 and the material of the covering 71 may be the same or different. The material of the support 2 and the covering 71 may be any material that does not itself participate in the reaction, and any material that can perform a nucleic acid reaction such as an amplification reaction there may be used. For example, it may be arbitrarily selected from silicon, glass, resin and metal.

  The support 2 and the covering 71 may be formed according to the material. For example, it may be performed using a molding method known per se, such as etching, extrusion molding, injection molding and / or stamping.

  In this embodiment, an example in which the primer set 6 and the probe nucleic acid 7 are immobilized on the bottom surface 42 of the flow channel 41 of the array type primer probe chip 1 having one wavy line-shaped flow channel is shown. However, the shape, number and arrangement of the flow paths are not limited to this. The surface on which the primer set 6 and the probe nucleic acid 7 are immobilized may be any surface constituting the flow path. For example, such a surface may be a primer set 5 and / or a probe nucleic acid set that is circular with respect to the bottom surface of the channel, at least one side surface, a plurality of side surfaces and / or the ceiling surface, or all surfaces constituting the channel. May be fixed.

  The kind of primer set 6 immobilized on the channel 41, the kind of probe nucleic acid 7 corresponding to one primer set 6, and the number thereof may be arbitrarily selected.

  The distance to the probe immobilization region 5 corresponding to one primer immobilization region 4 may be 0.1 mm to 50 mm, 0.5 mm to 20 mm, and preferably 1 mm to 10 mm.

  In the above example, the array type primer probe chip 1 is formed by using the covering body 71 in which the channel 41 is formed and the substrate-like support body 2. However, the flow path 41 may be defined by a recess formed on the support body 2 side and a substrate-like covering body 71. For example, a first substrate in which a flow path is formed by forming a recess or a groove in advance may be used as the support 2, and the second substrate as the covering 71 may be bonded to the support 2. In that case, a plurality of primer sets are independently and releasably immobilized on at least a part of the wall surface of the flow path, and a plurality of probe nucleic acids corresponding to one primer set are independently immobilized. It becomes. Thereafter, the array-type primer probe chip 1 as shown in FIG. 9 may be manufactured by covering with silicon rubber as the second substrate.

  FIG. 10 schematically shows the state of the nucleic acid reaction in the channel 41 of the array type primer probe chip 1.

  FIG. 10A shows a state in which one primer immobilization region 4 and six probe immobilization regions 5 corresponding thereto are arranged along the flow direction of the channel 41. The primer immobilization region 4 is disposed on one side surface of the channel 41. The six probe immobilization regions 5 are disposed on the other side surface of the channel 41. The primer immobilization region 4 is disposed so as to face the first probe immobilization region 5a disposed closest to the injection portion 72 opened near the end of the flow path 41. The second probe immobilization region 5b, the third probe immobilization region 5c, the fourth probe immobilization region 5d, the fifth probe immobilization region 5e, and the sixth probe immobilization region 5f are primer immobilization regions. 4 and the first probe immobilization region 5 a are disposed downstream, and are disposed downstream along the flow direction of the flow path 41.

  FIG. 10B shows a state after the reaction solution 8 is added to the flow path 41 from the injection portion 72. When contacted with the reaction solution 8, the primer set immobilized on the primer immobilization region 4 is released and diffuses. When the target sequence to be amplified by the primer set is present in the reaction solution 8, an amplification reaction occurs. The free and diffused state of the primer set and / or the state in which the amplification reaction has occurred is schematically indicated by regions 9 and 10. When the target sequence to be hybridized with the probe nucleic acid 7a immobilized in the first probe immobilization region 5a is present in the amplification products contained in the regions 9 and 10, the probe nucleic acid 7a and the amplification product Hybridization occurs between them.

  As time elapses, the regions 9 and 10 diffuse and / or move downstream of the flow path 41 due to the diffusion action and / or the flow action of the reaction liquid present in the flow path 41. A state in which such diffusion and / or movement of the regions 9 and 10 occurs is schematically shown in FIG.

  As the amplification products existing in the regions 9 and 10 diffuse and / or move, the amplification products contact with the second probe nucleic acid 7b to the sixth probe nucleic acid 7f. As a result, when the target sequence to which these probe nucleic acids are to be hybridized is included in the amplification product, the second probe nucleic acid 5b, the third probe nucleic acid 5c, the fourth probe nucleic acid 5d, the fifth probe nucleic acid Hybridization occurs between the probe nucleic acid 5e and / or the sixth probe nucleic acid 5f and the amplification product. In the method for detecting the target nucleic acid, the hybridized signal generated in this way is detected.

  In the above example, the primer immobilization region 4 and the first probe immobilization region 5a are opposed to each other and arranged at the same position on the flow direction axis of the channel 41. However, the primer immobilization region 4 may be arranged upstream (closer to the injection part) than any corresponding probe immobilization region. In the above example, any probe immobilization region 5 is disposed on the other side surface opposite to the side surface on which the primer immobilization region 4 is disposed, but the present invention is not limited to this. If the probe immobilization region 5 is in the vicinity of the primer immobilization region 4 and arranged downstream of the primer immobilization region 4, any inner surface, for example, the bottom surface, one or more side surfaces, and / or Or you may arrange | position on a ceiling surface. For example, the probe immobilization region 5 may be disposed on the same side as the primer immobilization region 4.

  A primer set 6 is releasably immobilized in the primer immobilization region 4. Different types of probe nucleic acids 7 are immobilized on the six probe immobilization regions 5.

  According to such an embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field. This makes it possible to perform a target inspection in a shorter time than in the past. In addition, the possibility that a sample may be mixed is reduced.

  In addition, since the reaction can be performed in the flow path, an array type primer probe chip with better operability is provided.

  Further, by providing the channel 41, it is possible to spread the reaction solution to all the primer sets 6 and the probe nucleic acids 7 more easily and in a short time.

<Eighth Embodiment>
The array-type primer probe chip according to the eighth embodiment will be described with reference to FIGS.

(1) Chip Material First, an example of the structure and manufacturing method of the chip material of an array-type primer probe chip that detects a hybridization signal by electrochemical detection will be described with reference to FIGS. 11 (a) and 11 (b). 11A is a plan view of the chip material, and FIG. 11B is a cross-sectional view taken along line BB of the chip material of FIG.

  The chip material 111 includes, for example, four electrodes 113a to 113d arranged on a rectangular substrate 112 along the longitudinal direction thereof. Each of the electrodes 113a to 113d has a structure in which a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order. Each of the electrodes 113a to 113d has a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118. The insulating film 119 is covered on the substrate 112 including the electrodes 113a to 113d. The circular window 120 is opened at the insulating film 119 portion corresponding to the large rectangular portion 116. The rectangular window 121 is opened at a portion of the insulating film 119 corresponding to the small rectangular portion 117. The large rectangular portion 116 exposed from the circular window 120a of the electrode 113a functions as the first working electrode 122a. The large rectangular portion 116 exposed from the circular window 120b of the electrode 113b functions as the second working electrode 122b. The large rectangular portion 116 exposed from the circular window 120 c of the electrode 113 c functions as the counter electrode 123. The large rectangular portion 116 exposed from the circular window 120d of the electrode 113d functions as the reference electrode 124. The small rectangular portion 117 exposed from the rectangular window 121 of the electrodes 113a to 113d functions as a prober contact portion.

  Such a chip material can be manufactured by the following method.

  First, a first metal thin film and a second metal thin film are deposited on the substrate 112 in this order by, for example, a sputtering method or a vacuum evaporation method. Subsequently, these metal thin films are sequentially selectively etched using, for example, a resist pattern as a mask, and a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order, for example, four electrodes 113a to 113a. 113 d is formed along the longitudinal direction of the substrate 112. These electrodes 113 a to 113 d have a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118.

  Next, an insulating film 119 is deposited on the substrate 112 including the electrodes 113a to 113d by, for example, a sputtering method or a CVD method. Subsequently, the insulating film 119 portion corresponding to the large rectangular portion 116 of each of the electrodes 113a to 113d and the insulating film 119 portion corresponding to the small rectangular portion 117 are selectively etched using the resist pattern as a mask to form the large rectangular portion 116. A circular window 120 is opened in the corresponding insulating film 119 portion, and a rectangular window 121 is opened in the insulating film 119 portion corresponding to the small rectangular portion 117. Thereby, the above-described chip material 111 is produced.

  The substrate 112 is made of glass or resin such as Pyrex (registered trademark) glass, for example.

  The first metal thin film functions as a base metal film for bringing the second metal thin film into close contact with the substrate 112, and is made of, for example, Ti. The second metal thin film is made of, for example, Au.

  Examples of etching when patterning the first and second metal thin films include plasma etching using an etching gas or reactive ion etching.

  Examples of the insulating film 119 include a metal oxide film such as a silicon oxide film and a metal nitride film such as a silicon nitride film.

  Examples of etching when patterning the insulating film 119 include plasma etching using an etching gas or reactive ion etching.

(2) Array-type Primer Probe Chip Next, an example of the configuration and manufacturing method of an array-type primer probe chip in which a primer set and a probe nucleic acid are fixed to the chip material 111 manufactured in (1) above is shown in FIG. This will be described with reference to FIG. FIG. 12A is a plan view of the array-type primer probe chip 1, and FIG. 12B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG.

  The first working electrode 122a of the electrode 113a formed on the chip material 111 is defined as a first probe immobilization region 201a, and the first probe immobilization region 201a includes a first sequence that includes a complementary sequence of the first target sequence. The probe nucleic acid 202a is fixed. A plurality of the first probe nucleic acids 202a to be immobilized are immobilized as one probe nucleic acid group. Similarly, the second working electrode 122b of the electrode 113b is used as a second probe immobilization region 201b, and a complementary sequence of a second target sequence different from the first target sequence is provided in the second probe immobilization region 201b. The contained second probe nucleic acid 202b is fixed.

  Examples of the method for immobilizing the probe nucleic acids 202a and 202b to the probe immobilization region 201 include a method for introducing a thiol group at the 3 ′ end into the first probe nucleic acid 202a for the chip material 111 having a gold electrode. .

  Next, the first primer immobilization region 203a is disposed in the vicinity of the first working electrode 122a, and the second primer immobilization region 203b is disposed in the vicinity of the second working electrode 122b. The first primer set 204a is fixed on the first primer fixing region 203a, and the second primer set 204b is fixed on the second primer fixing region 203b. Thereby, the array type primer probe chip 1 is produced.

  The first primer set 204a has a sequence designed to amplify the first target sequence, and the second primer immobilization region 204b is a second target sequence comprising a sequence different from the first target sequence. Has a sequence designed to amplify.

  The first and second primer sets 204a and 204b are immobilized on the first and second primer immobilization regions 203a and 203b, respectively. For example, the primer set 203 is contained in a liquid such as water, a buffer solution, or an organic solvent. Then, it is dropped, and then, for example, it is left for 10 minutes in the case of room temperature until drying under an appropriate temperature condition such as room temperature.

(3) Array type primer probe chip at the time of use The usage method of the array type primer probe chip 1 produced in said (2) is demonstrated, referring FIG. 13 and FIG.

  FIG. 13A is a plan view of the array-type primer probe chip 1 in use, and FIG. 13B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. It is.

  When the array type primer probe chip 1 of the present embodiment is used, the first working electrode 122a, the second working electrode 122b, the counter electrode 123, the reference electrode 124, and the first primer formed on the electrodes 113a to 113d, respectively. The reaction solution is maintained so that the immobilization region 203a and the second primer immobilization region 203b are included in the same one reaction field. Therefore, the covering 301 is mounted on the array type primer probe chip 1 before using the array type primer probe chip 1. The covering 301 is a rectangular parallelepiped that is hollow and has one surface open. The covering 301 is made of, for example, a silicon resin such as silicon rubber and / or a resin such as a fluororesin, and is made of such a resin by, for example, extrusion molding, injection molding or stamping and / or, for example, It is formed by any known resin molding method such as bonding of each member with an adhesive. After the covering 301 is mounted, the reaction solution 302 is added to the space defined by the array type primer probe chip 1 and the covering 301. The reaction solution 302 includes a template nucleic acid 303.

  In the array-type primer probe chip 1 to which the covering 301 is attached, the small rectangular portions 117 exposed from the rectangular windows 121 of the electrodes 113a to 113d are exposed.

  Examples of the method of attaching the covering 301 to the array type primer probe chip 1 include, for example, pressure bonding, adhesion with an adhesive, and the like.

  The reaction solution 302 is added after the covering 301 is mounted on the array type primer probe chip 1.

  An example of a method of adding a liquid such as a reaction solution to a space formed by the array-type primer probe chip 1 and the covering 301 is, for example, that an opening is provided in advance in a part of the covering 301 and the opening Or using a syringe having a sharp tip such as a needle, piercing the covering body 301 with the needle, inserting the needle into a part of the covering body 301, and the like. Is mentioned.

  The reaction solution 302 is used when a sample, an amplification reagent, for example, an enzyme such as a polymerase, a primer, and a substrate such as oxynucleoside triphosphate, which are necessary for forming a new polynucleotide chain, and reverse transcription simultaneously. Includes a reverse transcriptase and its necessary substrates, as well as buffers such as salts to maintain an appropriate amplification environment and an intercalator that recognizes a double stranded nucleic acid such as Hoechst 33258 and generates a signal. including. When the template nucleic acid containing the target sequence to be amplified by the primer set immobilized on the specific primer immobilization region is present in the sample to be examined, the primer immobilization region and the corresponding probe immobilization region are included. An amplification product is formed in the reaction field. This is schematically shown in FIG.

  FIG. 14A schematically shows a state in which an amplification product is formed in the region 401 of the reaction solution 302 that is a reaction field. FIG. 14A is a plan view of the array-type primer probe chip 1 in use, and FIG. 14B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. It is. As described above, the sample added in FIG. 13 contains a nucleic acid containing a sequence that can be bound by the second primer set 204b. Therefore, as shown in FIGS. 14 (a) and 14 (b). The second primer set 204b is released and diffused in the region 401, and after encountering the template nucleic acid 303, an amplification reaction occurs and an amplification product is formed. The amplification product by the second primer set 204b diffuses around the second primer immobilization region 203b and reaches the second probe immobilization region 201b. When the reached amplification product includes the target sequence, the second probe nucleic acid 202b and the amplification product are hybridized to form a double-stranded nucleic acid. The intercalator contained in the reaction solution 302 is combined with this double-stranded nucleic acid to generate a hybrid signal.

  The hybrid signal is generated by, for example, bringing a prober into contact with the small rectangular portion 117 exposed from each rectangular window 121 of the electrodes 113a to 113d and measuring the current response of an intercalator such as Hoechst 33258.

  By using an array-type primer probe chip that utilizes electrochemical detection, the target nucleic acid contained in the amplification product can be detected after the target nucleic acid contained in the sample has been amplified more easily and in a short time. Is possible.

<Method of detecting target nucleic acid>
A method for amplifying a plurality of target nucleic acids using the array type primer probe chip as described above as an example and detecting a target nucleic acid using a hybridization signal as an index is also provided as a further embodiment.

The method for detecting the target nucleic acid comprises, for example, the following (a) to (g);
(A) At least one surface of a support configured to support one reaction field in a liquid phase, and when the reaction field is formed by the liquid phase, the surfaces contacting the reaction field are mutually Immobilizing a plurality of types of primer sets for amplifying a plurality of types of target nucleic acids in each of a plurality of independent immobilization regions in a releasable manner,
(B) The probe-immobilized region at or near the plurality of primer-immobilized regions includes a complementary sequence of the target sequence for each type so that a hybridization signal can be detected independently for each probe-immobilized region. Immobilizing at least one probe nucleic acid;
(C) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(D) bringing a sample into the reaction field;
(E) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field;
(F) independently detecting the presence or amount of hybridization between the amplification product obtained by the amplification reaction and the probe nucleic acid for the amplification product obtained by each of the primer sets;
(G) detecting or measuring the presence of the target nucleic acid containing the target sequence based on the result of (f).

  In addition, multiple types of primer sets designed to amplify multiple types of target nucleic acids are released to at least one surface of a support such as a substrate on which a specific container, tube, dish or flow path is formed. A method for detecting a target nucleic acid comprising the step of immobilizing and / or the step of immobilizing one or more types of probe nucleic acids to a probe immobilization region is also provided as a further embodiment.

  Such a method for detecting a target nucleic acid may comprise, for example, the following steps: releasably fixing a plurality of types of primer sets to at least one surface of a desired support, wherein The plurality of types of primer sets are designed to amplify a plurality of types of target nucleic acids, respectively, and include at least a complementary sequence of the target sequence at the position of the plurality of primer immobilization regions or the probe immobilization region in the vicinity thereof. Immobilizing one type of probe nucleic acid, wherein a plurality of probe nucleic acids are immobilized for each type so that a hybridization signal can be independently detected for each probe immobilization region; The reaction solution is added so as to be included in one reaction field, where the reaction solution is a reagent necessary for amplification, for example, an enzyme such as a polymerase, a Necessary for the formation of a new polynucleotide chain starting from the mer, if oxynucleoside triphosphate, etc., and reverse transcription are performed simultaneously, reverse transcriptase and the necessary substrate, etc. Including a buffer such as salts for maintaining the environment: bringing the sample into the reaction field, where the bringing in may be performed, for example, by adding to the reaction solution or the array-type primer probe chip; Adjusting the reaction environment, where the adjustment may be to achieve a suitable environment for the amplification reaction, such as temperature control by heating or cooling the support on which the primer is immobilized; multi-nucleic acid amplification reaction And the presence or absence of hybridization between the amplification product generated by the multi-nucleic acid amplification reaction and at least one type of probe nucleic acid, and Or amount detection and / or measuring that.

  A specific amplification reaction may be performed using a technique known per se according to the type of the amplification reaction.

  Specific detection means include known hybrid signal detection means, for example, detection and / or measurement of fluorescence intensity using a fluorescent label, or method of detecting and / or measuring current response using an intercalator May be used.

  Furthermore, a step of releasably fixing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on the surface of a substrate such as a microbead, a plate piece or a rod, and a plurality of primer immobilizations Immobilizing at least one type of probe nucleic acid containing a complementary sequence of the target sequence for each type so that a hybridization signal can be independently detected for each probe-immobilized region in the probe-immobilized region at or near the region. A method for detecting a target nucleic acid comprising: is also provided as a further embodiment.

  Such a nucleic acid detection method includes, for example, releasably fixing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on at least one surface of a desired substrate. And at least one type including a complementary sequence of the target sequence for each type in the probe-immobilized region at or near the position of the plurality of primer-immobilized regions so that the hybridization signal can be detected independently for each probe-immobilized region A substrate, such as oxynucleoside triphosphate, which is necessary for forming a new polynucleotide chain starting from a reagent necessary for amplification, for example, an enzyme such as a polymerase or a primer. When reverse transcription is performed at the same time, reverse transcriptase and its necessary substrates, such as salts for maintaining an appropriate amplification environment In a reaction solution containing a buffer such as; adding a reaction solution containing a buffer such as salts to maintain an appropriate amplification environment; and adding the sample to the reaction field by addition to the reaction solution Bringing in; adjusting the reaction environment suitable for the amplification reaction, such as temperature adjustment by heating or cooling the reaction solution; thereby carrying out the multi-nucleic acid amplification reaction; amplification products generated by the multi-nucleic acid amplification reaction Detecting and / or measuring the presence and / or amount of hybridization between and at least one probe nucleic acid.

  For example, according to the array-type primer probe chip shown in the embodiment, amplification of a plurality of types of target sequences can be performed independently and simultaneously without receiving interference from different sequences. Furthermore, the presence or absence and / or the amount of the target nucleic acid can be detected and / or measured for the amplification product generated by the amplification reaction in the same reaction field where the amplification reaction is performed simultaneously with or subsequent to the amplification reaction. it can.

  In the prior art, when multiplex amplification is performed using a plurality of types of primers in one container, there is a problem that the reaction efficiency is biased and the number of types is limited. In other words, necessary enzymes and dNTPs may be intermingled between different types of primers. In addition, the reaction specificity and / or reaction efficiency may vary depending on the sequence of the target sequence and the sequence of the primer. In that case, the amplification reaction start point varies depending on the type of primer, only the amplification for some primer sets is started and advanced, or the amplification for some primer sets is not sufficiently achieved, etc. Problems arise. Such conventional problems are also solved by the embodiments disclosed herein.

  That is, when an amplification reaction is performed using the array-type primer probe chip illustrated in the embodiment, the amplification reaction proceeds only in the vicinity of the fixed amplification reagent. Therefore, it is possible to independently carry out amplification reactions of various targets without interfering with each other's amplification reaction while being in the same container and / or the same solution. Moreover, the presence and / or amount of the target nucleic acid can be detected and / or measured in the same reaction vessel as that in which the amplification reaction was performed simultaneously or subsequently with such an amplification reaction. In such a method, a different primer set may be added after individual amplification reactions have progressed to some extent. You may use combining the array-type primer probe chip of the container form shown in 1st Embodiment, and said array-type primer probe chip carrier.

[Example]
<Example 1>
Example 1-1
Below, the example which produced and used the array-type primer probe chip | tip for electrochemical detection is described. An array-type primer probe chip for electrochemical detection includes a primer set fixed to a primer fixing region and a probe DNA as a probe nucleic acid fixed to a probe fixing region in the vicinity of the primer fixing region. The probe immobilization region was composed of electrodes and was used as a sensor for detecting a current response generated depending on the presence of hybridization.

(1) Production of Chip Material A schematic diagram of a chip material for an array type primer probe chip is shown in FIG. Titanium and gold thin films were formed on the Pyrex glass surface by sputtering. Thereafter, titanium and gold electrodes were formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and a circular window and a rectangular window were opened in the insulating film by an etching process to expose the working electrode, the counter electrode, the reference electrode, and the prober contact portion. This was used as a chip material for an array type primer probe chip.

(2) Preparation of array type primer probe chip First, probe DNA was fixed on the working electrode. Table 1 shows the base sequence of the probe DNA used.

  A probe DNA solution containing 3 μM each of probe DNA (A) and probe DNA (B) was prepared, and 100 nL of this solution was spotted on the working electrode. It dried at 40 degreeC and wash | cleaned with the ultrapure water. Thereafter, ultrapure water remaining on the surface of the working electrode was removed, and the probe DNA was immobilized on the working electrode of the chip material.

Next, primer DNA used as a primer set was prepared. The primer DNA to be used is a primer set for amplification by a loop-mediated isal amplification (LAMP) method. Table 2 shows the base sequences of the primer DNAs used.

  For the primer DNA (Set A), 200 μM FIP, BIP, F3, B3, and LPF were prepared, each containing 0.1 μL, 0.1 μL, 0.0125 μL, 0.00125 μL, and 0.05 μL. 275 μL of solution was used. These were immobilized on the working electrode, which is a primer immobilization region in the vicinity of the corresponding probe DNA (A). Specifically, 0.275 μL of each prepared solution was spotted on the corresponding nearby working electrode to which the probe DNA (A) was fixed. This was dried at 63 ° C. for 5 minutes. Thereby, an array type primer probe chip was obtained.

(3) Preparation of LAMP reaction solution Table 3 shows the composition of the LAMP reaction solution.

Compositions (1) to (4) commonly include Bst DNA polymerase and reaction mix. Distilled water (that is, DW) was added to these so that the total amount was 50 μL together with the template solution described later. The composition (1) includes a template B that is detected by hybridization with the probe DNA (B) in which an amplification reaction by the LAMP method is caused by the primer DNA (set B). Composition (2) does not contain a template. Composition (3) contains template A. Template A undergoes an amplification reaction by the LAMP method with the primer DNA (set A) and hybridizes with the probe DNA (A). Composition (1) includes both template A and template B. These were used as LAMP reaction solutions. Templates A and B are synthetic oligo DNAs having the base sequences shown in Table 4.

(4) LAMP amplification reaction on array type primer probe chip and detection of target nucleic acid by probe DNA A covering was attached to the array type primer probe chip as schematically shown in FIG. The space constituted by the array type primer probe chip and the covering includes an electrode as a probe immobilization region and a primer DNA immobilization region. This space functions as a reaction vessel. The covering is a molded silicon rubber. The LAMP reaction solution was injected into the reaction vessel through a hole provided in advance in the silicon rubber. Then, the hole of the covering was covered. This was set | placed on the plate set to 63 degreeC, and LAMP reaction was performed for 60 minutes.

  As schematically shown in FIG. 14, when a LAMP reaction solution contains a template that can bind to a specific primer and contains the target sequence to be amplified, such specific primer is immobilized. The LAMP reaction proceeds locally at the location. The resulting LAMP product hybridizes with the nearby probe DNA.

  After the LAMP reaction for 60 minutes, a hybridization reaction was performed at 45 ° C. for 10 minutes, and washing was performed at 45 ° C. for 10 minutes. Thereafter, the washing solution was removed and a 75 μM Hoechst 33258 solution was injected. The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecules specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The series of reactions described above was carried out using an automatic DNA test apparatus described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008.

(5) Detection results The detection results are shown in Table 5.

  When the LAMP reaction solution composition (1) containing the template B was added, a current value of 70 nA was obtained. That is, the LAMP reaction with the primer DNA (set B) immobilized in the vicinity of the working electrode 2 proceeded, and the resulting LAMP product reacted with the probe DNA (B).

  On the other hand, no current value was obtained with the primer DNA (set A) immobilized in the vicinity of the working electrode 2. That is, the LAMP reaction did not progress.

  From these two numerical values, it was possible to determine that the template B was contained in the LAMP reaction solution.

  In addition, when the LAMP reaction solution composition (2) not containing the template was added, no current value was obtained. That is, the LAMP amplification reaction by the primer DNA (set A) and the primer DNA (set B) immobilized in the vicinity of the working electrodes 1 and 2 did not proceed.

  From the above results, it was shown that the template contained in the LAMP reaction solution can be detected and the sequence thereof can be identified using the array-type primer probe chip described in this example.

Example 1-2
In the following, an experiment was conducted to prove that 10 types of LAMP multi-amplification can be performed simultaneously in one container. This will be described below.

(1) Production of multi-amplification container As a multi-amplification container, a reaction container was prepared in which only a plurality of types of primers were immobilized independently and advantageously.

The base sequences of the primers used are shown in Table 6-1 and Table 6-2.

  For each primer set, a TE solution containing 40 pmol FIP, 40 pmol BIP, 40 pmol F3, 5 pmol B3, and 20 pmol LP was prepared. 0.6 μL of TE solution was spotted on the glass substrate surface. It was left at room temperature for 10 minutes. Thereby, the primer was fixed by drying. A silicon rubber having a channel formed in advance was filled on the substrate on which the primer was dried and fixed. In this way, a multi-amplification container having a primer spotted in the flow path was produced. This has the same configuration as the array-type primer probe chip of the embodiment shown in FIG. 7 except that it does not contain probe nucleic acid (FIG. 7).

(2) Preparation of LAMP reaction solution for multi-amplification container and LAMP reaction Table 2 shows the composition of the LAMP reaction solution for multi-amplification container.

  Template No. contained in this reaction solution The base sequences 1 to 10 are shown in Table 8-1, Table 8-2, Table 8-3, Table 8-4, Table 8-5, Table 8-6, Table 8-7, Table 8-8, Table 8-. 9 and Table 3-10.

  50 μL of a multi-amplification container LAMP reaction solution was injected into the flow path of the multi-amplification container, and placed on the hot plate so that the glass surface was in contact with the hot plate set at 63 ° C., and the LAMP reaction was performed for 1 hour. .

(3) Preparation of LAMP reaction solution for PCR tube and LAMP reaction Table 9 shows the composition of the LAMP reaction solution for PCR tube. This reaction solution was set in a thermostat set at 63 ° C., and a LAMP reaction was performed for 1 hour.

(4) Preparation of DNA chip for detection of LAMP amplification product The probe nucleic acids (3′-end SH-labeled synthetic oligos) shown in Table 10 were synthesized.

  Probe nucleic acid No. No. 1 is primer set No. 1 is a probe nucleic acid for detecting the LAMP amplification product obtained from No. 1. 2 to 10 are primer set Nos. A probe nucleic acid for detecting a LAMP amplification product obtained from 2 to 10. A DNA chip was prepared by spotting 100 nL of a solution containing 3 uM of each probe nucleic acid on the electrode and drying and fixing the solution. As the DNA chip and the DNA chip measuring apparatus, those described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008 were used.

(5) Detection of LAMP amplification product The LAMP amplification product amplified in the multi-amplification container and the LAMP amplification product amplified in the PCR tube were detected with a DNA chip, and the amplification product amplified in each amplification container and tube was confirmed. . The results are shown in Table 11.

  When PCR tubes were used, only 3 out of 10 types were positive and the other 7 types were negative. On the other hand, when the multi-amplification container was used, all 10 types became positive. This showed that 10 types of multi-amplification reactions were possible. In the PCR tube, primer set No. 1-10 and mold no. 1-10 are included, in which the LAMP amplification reaction proceeds. It was thought that when several types of LAMP amplification reactions were started among the 10 types of primer sets, enzymes (Bst DNA Polymerase) and the like were consumed for the reaction, and the efficiency of other types of LAMP amplification reactions decreased. . On the other hand, in the multi-amplification container, primer set No. 1-10 and mold no. 1-10 are included, in which the LAMP amplification reaction proceeds. By spotting the primer, the LAMP amplification reaction was started at each spot position in the flow path, and it was expected that the amplification reaction by various primers proceeded independently.

Example 1-3
Hereinafter, an example of an array type primer probe chip in which a probe nucleic acid is immobilized on a probe immobilization region present downstream of the reaction solution injection port from the primer immobilization region will be described.

(1) Production of chip material A chip material similar to the chip material shown in Fig. 11 was formed except that 10 working electrodes were included. First, a titanium and gold thin film was formed on the surface of Pyrex glass by sputtering. Thereafter, an electrode pattern of titanium and gold was formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and electrodes, that is, a working electrode, a counter electrode, a reference electrode, and a probe electrode were exposed by an etching process. This was used as a chip material for a multi-nucleic acid amplification detection reaction tool.

(2) Production of multinucleic acid amplification detection reaction tool Probe DNA was immobilized on the working electrode of the chip material produced as described above. The base sequence of the probe DNA used is represented by SEQ ID NO: 2 shown in Table 1 and is a probe DNA (B) having the following sequence: TTTGGTGCAATGGATTTTACTACATTACAAGCTA-SH.

  The probe DNA (B) was immobilized as follows. A probe DNA solution containing 3 μM of probe DNA (B) was prepared, and 100 nL of this solution was spotted on all working electrodes. After drying at 40 ° C., it was washed with ultrapure water. Thereafter, ultrapure water remaining on the surface of the working electrode was removed, and a DNA chip in which the probe DNA was immobilized on the electrode of the chip material was produced.

  Next, a covering made of a silicon rubber plate was prepared as described above. On one surface of the covering, a groove is formed at a position corresponding to the probe fixing region.

  The primer set was fixed to the bottom surface of the groove of the covering. The immobilization region of the primer set was adjusted so as to be a position close to one of the two through holes present in the covering.

  First, primer DNA to be used was prepared. The primer DNA to be used is a primer set for amplification by a loop-mediated isal amplification (LAMP) method. The base sequence of the primer DNA is the same as that shown in Table 2 as primer set B.

  200 μM FIP, BIP, F3, B3 and LP are prepared as the primer DNAs of the primer set B, respectively, and 0.02 μL, 0.02 μL, 0.0025 μL, 0.0025 μL and 0.01 μL are included in each of 0. 055 μL of solution was prepared.

  Specifically, 0.055 μL of the primer mixed solution was spotted on the bottom of the groove of the silicon rubber that is the covering, and dried at 40 ° C. for 2 minutes. Two through holes are opened at the two ends of the silicon rubber groove, which is a covering, and the spot is either of the two through holes when the covering is attached to the DNA chip. It was performed so as to be in a position facing the probe DNA adjacent to the. The covering and the chip material prepared above were bonded so that the groove portion of the covering and the surface on which the probe DNA was immobilized faced each other. Thereby, a nucleic acid amplification detection reaction tool was obtained.

(3) Preparation of LAMP reaction solution The LAMP reaction solution used was prepared with the composition shown in Table 3.

  The LAMP reaction solution of composition (1) contains a reaction mix such as deoxyribonucleic acid necessary for the LAMP reaction and template B shown in Table 4, and distilled water (ie, DW) so that the total amount becomes 50 μL. Was added. This was used as a LAMP reaction solution.

  The template B is LAMP amplified by the primer DNA (set B) that is dried and fixed in the groove of the covering. The resulting amplification product hybridizes with the probe DNA (B).

(4) LAMP amplification reaction on array type primer probe chip and detection of target nucleic acid by probe DNA One of the two through holes provided in the silicon rubber plate as a coating was used as an inlet. A flow path constituted by a groove provided on the silicon rubber plate and one surface of the chip material was used as a reaction part. Reaction was performed in the reaction part. The LAMP reaction solution was injected into the reaction part from the inlet. Immediately thereafter, a multi-nucleic acid amplification detection reaction tool was installed in the automatic DNA testing apparatus. A LAMP reaction was performed at 64 ° C. for 60 minutes on a Peltier in an automatic DNA testing apparatus.

  After the LAMP reaction for 60 minutes, a hybridization reaction was performed at 45 ° C. for 10 minutes, and washing was performed at 30 ° C. for 3 minutes. Thereafter, the washing solution was removed and a 20 μM Hoechst 33258 solution was injected from the inlet.

  The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecules specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The series of reactions described above was carried out using an automatic DNA test apparatus described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008.

(5) Detection results The detection results are shown in FIG.

  For the test, as described above, a DNA chip having the same type of probe DNA immobilized thereon was used. A LAMP reaction solution was injected into the reaction part of this DNA chip from the inlet, and amplification and detection reactions were performed in the reaction field. As a result, the highest current value of about 50 nA was obtained at the probe-immobilized electrode at the fourth position from the inlet.

  As a result, since the primer DNA (set B) was immobilized at a position close to the inlet, the primer DNA was eluted together with the injection of the LAMP reaction solution, and the LAMP reaction proceeded downstream from the immobilized position. Alternatively, it is considered that the amplified product after the LAMP reaction was eluted downstream from the immobilization position.

  The above results show that the position of the injection part is the most upstream of the flow path, and is higher from the probe nucleic acid immobilized in the probe immobilization region disposed about 1 mm to about 10 mm downstream from the position of the primer immobilization region. It shows that a current value was obtained.

3. Use of Thickener <Ninth Embodiment>
When the reaction is performed using the multi-nucleic acid reaction tool, a thickener may be present in the reaction solution. If a thickener is included in the reaction solution, it remains in local diffusion after the immobilized primer is released. The thickener is preferably a substance that does not inhibit the amplification reaction. Details of the thickener will be described later.

  In order to achieve the amplification reaction more efficiently, means for controlling the rate of addition of the reaction solution to the reaction field is also effective. For example, the flow rate of the reaction solution flowing over the primer immobilization position is preferably 1 mm / second or more, and more preferably 10 mm / second or more. Thereby, since the release of the immobilized primer set is affected, the primer set can be diffused more locally. However, the flow rate of the reaction liquid flowing on the primer immobilization position can take an arbitrary value depending on the shape and size of the reaction part constituted by the support and other members that define the reaction field.

  For example, when performing a multi-amplification reaction using the embodiment, it is possible to achieve a multi-amplification reaction more efficiently by including a thickener in the reaction solution to be used.

  For the method of adding a thickener to the reaction solution, a thickener may be added to the reaction solution itself, or when the primer set is fixed to the support, the solution for fixing the primer set is thickened. By including the agent, the thickener may be fixed to the primer fixing region together with the primer set to be used. Alternatively, after the primer is fixed, the primer fixing region may be further coated with a thickener. Furthermore, means such as attaching a film-like thickener may be used, and there is no particular limitation.

  By including a thickener in the reaction solution, it is possible to reduce the rate at which the fixed primer set is released into the reaction solution, that is, the elution rate. Thereby, the primer set remains localized for a longer time. As a result, multi-amplification with a plurality of primer sets can be achieved more efficiently without being influenced by the reaction efficiency of each fixed primer set.

  The thickener is preferably a substance having a specific viscosity greater than that of the primer and does not inhibit the nucleic acid amplification reaction. Examples of thickeners are derived from seeds such as beans, such as thickeners derived from sap, such as almond gum, Elemi resin, dammar resin, gum arabic, Karaya gum, tragacanth gum, arabinogalactan, gati gum and peach resin Thickeners such as ama seed gum, guar gum enzyme degradation product, tamarind seed gum, cassia gum, silum seed gum, tara gum, carob bean gum = locust bean gum, mackerel mugwort seed gum, triacanthus gum, guar gum and sesbania gum Thickeners derived from fruits, leaves, rhizomes, etc., such as aloe vera extract, Kidachi aloe extract, pectin, okra extract and troaroao Such as aeromonas gum, enterobacter gum, natto gum, aureobasidium broth, curdlan, pullulan, azotobacter vinelandie gum, xanthan gum, macrohomopsis gum, welan gum, gellan gum, lamb zan gum, erwinia Mitsiensis gum, sclerogum, levan, Enterobacter simanas gum and dextran, and other thickening stabilizers such as yeast cell membrane, chitin, oligoglucosamine, microfibrous cellulose, chitosan, microcrystalline cellulose and glucosamine. Furthermore, the thickener is generally used as a food or drink additive, and may be, for example, agar, soybean polysaccharide, nata de coco, starch, konjac potato extract, gelatin or the like. Preferred are agarose such as agarose and / or gelatin, polyethylene glycol and the like, but are not limited thereto.

  When adding a thickener to a reaction liquid, you may melt | dissolve a thickener directly with respect to the solvent for producing a reaction liquid at the time of preparation of a reaction liquid. Alternatively, first, a thickener solution prepared by dissolving a thickener in a solvent is prepared. Separately, a reaction solution is prepared by dissolving other components necessary for the reaction in a solvent. You may mix the obtained thickener solution and reaction liquid. The solvent may be water, brine, buffer solution, and the like.

  When adding a thickener to the solution which fixes a primer set, it is preferable that the density | concentration of a thickener is a state which is a liquid at room temperature (25 degreeC), and can be dripped at a reaction field. The concentration of the thickener may be in the range of about 30% to 0.01% final concentration, for example. For example, in the case of agarose, the final concentration may be in the range of 10% to 0.01%, more preferably in the range of 5% to 0.05%, and mixing in the range of 3% to 0.1%. Is more preferable. The same may be applied when a thickener is fixed to the primer fixing region.

  Moreover, when adding a thickener to a reaction liquid, it is preferable that the density | concentration of a thickener is a liquid at room temperature (25 degreeC). The concentration of the thickener may be in the range of about 30% to 0.01% final concentration, for example. For example, in the case of agarose, the final concentration may be in the range of 10% to 0.01%, and more preferably in the range of 5% to 0.1%.

  In the conventional method, in the case of preparing a reaction container for amplifying each target gene, the number of necessary reaction containers and the amount of work at the time of testing increase as the number of target genes increases. In addition, when reagents for detecting all target genes are put in one reaction container, the number of target genes is limited due to factors such as bias in amplification reaction efficiency. In addition, in the case of conventional multi-amplification, when multiple types of primer sets are eluted and diffused, there is a risk that amplification efficiency decreases due to non-specific binding between primers, or only primer sets with good characteristics preferentially amplify. There is a decrease in sensitivity of amplification reaction, restriction of amplification type, and the like. Such a problem is also solved by this embodiment.

<Tenth Embodiment>
Although the example which contains a thickener in the reaction liquid was shown above, you may fix a thickener to the area | region where a primer set is fixed at least without including a thickener in a reaction liquid. An example of fixing the thickener to the tenth embodiment will be described below.

  When the thickener is fixed on the support, it may be before fixing the primer set, at the same time as fixing the primer set, or after fixing the primer set. Preferably, the thickener is fixed to the support simultaneously with the fixing of the primer set or after the fixing of the primer set.

  When the thickener is fixed to the support simultaneously with the fixing of the primer set, the thickener may be dissolved in the solution in which the primer set is dissolved. Alternatively, first, a thickener solution may be prepared by dissolving a thickener in a solvent and mixed with a separately prepared primer set fixing solution. What is necessary is just to fix the solution containing the obtained primer set and thickener by dripping and drying.

  If the thickener solution is fixed before or after fixing the primer set, the solution containing the thickener is dropped, sprayed, printed, applied by brush or dipped in the thickener solution, and then dried. What is necessary is just to fix to the support body surface containing a primer fixed area | region by the above.

  As a method for drying a thickener solution or a solution containing a primer set and a thickener, heat drying may be performed by heating at a temperature of room temperature or higher using a heat block, a hot plate, an incubator, or the like. Alternatively, it may be left at room temperature and dried naturally. Alternatively, vacuum drying or freeze drying may be performed. For example, when agarose is used, it is preferable that heat drying is performed and the state after drying is a film.

  The concentration of the thickener in the thickener solution used for fixing may be a state in which the state at the time of fixing is liquid at room temperature (25 ° C.) and can be dropped onto the support. The density | concentration of the thickener at the time of fixing a thickener should just be the range of about 30%-0.01% of final concentration, for example. For example, in the case of agarose, the final concentration may be in the range of 10% to 0.01%, more preferably in the range of 5% to 0.05%, and mixing in the range of 3% to 0.1%. Is more preferable.

  Further, the thickener solution used for fixation may contain a primer set, and may contain other substances necessary for the amplification reaction in addition to the primer set.

  By using a thickener, the sensitivity reduction in the multi-amplification reaction, the limitation on the kind of amplification, etc. are eliminated.

<Eleventh embodiment>
A further example of an array type primer probe chip to which a thickener is fixed will be described with reference to FIG.

  FIG. 16 is a plan view showing a further example of an array type primer probe chip. The array-type primer probe chip 1 described in FIG. 16 is an example in which a substrate is used as the support 2. A plurality of primer immobilization regions 4 independent from each other are arranged on one surface 503 of the support 2. A plurality of probe immobilization regions 5 are arranged adjacent to the plurality of primer immobilization regions 4 and corresponding to the respective primer immobilization regions 4.

  One type of primer set 6 is releasably immobilized in each primer immobilization region 4. That is, a plurality of primer sets 6 for each type are immobilized on each of the plurality of primer immobilization regions 4. The configuration of the primer set 6 included in each primer immobilization region 4 includes different types of primers necessary for amplifying one type of specific target nucleic acid, as in the first embodiment. Furthermore, a thickener 504 is fixed to the primer fixing region 4 by coating so as to cover the primer set 6.

  One type of probe nucleic acid 7 is immobilized in each probe immobilization region 5. That is, a plurality of probe nucleic acids 7 are immobilized in each of the plurality of probe immobilization regions 5 for each type.

  Amplification using the eleventh embodiment may be performed after forming a reaction field by placing a reaction solution on at least the region where the primer set 6 and the probe nucleic acid 7 of the support 2 are immobilized.

  The primer immobilization region 4 may be disposed on a concave surface formed in advance on one surface of the support 2 or an inner wall of a flow path formed by the concave portion and / or the convex portion.

  When the substrate is used as the support 2, the material may be a material that can perform an amplification reaction there, rather than a material that itself does not participate in the reaction. For example, it may be arbitrarily selected from silicon, glass, resin and metal. In addition, the primer set 6 and the probe nucleic acid 7 may be immobilized on the support 2 in the same manner as in the first embodiment.

  Furthermore, the array type primer probe chip 1 of the eleventh embodiment may be arranged in a container capable of maintaining it, and a reaction field may be formed by adding a reaction solution into the container. In this case, the primer set 6 may be releasably fixed to at least both surfaces of the support 2, and the thickener 504 may be fixed by coating so as to cover the primer set 6. As a result, it is possible to immobilize more types of primer sets 6 on the array-type primer probe chip 1 and amplify more target sequences. In this aspect, the array-type primer probe chip 1 may be any support 2 capable of independently immobilizing a plurality of primer sets 6 and corresponding probe nucleic acids 7 on at least one surface. It may be a body. The material of the support 2 and the primer immobilization method in this case may be the same as those in the first embodiment, the second embodiment, and the like. Such an array-type primer probe chip has a plurality of primer-immobilized regions arranged independently of each other on the substrate and at least one surface thereof, and a probe-immobilized region arranged in the vicinity of each of the plurality of primer-immobilized regions. A region, a plurality of primer sets that are releasably immobilized on each of the plurality of primer immobilization regions, a thickener coated so as to cover the plurality of primer sets, and a type in the probe immobilization region It may be provided as an array-type primer probe carrier comprising a plurality of types of probe nucleic acids immobilized every time. In this case, the practitioner may arbitrarily select the size and shape of the substrate. For example, the shape may be a plate shape, a spherical shape, a rod shape, or a part thereof.

  The thickener may be fixed at the same time as the primer set or before the primer set is fixed. Moreover, you may make a reaction liquid contain a thickener at the time of reaction, without fixing a thickener.

  By using a thickener, the sensitivity reduction in the multi-amplification reaction, the limitation on the kind of amplification, etc. are eliminated.

<Twelfth Embodiment>
A further example of a container-type multi-nucleic acid reaction device having a thickener fixed thereon will be described with reference to FIGS. 17 (a), (b) and (c).

  FIG. 17A is a perspective view of the multi-nucleic acid reaction tool 1. The multi-nucleic acid reaction tool 1 shown in FIG. 17A has a container-shaped support 2. A plurality of primer immobilization regions 4 independent from each other are arranged on the inner bottom surface 3 of the support 2. Furthermore, a thickener 504 is fixed to the primer fixing region 4 by coating so as to cover the primer set 6. A plurality of probe immobilization regions 5 are arranged adjacent to the plurality of primer immobilization regions 4 and corresponding to the respective primer immobilization regions 4.

  FIG. 17B is an enlarged schematic view of the primer immobilization region 4. As shown therein, one kind of primer set 6 is fixed to one primer fixing region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are fixed for each type. Furthermore, a thickener 504 is fixed to the primer fixing region 4 by coating so as to cover the primer set 6.

  Plural types of primer sets 6 are prepared for amplifying a plurality of target nucleic acids. One primer set 6 for amplifying one specific target nucleic acid is fixed to one primer fixing region 4. For example, in the case of a PCR amplification reaction tool, one primer-immobilized region 4 includes a plurality of forward primers and reverse primers necessary for amplifying one type of specific target nucleic acid. In the case of a reaction tool for LAMP amplification, the FIP primer, BIP primer, and F3 primer, B3 as necessary, for amplifying one specific target nucleic acid in one primer immobilization region 4. A plurality of primers and LP primers are included.

  The primer set 6 is immobilized in the primer immobilization region 4 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 6 to the primer immobilization region 4 can be achieved, for example, by dropping a solution containing one set of primer sets onto one primer immobilization region 4 and then drying. Further, similarly, a solution containing a desired primer set 6 may be dropped and dried for each of the other primer immobilization regions 4 to fix a desired number of primer sets 6 to the support 2. As a result, the primer set 6 is fixed to all the primer fixing regions 4 arranged independently on one surface of the support 2. However, immobilization of the primer set 6 to the immobilization region 4 may be performed in a state where it can be released in contact with a liquid phase for providing a reaction field. Therefore, any immobilization method known per se capable of such immobilization may be used. In the method of dropping a solution containing the primer set 6, the solution containing the primer set 6 may be, for example, water, a buffer solution or an organic solvent.

  The plurality of primer immobilization regions 4 arranged on the support 2 may be arranged independently of each other. Independently arranged means arranged at intervals that do not prevent amplification initiated and / or advanced for each primer set in the reaction field. For example, the adjacent primer immobilization regions 4 may be arranged in contact with each other, may be arranged in the vicinity of each other at a slight distance, or fixed in a commonly used detection device such as a so-called DNA chip. The probe nucleic acids may be arranged at the same distance from each other at the same distance.

  The liquid phase for providing the reaction field only needs to be a liquid phase that allows the amplification reaction to proceed after the immobilized primers are released. For example, the reaction phase is a reaction liquid necessary for the desired amplification. Good.

  The container-shaped support 2 may be, for example, a tube, a well, a chamber, a flow path, a cup and a dish, and a plate including a plurality of them, such as a multiwell plate. The material of the support 2 may be any material that does not itself participate in the reaction, and may be any material that can perform the amplification reaction there. For example, it may be arbitrarily selected from silicon, glass, resin and metal. The support 2 in the form of a container may use any commercially available container.

  FIG. 17 shows an example in which the primer immobilization region 4 is disposed on the inner bottom surface 3 of the support 2, but the present invention is not limited to this and may be disposed on at least a part of the inner side surface of the support 2. , The bottom surface and the inner side surface, or any or all of the ceiling surface defined by the covering.

  FIG. 17C is an enlarged view of the probe immobilization region 5 arranged in the vicinity of the primer immobilization region 4. A plurality of probe nucleic acids 7 including a complementary sequence of a desired sequence to be detected are immobilized in the probe immobilization region 5.

  The desired sequence to be detected may be the target sequence. The probe immobilization region 5 is arranged so that a hybridization signal between the probe nucleic acid 7 and the target sequence chain can be detected independently between the plurality of probe immobilization regions 5.

  For fixing the probe nucleic acid 7 to the probe immobilization region 5, any general technique for fixing the probe nucleic acid 7 to the substrate surface in a so-called DNA chip known per se may be used. The primer set 6 may be immobilized after the probe nucleic acid 7 is immobilized, the probe nucleic acid 7 may be immobilized after the primer set 6 is immobilized, and the primer set 6 and the probe nucleic acid 7 are immobilized. Immobilization may be performed simultaneously.

  For example, when the distance between the probe immobilization region 5 and the primer immobilization region 4 is 0 μm, it is understood that the probe immobilization region 5 and the primer immobilization region 4 are at the same position on the surface of the support 2. May be. Further, the probe immobilization region 5 may be included in the primer immobilization region 4, and the primer immobilization region 4 may be included in the probe immobilization region 5.

  The number of primer-immobilized regions 4 and probe-immobilized regions 5 arranged on one array-type primer probe chip 1 may be the same or different. That is, the same number of probe immobilization regions 5 may be arranged so as to correspond to all the primer immobilization regions 4, and the number of primer immobilization regions 4 may be larger than the number of probe immobilization regions 5, The number of primer immobilization regions 4 may be smaller than the number of probe immobilization regions 5. Further, a positive control and / or a negative control for confirming the amplification reaction state or for confirming the state of the hybridization reaction may be included. Such positive control and / or negative control may be provided for the primer set 6 and / or the probe nucleic acid 7.

  In the above example, only the primer set 6 was immobilized on the support 2 for the amplification reaction. However, the present invention is not limited to this, and other components necessary for amplification may be immobilized on the support 2 together with the primer set 6 under the condition that the primer set 6 is immobilized on each immobilization region for each type. Good. Other components necessary for amplification may be, for example, enzymes such as polymerases, reverse transcriptases, substrates, substrates and / or buffers. In that case, the substance to be fixed may be contained in a desired liquid medium together with the primer set 6 and fixed by dropping, drying, or the like in the same manner as described above. In such an array-type primer probe chip 1, when an amplification reaction is performed, the composition of the reaction solution added thereto may be selected according to the immobilized components.

  Moreover, although the example which fixes a thickener to the support body 2 was shown in the above-mentioned example, a thickener may be included in a reaction liquid instead of fixation, and it reacts after fixing a thickener. It may be included in the liquid.

  The support 2 is not limited to a container shape, and may be a plate shape, a spherical shape, a rod shape, or a part thereof, as described above, and the size and shape of the substrate can be arbitrarily determined by the practitioner. You may choose. Moreover, it is also preferable to comprise the support body 2 using the board | substrate which has a flow path.

<13th Embodiment>
A further example of a chip-type multi-nucleic acid reaction tool, that is, an array-type primer probe chip will be described with reference to FIG. An array type primer probe chip 1 shown in FIG. 18 is an example using a support 2 having a flow path. 18A is a plan view of the array-type primer probe chip, and FIG. 18B is a cross-sectional view of the array-type primer probe chip in FIG. 18A taken along line mm ′.

  The array-type primer probe chip 1 has a channel 41 formed inside the array-type primer probe chip 1. A plurality of independent primer immobilization regions 4 arranged on the inner bottom surface of the channel 41 are arranged.

  Specifically, the array type primer probe chip 1 includes a support 2 and a covering 71. The covering 71 has a recess 601 that defines the flow path. The primer immobilization region 4 is disposed on the surface of the support 2 that contacts the inside of the flow path 41.

  In the plurality of primer immobilization regions 4, a plurality of primer sets 6 are immobilized in a releasable manner for each type. Furthermore, a thickener 504 is fixed to the primer fixing region 4 by coating so as to cover the primer set 6.

  Corresponding to each primer immobilization region 4, a probe immobilization region 5 is arranged in the vicinity of each primer immobilization region 4. In the probe immobilization region 5, a plurality of probe nucleic acids 7 are immobilized for each type.

  The array type primer probe chip 1 may be manufactured using, for example, a first substrate and a second substrate for the covering 71. First, a mixture of a plurality of primer sets 6 and a thickener 504 is releasably immobilized on a predetermined primer immobilization region 4 of the first substrate. The primer set 6 is immobilized for each type. This immobilization can be performed as described above. Further, a plurality of probe nucleic acids 7 are immobilized on a predetermined probe immobilization region 5 in the vicinity of the primer immobilization region 4.

  On the other hand, a recess 601 is formed on the second substrate so as to correspond to the shape of the desired flow path 41. The concave portion 601 can be formed by a method known per se according to the material of the second substrate to be used. Further, the arrangement of the primer immobilization region 4 is determined so as to be included in the flow path 41 formed by the recess 601 formed in the second substrate. Next, the first substrate and the second substrate are integrated. By integration, a reaction portion having a flow path shape is formed by the inner wall of the recess 601 and the surface of the first substrate on the second substrate side. At this time, the concave portion 601 of the second substrate is integrated so as to face the first substrate side. Further, a through hole (not shown) may be provided in a part of the recess 601 of the second substrate. This through hole may be used as an inlet / outlet of a reaction liquid to / from the channel 41.

  The material of the first substrate and the material of the second substrate may be the same or different. Further, the material of the first substrate and the second substrate may be any material that does not itself participate in the reaction, and any material that can perform an amplification reaction there may be used. For example, it may be arbitrarily selected from silicon, glass, resin and metal.

  In this embodiment, although the example which fixed the primer set 6 to the inner bottom face of the flow path 41 of the support body 2 which has the flow path 41 was shown, arrangement | positioning and shape of the flow path 41 are not limited to this. . Further, the surface on which the primer set 6 and the probe nucleic acid 7 are immobilized may be any surface that defines the flow channel 41, and the primer set 6 and the probe nucleic acid 7 are immobilized on all surfaces that define the flow channel 41. It may be fixed to a plurality of surfaces.

  Alternatively, a plurality of the primer-immobilized regions 4 arranged on a part of the wall surface of the first substrate on which the flow channel 41 is formed by forming a concave portion, a convex portion, or a groove in advance. Primer set 6 may be independently and releasably immobilized. Furthermore, a plurality of probe nucleic acids 7 may be independently immobilized on the probe immobilization region 5 arranged at a position corresponding to such a primer immobilization region 4. Thereafter, an array type primer probe chip may be manufactured by attaching a lid such as silicon rubber.

  The length of the primer used may be as described above.

  According to the above-described embodiment, it is possible to amplify a plurality of types of target sequences independently and simultaneously in one reaction field. Furthermore, following this amplification, it is possible to detect the plurality of obtained amplification products independently and simultaneously in one reaction field.

  By using a thickener, the sensitivity reduction in the multi-amplification reaction, the limitation on the kind of amplification, etc. are eliminated.

<Fourteenth embodiment>
The array type primer probe chip according to the eleventh embodiment will be described with reference to FIGS.

(1) Chip Material First, an example of the structure and manufacturing method of the chip material of an array type primer probe chip that detects a hybridization signal by electrochemical detection will be described with reference to FIGS. 19 (a) and 19 (b). FIG. 19A is a plan view of the chip material 111, and FIG. 19B is a cross-sectional view taken along line BB of the chip material 111 in FIG. 19A.

  The chip material 111 includes, for example, four electrodes 113a to 113d arranged on a rectangular substrate 112 along the longitudinal direction thereof. Each of the electrodes 113a to 113d has a structure in which a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order. Each of the electrodes 113a to 113d has a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118. The insulating film 119 is covered on the substrate 112 including the electrodes 113a to 113d. The circular window 120 is opened at the insulating film 119 portion corresponding to the large rectangular portion 116. The rectangular window 121 is opened at a portion of the insulating film 119 corresponding to the small rectangular portion 117. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113a functions as the first working electrode 122a. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113b functions as the second working electrode 122b. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113 c functions as the counter electrode 123. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113d functions as the reference electrode 124. The small rectangular portion 117 exposed from the rectangular window 121 of the electrodes 113a to 113d functions as a prober contact portion.

  Such a chip material 111 can be manufactured by the following method.

  First, a first metal thin film and a second metal thin film are deposited on the substrate 112 in this order by, for example, a sputtering method or a vacuum evaporation method. Subsequently, these metal thin films are sequentially selectively etched using, for example, a resist pattern as a mask, and a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order, for example, four electrodes 113a to 113a. 113 d is formed along the longitudinal direction of the substrate 112. These electrodes 113 a to 113 d have a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118.

  Next, an insulating film 119 is deposited on the substrate 112 including the electrodes 113a to 113d by, for example, a sputtering method or a CVD method. Subsequently, the insulating film 119 portion corresponding to the large rectangular portion 116 of each of the electrodes 113a to 113d and the insulating film 119 portion corresponding to the small rectangular portion 117 are selectively etched using the resist pattern as a mask to form the large rectangular portion 116. A circular window 120 is opened in the corresponding insulating film 119 portion, and a rectangular window 121 is opened in the insulating film 119 portion corresponding to the small rectangular portion 117. Thereby, the above-described chip material 111 is produced.

  The substrate 112 is made of glass or resin such as Pyrex (registered trademark) glass, for example.

  The first metal thin film functions as a base metal film for bringing the second metal thin film into close contact with the substrate 112, and is made of, for example, Ti. The second metal thin film is made of, for example, Au.

  Examples of etching when patterning the first and second metal thin films include plasma etching using an etching gas or reactive ion etching.

  Examples of the insulating film 119 include a metal oxide film such as a silicon oxide film and a metal nitride film such as a silicon nitride film.

  Examples of etching when patterning the insulating film 119 include plasma etching using an etching gas or reactive ion etching.

(2) Array-type Primer Probe Chip Next, an example of the configuration and manufacturing method of an array-type primer probe chip in which a primer set and a probe nucleic acid are fixed to the chip material 111 manufactured in (1) above is shown in FIG. This will be described with reference to FIG. FIG. 20A is a plan view of the array-type primer probe chip 1, and FIG. 20B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG.

  The first working electrode 122a of the electrode 113a formed on the chip material 111 is defined as a first probe immobilization region 201a, and the first probe immobilization region 201a includes a first sequence that includes a complementary sequence of the first target sequence. The probe nucleic acid 202a is immobilized. A plurality of the first probe nucleic acids 202a to be immobilized are immobilized as one probe nucleic acid group. Similarly, the second working electrode 122b of the electrode 113b is used as a second probe immobilization region 201b, and a complementary sequence of a second target sequence different from the first target sequence is provided in the second probe immobilization region 201b. The contained second probe nucleic acid 202b is immobilized.

  Examples of the method of immobilizing the probe nucleic acids 202a and 202b to the probe immobilization region 201 include a method of introducing a thiol group into the probe nucleic acids 202a and 202b at the 3 ′ end for the chip material 111 having a gold electrode. .

  Next, the first primer immobilization region 203a is disposed in the vicinity of the first working electrode 122a, and the second primer immobilization region 203b is disposed in the vicinity of the second working electrode 122b. The first primer set 204a and the thickener 205 are releasably immobilized on the first primer immobilization region 203a, and the second primer set 204b and the thickener are immobilized on the second primer immobilization region 203b. 205 is fixed releasably. Thereby, the array type primer probe chip 1 is produced.

  The first primer set 204a has a sequence designed to amplify the first target sequence, and the second primer set 204b amplifies a second target sequence consisting of a sequence different from the first target sequence. Having an array designed to

  Immobilizing the first and second primer sets 204a and 204b to the first and second primer immobilization regions 203a and 203b, respectively, can be achieved by, for example, applying the primer set to a liquid such as water, a buffer solution, or an organic solvent. It is carried out by dropping it and then leaving it for 10 minutes in the case of room temperature, for example, until drying under an appropriate temperature condition such as room temperature.

  The thickener 205 may be fixed in the same manner as in the tenth embodiment, or the thickener 205 may not be fixed and may be only present in the reaction solution.

(3) Array-type primer probe chip at the time of use The usage method of the array-type primer probe chip produced in said (2) is demonstrated referring FIG. 21 and FIG.

  21A is a plan view of the array-type primer probe chip in use, and FIG. 21B is a cross-sectional view taken along line BB of the array-type primer probe chip in FIG. .

  When the array type primer probe chip 1 of the present embodiment is used, the first working electrode 122a, the second working electrode 122b, the counter electrode 123, the reference electrode 124, and the first primer formed on the electrodes 113a to 113d, respectively. The reaction solution is maintained so that the immobilization region 203a and the second primer immobilization region 203b are included in the same one reaction field. Therefore, the covering 301 is mounted on the array type primer probe chip 1 before use. The covering 301 is made of, for example, a silicon resin such as silicon rubber and / or a resin such as a fluororesin, which is known per se such as extrusion molding, injection molding or stamping and / or adhesion with an adhesive. Molded by the resin molding method. After the covering 301 is mounted, the reaction solution 302 containing the template nucleic acid 303 is added to the space formed by the array type primer probe chip 1 and the covering 301.

  In the array-type primer probe chip 1 to which the covering 301 is attached, the small rectangular portions 117 exposed from the rectangular windows 121 of the electrodes 113a to 113d are exposed.

  Examples of means for attaching the covering 301 to the array type primer probe chip 1 include means such as pressure bonding and adhesion with an adhesive.

  Next, the reaction solution 302 is added after the covering 301 is attached to the array type primer probe chip 1.

  For example, a method of adding a liquid such as a reaction solution to the space formed by the array-type primer probe chip 1 and the covering 301 includes, for example, providing an opening in a part of the covering 301 in advance and adding from the opening Alternatively, it may be added by being inserted into a part of the covering 301 using an injector having a sharp tip such as a needle.

  The reaction solution 302 is composed of a sample, a thickener, an amplification reagent, for example, an enzyme such as a polymerase, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, reverse transcription, and the like. Are simultaneously recognized, the reverse transcriptase and the necessary substrate, and other buffers such as salts for maintaining an appropriate amplification environment and double-stranded nucleic acid such as Hoechst 33258 are recognized and signaled. May include an intercalator that produces When the template nucleic acid containing the target sequence to be amplified by the primer set immobilized on the specific primer immobilization region is present in the sample to be examined, the primer immobilization region and the corresponding probe immobilization region are included. An amplification product is formed in the reaction field. This is schematically shown in FIG.

  FIG. 22A schematically shows a state in which an amplification product is formed in the region 401 in the reaction field provided by the reaction solution 302. 22 (a) is a plan view of the array-type primer probe chip 1 in use, and FIG. 22 (b) is a cross-sectional view taken along line BB of the array-type primer probe chip in FIG. 22 (a). is there. As described above, the sample added in FIG. 21 contained a nucleic acid containing a sequence that can be bound by the second primer set 204b. Therefore, as shown in FIGS. 22 (a) and 22 (b). In the region 401, the second primer set 204b is released and diffused. In the region 401, the second primer set 204b that has encountered the template nucleic acid undergoes an amplification reaction, whereby an amplification product is formed. The amplification product by the second primer set 204b diffuses around the second primer immobilization region 203b and reaches the second probe immobilization region 201b. When the reached amplification product contains the target sequence, the second probe nucleic acid 202b and the amplification product are hybridized to form a double-stranded nucleic acid. The intercalator contained in the reaction solution 302 is combined with this double-stranded nucleic acid to generate a hybrid signal.

  The hybrid signal is generated by, for example, bringing a prober into contact with the small rectangular portion 117 exposed from each rectangular window 120 of the electrodes 113a to 113d and measuring the current response of an intercalator such as Hoechst 33258.

  By using the array-type primer probe chip 1 that utilizes electrochemical detection, the target nucleic acid contained in the amplification product can be detected more easily after amplifying the target nucleic acid contained in the sample in a shorter time. Is possible.

(4) Method for detecting target nucleic acid A method for amplifying a plurality of target nucleic acids using an array-type primer probe chip as described above as an example, and detecting the target nucleic acid using a hybridizing signal as an index is a further embodiment. Offered as.

  In addition, multiple types of primer sets designed to amplify multiple types of target nucleic acids are released to at least one surface of a support such as a substrate on which a specific container, tube, dish or flow path is formed. A method for detecting a target nucleic acid comprising the step of immobilizing and / or the step of immobilizing one or more types of probe nucleic acids to a probe immobilization region is also provided as a further embodiment.

  Such a method for detecting a target nucleic acid, for example, releasably immobilizes a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on at least one surface of a desired support. And at least one containing a complementary sequence of the target sequence for each type so that a hybridization signal can be independently detected for each probe-immobilized region at or near the position of a plurality of primer-immobilized regions. Fixing various types of probe nucleic acids and forming a new polynucleotide chain starting from reagents necessary for amplification such as polymerase and other enzymes and primers so that multiple types of primer sets are included in one reaction field When using reverse transcription at the same time as a substrate such as deoxynucleoside triphosphate required for Add a reaction solution containing a buffer such as a substrate to maintain an appropriate amplification environment, such as a substrate, and bring the sample into the reaction field, for example, by adding it to the reaction solution or an array-type primer probe chip. Adjusting the reaction environment suitable for the amplification reaction, such as temperature adjustment by heating or cooling the support on which the primer is immobilized, thereby performing a multi-nucleic acid amplification reaction, and a multi-nucleic acid amplification reaction And / or measuring the presence / absence and / or amount of hybridization between the amplification product generated by step 1 and at least one type of probe nucleic acid. The reaction solution may contain a thickener.

  A specific amplification reaction may be performed using a technique known per se according to the type of the amplification reaction.

  Specific detection means include known hybrid signal detection means, for example, detection and / or measurement of fluorescence intensity using a fluorescent label, or method of detecting and / or measuring current response using an intercalator May be used.

  Furthermore, a step of releasably fixing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on the surface of a substrate such as a microbead, a plate piece or a rod, and a plurality of primer immobilizations Immobilizing at least one type of probe nucleic acid containing a complementary sequence of the target sequence for each type so that a hybridization signal can be independently detected for each probe-immobilized region in the probe-immobilized region at or near the region. A method for detecting a target nucleic acid comprising: is also provided as a further embodiment.

  Such a nucleic acid detection method includes, for example, releasably fixing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on at least one surface of a desired substrate. And at least one kind including a complementary sequence of the target sequence for each kind so that the hybridization signal can be detected independently for each probe immobilization area at the position of the plurality of primer immobilization areas or in the vicinity of the probe immobilization area. Fixing the probe nucleic acid, and a substrate, a reagent necessary for amplification, for example, an enzyme such as a polymerase, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, When reverse transcription is performed at the same time, reverse transcriptase and its necessary substrates, such as salts to maintain an appropriate amplification environment Place the sample in the reaction field by placing it in a reaction solution containing a buffer, adding a reaction solution containing a buffer such as salts to maintain an appropriate amplification environment, and adding to the reaction solution. Adjusting the reaction environment suitable for the amplification reaction, such as bringing in and adjusting the temperature by heating or cooling the reaction solution, thereby performing the multi-nucleic acid amplification reaction, and the amplification product generated by the multi-nucleic acid amplification reaction And / or measuring the presence / absence and / or amount of hybridization between and at least one probe nucleic acid. Moreover, a thickener may be contained in the reaction solution.

  For example, according to the multi-nucleic acid amplification detection reaction tool shown in the embodiment, amplification of a plurality of types of target sequences can be performed independently and simultaneously without receiving interference from different sequences. Furthermore, the presence or absence and / or the amount of the target nucleic acid can be detected and / or measured for the amplification product generated by the amplification reaction in the same reaction field where the amplification reaction is performed simultaneously with or subsequent to the amplification reaction. it can. Moreover, the amplification reaction performed in parallel about several types of target sequence is efficiently performed by application of a thickener.

  Further, instead of adding the thickener to the reaction solution, the thickener may be fixed to the support as described above. Alternatively, the thickener may only be present in the reaction solution or may be provided by being fixed to the support.

  Furthermore, in order to achieve the amplification reaction more efficiently, it is preferable to inject the reaction solution into the reaction field at an injection speed of 25 mm / second or more. Thereby, the release of the immobilized primer set is affected, allowing a more localized spread of the primer set.

  In the prior art, when multiplex amplification is performed using a plurality of types of primers in one container, there is a problem that the reaction efficiency is biased and the number of types is limited. In other words, necessary enzymes and dNTPs may be intermingled between different types of primers. In addition, the reaction specificity and / or reaction efficiency may vary depending on the sequence of the target sequence and the sequence of the primer. In that case, the amplification reaction start point varies depending on the type of primer, only the amplification for some primer sets is started and advanced, or the amplification for some primer sets is not sufficiently achieved, etc. Problems arise. Such conventional problems are also solved by the embodiments disclosed herein.

  In other words, when the amplification reaction is performed using the multi-nucleic acid amplification detection reaction tool illustrated in the embodiment, the amplification reaction proceeds only in the vicinity of the fixed amplification reagent, so that it is in the same container and / or in the same solution. However, it is possible to independently proceed with the amplification reaction of various targets without interfering with each other's amplification reaction, and in the same reaction vessel where the amplification reaction was performed simultaneously or subsequently with such an amplification reaction. The presence and / or amount of nucleic acid can be detected and / or measured. Furthermore, after individual amplification reactions have progressed to some extent, different primer sets may be added. The container-shaped multinucleic acid amplification detection reaction tool shown in the first embodiment and the above-described multinucleic acid amplification detection reaction You may use it in combination with a tool.

  When detecting a plurality of target genes, a method for preparing a reaction container for amplifying each target gene, a method for performing a multi-nucleic acid amplification reaction by putting reagents for detecting all target genes in one reaction container , Either. In the case of preparing a reaction container for amplifying each target gene, the number of necessary reaction containers and the amount of work at the time of testing increase as the number of target genes increases. Moreover, when the reagent for detecting all the target genes is put into one reaction container, the amplification reaction efficiency is biased. By using a thickener, it is possible to prevent such a bias in the amplification reaction efficiency.

<Example 2>
Example 2-1
Using the thirteenth embodiment, the nucleic acid status of the primers was evaluated.

  A fluorescently labeled primer set was prepared. FIG. 23 shows an array type primer chip 1121 having the same structure as that of FIG. 18 except that the probe nucleic acid is not immobilized and the primer set is immobilized at three positions. The array-type primer chip 1121 is configured to include a support 1122, a primer set fixed at three positions (the support center 1123 and two corners 1124 and 1125), and all primer sets. And a flow path 1130 defined by the support body 1122 and the groove 1126 of the cover body 1127. The array type primer chip 1121 was manufactured as follows.

  The prepared primer set was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to a final concentration of 200 μM. A fixed solution was prepared by dissolving agarose in this solution to a final concentration of 0.3%. A glass substrate was used as the support 1122. This fixing solution is dropped at three points on the center of the support 1122 (indicated by 1123 in the figure) and two corners of the support 1122 (indicated by 1124 and 1125 in the figure) and left at room temperature. And dried. A silicon rubber plate having a groove 1126 formed on one surface thereof was used as the covering 1127. Through holes 1128 and 1129 are formed at two ends of the groove 1126 of the covering 1127, respectively. The covering body 1127 was bonded to the supporting body 1122 so that the region where the primer set and the agarose of the supporting body 1122 were fixed was included in the groove portion 1126 of the covering body 1127. Thereby, an array type primer chip 1121 was obtained. In this array-type primer chip 1121, a channel 1130 is formed by the groove portion 1126 of the covering body 1127 and the surface of the support body 1122.

  The two through holes of the covering 1127 were used as the inlet 1128 and the outlet 1129, respectively. PBS was added from the inlet 1128. Thereafter, the diffusion state of the primer was observed using the fluorescence intensity as an index.

  As a target, a control array type primer chip prepared by fixing only a fluorescently labeled primer set without fixing agarose in the same manner as described above was prepared. Similarly, TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was added to the control array type primer chip from the inlet A, and the nucleic acid state of the primer was observed using the fluorescence intensity as an index.

  The results are shown in FIGS. 24 (a) and (b). FIG. 24 (a) shows the results obtained with the control array primer chip in which only the primer is fixed. FIG. 24B shows the results obtained with an array-type primer chip in which a primer set and agarose are fixed.

  The distance of the control array type primer chip 1121 moved by addition of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was larger than that of the array type primer chip 1121 to which the primer set and agarose were fixed. From this result, it was revealed that the primer set and the agarose-fixed array-type primer chip enable more localized primer diffusion.

Example 2-2
An array type primer probe chip for electrochemical detection similar to that of the fourteenth embodiment was produced.

(1) Production of chip material A chip material for an array type primer probe chip as shown in FIG. 19 was formed. Titanium and gold thin films were formed on the Pyrex glass surface by sputtering. Thereafter, an electrode pattern of titanium and gold was formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and electrodes, that is, a working electrode, a counter electrode, a reference electrode, and a probe electrode were exposed by an etching process. This was used as a chip material for a multi-nucleic acid amplification detection reaction tool.

(2) Production of array-type primer probe chip Probe DNA was immobilized on the working electrode of the chip material produced as described above. Table 13 shows the base sequence of the probe DNA used.

  Thirteen types of probe DNAs (A), (B), (C), (D), (E), (F), (G), (H), (I), (J ), (K), (L) and (M) were immobilized on the chip material produced as described above. Probe DNA solutions each containing 3 μM of probe DNA were prepared. 100 nL of these solutions was spotted for each type on the working electrode. After drying at 40 ° C., it was washed with ultrapure water. Thereafter, ultrapure water remaining on the surface of the working electrode was removed, and a DNA chip in which the probe DNA was immobilized on the electrode of the chip material was produced.

  Next, a covering made of a silicon rubber plate was prepared as described above. On one surface of the covering, a groove is formed at a position corresponding to the probe fixing region.

  A plurality of primer sets were fixed to the inner bottom surface of the groove of the covering. The immobilization region of the primer set was adjusted so as to correspond to the position of the probe DNA immobilized earlier.

First, primer DNA to be used was prepared. The primer DNA to be used is a primer set for amplification by a loop-mediated isal amplification (LAMP) method. The base sequences of the primer DNA are shown in Tables 14A, 14B and 14C.

  Primer DNA (Set A), (Set B), (Set C), (Set D), (Set E), (Set F), (Set G), (Set H), (Set I), (Set J) ), (Set K), (Set L), and (Set M), 200 μM FIP, BIP, F3, B3, and LPF were prepared, respectively. A 0.6% agarose solution is added to a 0.100 μL solution containing 0.036 μL, 0.036 μL, 0.005 μL, 0.005 μL, and 0.018 μL of FIP, BIP, F3, B3, and LPF, respectively. 100 μL was mixed. This aqueous solution was fixed to the primer fixing region on the inner bottom surface of the groove portion of the silicon rubber as the covering.

  Specifically, 0.200 μL of each prepared solution was spotted on the bottom of the groove of the covering and dried at 40 ° C. for 2 minutes. Spotting was performed so that each probe DNA would be at a position facing the corresponding primer set when the covering was attached to the DNA chip. The covering and the chip material prepared above were bonded so that the groove portion of the covering and the surface on which the probe DNA was immobilized faced each other. Thereby, an array type primer probe chip was obtained. Two through holes are opened at two ends of the groove of the silicon rubber that is the covering.

(3) Preparation of LAMP reaction liquid The composition of the LAMP reaction liquid is shown in Table 15 to Table 18.

  Compositions (1) to (4) include Bst DNA polymerase and reaction mix in common, and distilled water (ie, DW) is added so that the total amount is 50 μL when combined with the template solution described below. It was used.

  Composition (1) includes template A, template C, template E, template G, template I, template K and template M.

  Template A is LAMP amplified by primer set A. The resulting amplification product hybridizes with the probe DNA (A). Template C is LAMP amplified by primer set C. The resulting amplification product hybridizes with the probe DNA (C). Template E is LAMP amplified by primer set E. The resulting amplification product hybridizes with the probe DNA (E). Template G is LAMP amplified by primer set G. The resulting amplification product hybridizes with the probe DNA (G). Template I is LAMP amplified by primer set I. The resulting amplification product hybridizes with probe DNA (I). Template K is LAMP amplified by primer set K. The resulting amplification product hybridizes with the probe DNA (K). Template M is LAMP amplified by primer set M. The resulting amplification product hybridizes with the probe DNA (M).

  Composition (2) includes template B, template D, template F, template H, template J and template L.

  Template B is LAMP amplified by primer set B. The resulting amplification product hybridizes with the probe DNA (B). Template D is LAMP amplified by primer set D. The resulting amplification product hybridizes with the probe DNA (D). Template F is LAMP amplified by primer set F. The resulting amplification product hybridizes with the probe DNA (F). Template H is LAMP amplified by primer set H. The resulting amplification product hybridizes with the probe DNA (H). Template J is LAMP amplified by primer set J. The resulting amplification product hybridizes with the probe DNA (J). The template L is LAMP amplified by the primer set L. The resulting amplification product hybridizes with the probe DNA (L).

  Composition (3) includes all of template A, template B, template C, template D, template E, template F, template G, template H, template I, template J, template K, template L and template M.

  Composition (4) does not contain a template.

Table 19A, Table 19B and Table 19C show the nucleotide sequences of Template A, Template B, Template C, Template D, Template E, Template F, Template G, Template H, Template I, Template J, Template K, Template L, and Template M. Shown in

(4) LAMP amplification reaction on array type primer probe chip and detection of target nucleic acid by probe DNA One of the two through holes provided in the silicon rubber plate as a coating was used as an inlet. A reaction was carried out using a flow path constituted by a groove provided on the silicon rubber plate and one surface of the chip material as a reaction part. The LAMP reaction solution was injected into the reaction part from the inflow port so that the reaction solution passed over the primer immobilization position at a flow rate of 25 mm / sec. Immediately thereafter, an array-type primer probe chip was placed in the DNA automatic testing apparatus. A LAMP reaction was performed at 64 ° C. for 60 minutes on a Peltier in an automatic DNA testing apparatus.

  After the LAMP reaction for 60 minutes, a hybridization reaction was performed at 55 ° C. for 10 minutes, and washing was performed at 30 ° C. for 3 minutes. Thereafter, the washing solution was removed, and 35.5 μM Hoechst 33258 solution was injected from the inlet.

  The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecules specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The above series of reactions was carried out using an automatic DNA testing apparatus described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008.

(5) Detection results Table 20 shows the detection results.

[Results of LAMP reaction solution composition (1)]
The results when the LAMP reaction solution composition (1) containing Template A, Template C, Template E, Template G, Template I, Template K, and Template M was added are as follows.

  Probe DNAs from which current values were obtained were probe DNA (A), probe DNA (C), probe DNA (E), probe DNA (G), probe DNA (I), probe DNA (K) and probe DNA (M )Met. 30 nA or more for all of these probes DNA (A), probe DNA (C), probe DNA (E), probe DNA (G), probe DNA (I), probe DNA (K) and probe DNA (M) The current value was obtained.

  Therefore, in the case of the LAMP reaction solution of composition (1), primer set A, primer set C, primer set E, primer set G, primer set I, primer set K and primer set M are fixed to the inner bottom surface of the silicone rubber. It became clear that the LAMP reaction by each proceeded locally and the resulting amplification product hybridized with the probe DNA.

  On the other hand, when the primer DNA fixed to the inner bottom surface of the silicone rubber was primer set B, primer set D, primer set F, primer set H, primer set J, primer set L, no current value was obtained.

  From these results, template A, template C, template E, template G, template I, template K, and template M contained in the LAMP reaction solution are amplified to primer sets corresponding to the array type primer probe chip of the embodiment. And hybridized with the corresponding probe nucleic acid.

  From this result, it can be determined that the LAMP reaction solution contained the templates A, C, E, G, I, K, and M.

[Results of LAMP reaction solution composition (2)]
In addition, the results when the LAMP reaction solution composition (2) containing Template B, Template D, Template F, Template H, Template J, and Template L was added are as follows.

  All probes of probe DNA (B), probe DNA (D), probe DNA (F), probe DNA (H), probe DNA (J) and probe DNA (L) are added by adding the reaction solution of composition (2). A current value of 30 nA or more was obtained.

  Therefore, the template B, the template D, the template F, the template H, the template J, and the template L are the primer DNAs fixed on the bottom surface of the silicone rubber, that is, the primer set B, the primer set D, the primer set F, and the primer set H. It was confirmed that the amplified products were locally amplified by the primer set J and the primer set L, respectively, and the resulting amplified product was hybridized to the corresponding probe DNA.

  On the other hand, no current value was obtained with primer DNA, primer set A, primer set C, primer set E, primer set G, primer set I, primer set K and primer set M) fixed on the bottom surface of the silicone rubber. .

  From this result, it is possible to determine that the LAMP reaction solution contained the templates B, D, F, H, J, and L.

[Results of LAMP reaction solution composition (3)]
The results when the LAMP reaction solution composition (3) containing all 13 types of templates are added are as follows.

  A current value of 30 nA or more was obtained for all the probe DNAs. Thereby, it became clear that the LAMP reaction by 13 kinds of primer DNAs fixed on the bottom surface of the silicon rubber proceeded locally, and the resulting LAMP product reacted with 13 kinds of probe DNAs.

  From this result, it can be determined that the LAMP reaction solution contained 13 types of templates.

  In addition, when the LAMP reaction solution composition (4) not containing the template was added, the LAMP amplification reaction by 13 types of primer DNAs fixed on the bottom surface of the silicone rubber did not proceed, and no current value was obtained.

  From the above results, it is shown that it is possible to detect a plurality of types of templates contained in the LAMP reaction solution and to identify the sequences using the array type primer probe chip described in this example. It was.

Example 2-3
The test was performed in the same manner as in Example 2-2 except that a primer mixed with water was used instead of the thickener. That is, the four types of LAMP reaction solutions were the same as the reagents used when the above thickener was added, and the same types of templates were used.

The results are shown in Table 21.

  In the LAMP reaction solution (4) containing no template, no current value was detected for any probe DNA.

  On the other hand, even if it is a result of adding the LAMP reaction liquids (1), (2) and (3), the current value derived from the template contained therein may not be obtained. This is because amplification of a part of the template contained in the LAPM reaction liquids (1), (2) and (3) was not obtained, so that no amplification product was generated by LAMP amplification, and thus hybridization with the probe DNA was performed. It is thought that this was because no occurred. Furthermore, the current value of the probe DNA from which the current value was obtained was also smaller than that obtained when the thickener was fixed together with the primer set.

  From these results, it was suggested that if a thickener is not added at the time of immobilizing the primer set, the elution range of the primer DNA is widened, and there is a high possibility that the amplification reaction to be originally obtained cannot be achieved.

  The following were shown from the results of Examples 2-2 and 2-3 described above. As described in this example, a plurality of types of templates contained in the LAMP reaction solution can be detected with higher accuracy by adding a thickener together with the fixation of the primer set in the array type primer probe chip according to the embodiment. It has been shown that it is possible and that its sequence can be identified.

Example 2-4
A thickener and a primer set were fixed to the chip material produced in Example 2-2 (1) described above.

  A method for preparing the thickener will be described. As a thickener, “Agarose-Super LM (melting temperature ≦ 60 ° C.)” manufactured by Nacalai Tesque was used. In the case of agarose, it has a specific melting temperature, gelation temperature, and remelting temperature depending on the molecular weight and structure, and it is necessary to set conditions according to the characteristics.

  After adding 0.6 g of agarose to 100 mL of DW and mixing well, it was completely dissolved by heating at 80 ° C. to prepare a 0.6% agarose solution. When mixing with the primer solution, it was again heated to 80 ° C. and completely dissolved, and then mixed with an equal amount of primer to prepare an agarose concentration of 0.3% final concentration.

  The final concentration 0.3% agarose mixed primer solution prepared as described above was dropped onto the support. Then, it was dried by heating for 2 minutes on a hot plate set to 40 ° C. After confirming that the dried state was on the film and was completely fixed, it was stored at −20 ° C. with the support until use.

4). Design of channel shape As a further embodiment, the array-type primer probe chip described here is used as a reaction device for performing a nucleic acid amplification reaction in a fine channel, or for detecting an amplification product after the amplification reaction. It may be provided as a detection device and a nucleic acid detection apparatus.

  With the development of genetic engineering in recent years, it is becoming possible to diagnose or prevent diseases caused by genes in the medical field. This is called genetic diagnosis, and it is possible to diagnose and predict a disease before the onset of the disease or at an extremely early stage by detecting a human genetic defect or change that causes the disease. In addition to the decoding of the human genome, research on the relationship between genotypes and epidemics is progressing, and treatment tailored to each individual's genotype (tailor-made medicine) is becoming a reality. Therefore, it is very important to easily detect genes and determine genotypes. Furthermore, since these gene tests often make a comprehensive judgment by detecting a plurality of types of genes, it is extremely important to simultaneously detect a plurality of types of target genes in a short time.

  As an apparatus for detecting a nucleic acid, a device called [mu] -TAS capable of sequentially performing a plurality of reactions involving a plurality of reagents in one device has been actively researched and developed. These include a reagent holding region, a reaction region, a sensor region, and the like, and are characterized by having a flow path connecting them.

  When multiple genes are detected simultaneously in one device, a method for preparing a plurality of separate amplification containers for amplifying a plurality of target genes, or a reaction container for amplifying all target genes Any of the methods for performing a multi-nucleic acid amplification reaction in the above-described case can be considered. However, when the number of target gene species to be detected increases, there is a problem that any method makes it difficult to make a device. In other words, in the method of preparing a plurality of separate amplification containers, it is necessary to prepare a large number of a plurality of amplification containers, which complicates the device. In the method of performing a multi-nucleic acid amplification reaction, the gene amplification reaction There is a problem that amplification efficiency is greatly reduced.

  As one of the means for solving the above problems, in each reaction field, a plurality of types of target nucleic acids are simultaneously amplified independently (in an independent region) using a plurality of types of primer sets, and each amplification product obtained There is a nucleic acid detection device that determines the presence or absence of a target nucleic acid by independently measuring the amount of the nucleic acid.

  However, a nucleic acid detection device that assumes that a plurality of types of target nucleic acids are simultaneously and independently amplified using a plurality of types of primer sets in one reaction field as described above has, for example, the following problems: is there.

  One of the problems is difficulty in maintaining the primer set in the amplification region of the nucleic acid detection device. In order to hold the primer set in the amplification region of the nucleic acid detection device, a solution containing the primer set is dropped onto each amplification region. However, this solution easily moves in the process of holding the primer set. Therefore, the nucleic acid detection device has a problem that the position where the primer set is held in each amplification region cannot be accurately defined.

  Another problem is the outflow of the primer set. The primer set previously held in the amplification region may flow out from the holding position to the adjacent amplification region when a solution containing the target nucleic acid is introduced into the amplification region.

  Another problem is inhibition of the amplification reaction due to the movement of the primer set and the amplification product. The primer set and the amplification product are diffused by the flow of the solution during the amplification reaction. If a non-target primer set and amplification product flow from another adjacent amplification region, the amplification reaction may be inhibited in the amplification region.

  Another problem is the inhibition of the amplification reaction due to the eluate from the protective film. The amplification reaction is performed in a region including the detection sensor of the amplification product. However, the eluate from the protective film of the sensor may inhibit the amplification reaction.

  By using the present embodiment, the multi-nucleic acid reaction tool can solve such problems and problems, thereby further improving the efficiency of nucleic acid amplification reaction, which is a further aspect. It becomes possible to provide a primer probe chip, for example, a nucleic acid reaction device, a nucleic acid detection device, and a nucleic acid detection apparatus.

<Fifteenth embodiment>
An example of a nucleic acid detection device 3001 according to the fifteenth embodiment will be described with reference to FIG. FIG. 25A is a plan view of an example of the nucleic acid detection device 3001. FIG. 25B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. The nucleic acid detection device 3001 is used to simultaneously and independently amplify a plurality of types of target nucleic acids using a plurality of types of primer sets 3031 in one reaction field.

  The nucleic acid detection device 3001 includes a support (second member) 3011 and a covering (first member) 3012. The support body 3011 has a substantially flat surface on the surface in contact with the covering body 3012. In the twelfth embodiment, the direction in which the support body 3011 and the covering body 3012 are arranged in this order is referred to as a stacking direction. The cover 3012 includes a groove 3121 on the surface (first surface) that contacts the support 3011. The groove portion 3121 is provided on a surface in contact with the support body 3011. The groove 312 is hermetically sealed by a cover 3012 and a support 3011 in contact with the cover 3012. The groove portion 3121 sealed with the covering body 3012 and the support body 3011 functions as a flow path for various solutions. As an example, the groove 3121 has a shape meandering in a curved shape from the inlet 3121a to the outlet 3121b of various solutions, but is not particularly limited. The groove 3121 is formed with substantially the same width from the inlet 3121a to the outlet 3121b. In the groove 3121, for example, a plurality of chambers (channel type chambers) 4121 are arranged at equal intervals. The chamber 4121 is used for performing a nucleic acid amplification reaction and reacting an electrode (sensor) described later with a nucleic acid sample. The chamber 4121 is formed in a shape recessed in the stacking direction from a region (part) other than the chamber 4121 in the groove 3121. That is, the depth of the chamber 4121 is formed deeper than the depth of the region other than the chamber 4121 in the groove 3121. In other words, the depth of the region other than the chamber 4121 in the groove 3121 is shallower than the depth of the chamber 4121. Accordingly, the cross-sectional area of the chamber 4121 is larger than the cross-sectional area of the region other than the chamber 4121 in the groove 3121. Note that the cross-sectional area of the chamber 4121 and the cross-sectional area of the region other than the chamber 4121 in the groove 3121 are cross-sectional areas based on a surface orthogonal to the surface of the covering 3012 where the groove 3121 is provided. The cross-sectional area of the region other than the chamber 4121 in the groove 3121 is preferably 90% or less of the cross-sectional area of the chamber 4121, but is not particularly limited. The chamber 4121 corresponds to the primer immobilization region 3021. For example, the primer immobilization region 3021 is formed on the upper surface portion of the chamber 4121 (a portion recessed in the stacking direction from the region other than the chamber 4121). The plurality of primer immobilization regions 3021 are arranged in the groove 3121 independently of each other.

  Note that the material of the support 3011 and the cover 3012 may be the same or different. In addition, the material of the support 3011 and the cover 3012 may be any material as long as the support 3011 and the cover 3012 themselves are not involved in the amplification reaction or the like. The support 3011 and the cover 3012 may be made of any material that can perform an amplification reaction in the groove 3121. The support body 3011 and the covering body 3012 may be arbitrarily selected from, for example, silicon, glass, resin, metal, and the like.

  Next, fixation (retention) of the primer set 3031 for amplifying the nucleic acid in the primer immobilization region 3021 will be described. The chamber 4121 holds the primer set 3031 on the channel wall surface. Note that the primer immobilization region 3021 corresponds to the position of the chamber 4121 and may be appropriately read as the chamber 4121. As shown in FIG. 25, the primer set 3031 is fixed in the vicinity of the upper surface in the stacking direction (primer immobilization region 3021) in the chamber 4121 so as to be released when it comes into contact with the liquid phase for providing the reaction field. The In the plurality of chambers 4121 (primer immobilization regions 3021), a plurality of primer sets 3031 are fixed for each type of target nucleic acid. That is, the plurality of chambers 4121 (primer immobilization regions 3021) hold a plurality of types of primer sets configured to amplify a plurality of types of target sequences, respectively. For the fixation of the primer set 3031, for example, only the covering 3012 is prepared so that the groove 3121 faces upward in the vertical direction, and a solution containing the primer set 3031 is dropped into one primer fixing region 3021 and then dried. Is possible. The method for holding the primer set 3031 is not limited to drying, and other methods such as freeze-drying may be used.

  As described above, since the chamber 4121 is formed deeper than the region other than the chamber 4121 in the groove portion 3121, the solution containing the primer set 3031 locally dropped on the primer immobilization region 3021 is the region other than the chamber 4121. It doesn't move easily. Therefore, adjacent primer immobilization regions 3021 can independently hold different primer sets 3031.

  Next, addition of a reaction solution to the nucleic acid detection device 3001 in which a plurality of primer sets 3031 are fixed to different primer fixing regions 3021 will be described. FIG. 26A is a plan view of an example of the nucleic acid detection device 3001. FIG. 26B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. FIG. 26 shows a state in which a reaction solution is added to the nucleic acid detection device 3001. The nucleic acid detection device 3001 shown in FIG. 26 is the same as FIG. 25 except that the reaction solution is added to the groove 3121.

  The reaction solution may contain components necessary for the nucleic acid amplification reaction. The reaction solution is not limited to these. For example, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from an enzyme such as a polymerase or a primer, and reverse transcription are simultaneously performed. In some cases, a buffer such as reverse transcriptase and a substrate necessary for the reverse transcriptase and salts for maintaining an appropriate amplification environment may be included.

  As shown in FIG. 26, after adding the reaction solution from the inlet 3121a, the primer set 3031 fixed to the primer fixing region 3021 starts to be released and diffused. A region where the primer is released and diffused is schematically shown as a primer release / diffusion region 3022 in FIG.

  When the reaction solution is introduced into the nucleic acid detection device 3001, it is desired that the primer set 3031 that is liberated and diffused does not easily flow out to the other adjacent primer immobilization region 3021. That is, the primer release / diffusion region 3022 desirably corresponds to the primer immobilization region 3021 (in other words, the chamber 4121).

  The nucleic acid detection device 3001 can greatly reduce the flow velocity in the vicinity of the portion where the primer set 3031 is fixed in the chamber 4121 (portion recessed in the stacking direction from the region other than the chamber 4121) when the reaction solution is introduced. . Therefore, the nucleic acid detection device 3001 can prevent the primer set 3031 from flowing out to other adjacent primer immobilization regions 3021. Therefore, the nucleic acid detection device 3001 according to the twelfth embodiment can make the primer sets 3031 of the adjacent primer immobilization regions 3021 independent.

  Further, even during the amplification reaction, the primer set 3031 and the generated amplification product in a certain amplification region (region corresponding to the primer-immobilized region 3021 (chamber 4121)) do not diffuse to other amplification regions and are independently It is desirable to be within the amplification region. In the nucleic acid detection device 3001, the depth (channel cross-sectional area) of the groove 3121 in the region other than the chamber 4121 is shallower (smaller) than the depth (channel cross-sectional area) of the chamber 4121. Therefore, the nucleic acid detection device 3001 can suppress the diffusion of the primer in the amplification region and the generated amplification product to the other amplification region during the amplification reaction. Therefore, the nucleic acid detection device 3001 according to the fifteenth embodiment can achieve amplification of a plurality of template sequences using a plurality of types of primer sets 3031 independently (locally) and simultaneously with high efficiency. is there.

  Specific detection means of the amplification product obtained locally includes detection means of a hybridization signal known per se, for example, detection and / or measurement of fluorescence intensity using a fluorescent label, or current using an intercalator This can be done using methods of detecting and / or measuring the response, and is not limited.

  Next, detection of a hybridization signal by the nucleic acid detection device 3001 will be described. FIG. 27A is a plan view of an example of the nucleic acid detection device 3001. FIG. 27B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. The nucleic acid detection device 3001 shown in FIG. 27 is the same as FIG. 25 except that the support 3011 includes the probe immobilization region 3111. For example, the probe immobilization region 3111 is disposed on the support 3011 at a position facing the primer immobilization region 3021 (chamber 4121). However, the probe immobilization region 3111 is not particularly limited and may be in any form within the chamber 4121. There may be. The probe immobilization region 3111 is a region where, for example, an electrode for detecting a hybridization signal (electrode for detecting a nucleic acid) is provided. That is, the nucleic acid detection electrode in the probe immobilization region 3111 is disposed at a position facing the surface of the covering 3012 that contacts the support 3011 and the groove 3121 (particularly the primer immobilization region 3021 (chamber 4121)). The

  In the probe immobilization region 3111, a plurality of probe nucleic acids including a complementary sequence of a desired sequence to be detected are immobilized. The nucleic acid detection device 3001 can obtain a hybridization signal in the probe immobilization region 3111 after performing an amplification reaction in the primer immobilization region 3021.

  Next, the case where a reaction solution is introduced into the nucleic acid detection device 3001 shown in FIG. 27 and an amplification reaction is performed will be described. FIG. 28A is a plan view of an example of the nucleic acid detection device 3001. FIG. 28B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. FIG. 28 shows a state in which a reaction solution is added to the nucleic acid detection device 3001. Note that the nucleic acid detection device 3001 shown in FIG. 28 is the same as FIG. 27 except that the reaction solution is added to the groove 3121. In the nucleic acid detection device 3001, when a reaction solution is introduced, if a template nucleic acid is present, the template nucleic acid is amplified by the corresponding free and diffused primer set 3031 to generate an amplification product 3032. The amplification product 3032 generated in the amplification reaction is locally generated in the chamber 4121 as shown in FIG. The nucleic acid detection device 3001 can obtain a hybridization signal by reacting the amplification product 3032 with the probe immobilized on the probe immobilization region 3111.

<Example 3 (part 1)>
Example 3-1
An example of nucleic acid detection using the nucleic acid detection device 3001 according to the fifteenth embodiment will be specifically described below. In Example 3-1, a packing 3012a made of silicon rubber was used as the covering 3012, and the primer set 3031 was fixed to the primer fixing region 3021 on the entire surface of the packing 3012a. FIG. 29 is a schematic diagram illustrating an example of a support 3011 according to Example 3-1. In Example 3-1, an array-type chip (substrate) 3011a for electrochemical detection using the probe-immobilized region 3111 as an electrode 3111a as the support 3011 is used, and a current response generated depending on the presence of hybridization is obtained. Used as a sensor to detect. The pad portion 3112a is for transmitting the hybridization signal of the electrode 3111a to the nucleic acid detection device (not shown) via the wiring 3113b. That is, the nucleic acid detection device detects nucleic acid based on the current value from each electrode 3111a.

(1) Preparation of nucleic acid detection device 1-1 Production of array chip for electrochemical detection The array chip 3011a for electrochemical detection is formed by sputtering a thin film of titanium and gold on the surface of Pyrex (registered trademark) glass. did. Thereafter, an electrode pattern of titanium and gold was formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and the electrode 3111a was exposed by an etching process.

Next, six types of nucleic acid probes (sequences A to E, NC) shown in Table 22 were immobilized on each electrode 3111a (AE, NC (negative control) in FIG. 29) on the chip material. A solution containing each nucleic acid probe was dropped onto each electrode 3111a, and then the excess nucleic acid probe was immobilized by washing away.

1-2 Preparation of Primer-Fixed Packing 3012a, Assembly of Device for Nucleic Acid Detection First, primer DNA used as the primer set 3031 was prepared. The primer DNA used is a primer set 3031 for amplification by a loop-mediated isothermal amplification (LAMP) method. Table 17 shows the base sequences of the primer DNAs used. The solution containing the primer DNA was spotted on the bottom surface of the packing 3012a facing the region where the corresponding probe DNA was fixed, and dried at 40 ° C. for 2 minutes. Thereby, primer-fixed packing 3012a was obtained. This packing 3012a was attached to the array type chip 30111a to obtain the nucleic acid detection device 3001 shown in FIG. FIG. 30A is a plan view of an example of the nucleic acid detection device 3001 according to Example 3-1. FIG. 30B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG.

(2) Detection of Nucleic Acid 2-1 Preparation of Template Solution As shown in Table 18, a template solution was prepared by mixing three types of templates of genes A, B, and D and a reagent for LAMP amplification.

The template solution used contained Bst DNA polymerase and reaction mix and was added with distilled water (ie, DW) so that the total amount was 50 μL. As shown in Table 19, the template solution was subjected to amplification reaction by the LAMP method with the primer DNA (set A) and hybridized with the probe DNA (A) to be detected, and the primer DNA (set B). ) Causes an amplification reaction by the LAMP method, and a template B detected by hybridization with the probe DNA (B) and the primer DNA (set D) cause an amplification reaction by the LAMP method, and the probe DNA (D And a template D detected by hybridization.

2-2 Addition of Template Solution 50 μL of the template solution prepared in 2-1 was added to the nucleic acid detection device 3001.

2-3 Nucleic Acid Reaction Under the conditions shown below, the flow channel region of the nucleic acid detection device 3001 was heated or cooled to perform various reactions. FIG. 31A is a plan view of an example of the nucleic acid detection device 3001 according to Example 3-1. FIG. 31B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. As shown in FIG. 31, when a template containing a target sequence to be amplified is contained in a LAMP reaction solution for a specific primer, the LAMP reaction proceeds locally at the location where the primer is fixed, and the resulting amplification The product 3032 hybridizes with the probe DNA in the vicinity thereof.

Nucleic acid amplification reaction: 64 ° C., 60 minutes Hybridization reaction: 50 ° C., 10 minutes Washing reaction: 30 ° C., 5 minutes Current detection reagent (Hoechst 33258) reaction: 25 ° C., 3 minutes.

2-4 Detection of Nucleic Acid The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecule specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The above series of reactions was carried out using an automatic DNA testing apparatus described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008.

Results The results obtained are shown in FIG. FIG. 32 is a graph showing the results of the nucleic acid amplification reaction according to Example 3-1. In the electrodes A, B, and D on which the probes A, B, and D corresponding to the genes A, B, and D to which the template was added were immobilized, a current value larger than that of the NC was obtained. On the other hand, the current values of the electrodes C and E corresponding to the genes C and E to which no template was added were about the same as those of the NC. This revealed that the template in the template solution could be reliably detected.

<Sixteenth Embodiment>
The sixteenth embodiment will be described in detail with reference to the drawings. Note that the same components as those described in the fifteenth embodiment are denoted by the same reference numerals and description thereof is omitted. An example of a nucleic acid detection device 3001 according to the sixteenth embodiment will be described with reference to FIG. FIG. 33A is a plan view of an example of the nucleic acid detection device 3001. FIG. 33B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. The nucleic acid detection device 3001 is used to simultaneously and independently amplify a plurality of types of target nucleic acids using a plurality of types of primer sets 3031 in one reaction field. The sixteenth embodiment differs from the fifteenth embodiment in the shape of the groove 3121.

  The groove 3121 is formed from the inlet 3121a to the outlet 3121b at substantially the same depth in the stacking direction. In the groove 3121, a plurality of chambers 4121 are arranged at predetermined intervals. The width of the chamber 4121 is formed to be wider than the width of the region other than the chamber 4121 in the groove 3121. In other words, the width of the region other than the chamber 4121 in the groove 3121 is narrower than the width of the chamber 4121. Note that the chamber 4121 has only to be wider than the region other than the chamber 4121 in the groove 3121, and may protrude in an arc shape in a direction perpendicular to the stacking direction or may protrude in a square shape. Accordingly, the cross-sectional area of the chamber 4121 is larger than the cross-sectional area of the region other than the chamber 4121 in the groove 3121. Note that the cross-sectional area of the chamber 4121 and the cross-sectional area of the region other than the chamber 4121 in the groove 3121 are cross-sectional areas based on a surface orthogonal to the surface of the covering 3012 where the groove 3121 is provided. The cross-sectional area of the region other than the chamber 4121 in the groove 3121 is preferably 90% or less of the maximum value of the cross-sectional area of the chamber 4121, but is not particularly limited. The chamber 4121 corresponds to the primer immobilization region 3021. The primer immobilization region 3021 is formed in the vicinity of the upper surface portion of the groove portion 3121 in the stacking direction. The plurality of primer immobilization regions 3021 are arranged in the groove 3121 independently of each other.

  As described above, since the chamber 4121 is formed wider than the region other than the chamber 4121 in the groove 3121, the solution containing the primer set 3031 locally dropped on the primer immobilization region 3021 is not included in the chamber 4121. It does not move easily into the area. Therefore, adjacent primer immobilization regions 3021 can independently hold different primer sets 3031.

  Next, addition of a reaction solution to the nucleic acid detection device 3001 in which a plurality of primer sets 3031 are fixed to different primer fixing regions 3021 will be described. FIG. 34A is a plan view of an example of the nucleic acid detection device 3001. FIG. 34B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. FIG. 34 shows a state where a reaction solution is added to the nucleic acid detection device 3001. The nucleic acid detection device 3001 shown in FIG. 34 is the same as FIG. 34 except that the reaction solution is added to the groove 3121. The reaction solution may be the same component as in the twelfth embodiment.

  As shown in FIG. 34, after adding the reaction solution from the inlet 3121a, the primer set 3031 fixed to the primer fixing region 3021 starts to be released and diffused. A region where the primer is released and diffused is schematically shown as a primer release / diffusion region 3022 in FIG. In the nucleic acid detection device 3001, the width (channel cross-sectional area) of the groove 3121 in the region other than the chamber 4121 is narrower (smaller) than the width (channel cross-sectional area) of the chamber 4121. Therefore, the nucleic acid detection device 3001 can suppress the diffusion of the primer in the amplification region and the generated amplification product to the other amplification region during the amplification reaction. Therefore, the nucleic acid detection device 3001 according to the thirteenth embodiment can achieve amplification of a plurality of template sequences using a plurality of types of primer sets 3031 independently (locally) and simultaneously with high efficiency. is there.

  In the example shown in FIGS. 33 and 34, the example in which the primer set 3031 is fixed to the channel wall surface in the vicinity of the upper surface of the chamber 4121 has been described. However, the present invention is not limited to this. FIG. 34 is a plan view of an example of the nucleic acid detection device 3001 showing other fixing locations of the primer set 3031 in the chamber 4121 (primer immobilization region 3021). The primer set 3031 may be fixed to the channel wall surface of the chamber 4121 orthogonal to the stacking direction. That is, the primer set 3031 is provided in a portion of the chamber 4121 that protrudes in a direction perpendicular to the stacking direction from the region of the groove 3121 other than the chamber 4121.

  As shown in FIG. 35, when the primer set 3031 is fixed in the chamber 4121, the nucleic acid detection device 3001 has a portion of the chamber 4121 where the primer set 3031 is fixed (in the groove 3121 other than the chamber 4121) when the reaction solution is introduced. The flow velocity in the vicinity of the portion protruding in the direction perpendicular to the stacking direction than the region can be greatly reduced. Therefore, the nucleic acid detection device 3001 can prevent the primer set 3031 from flowing out to other adjacent primer immobilization regions 3021. Therefore, the nucleic acid detection device 3001 according to the thirteenth embodiment can make the primer sets 3031 of the adjacent primer immobilization regions 3021 independent.

  In the example shown in FIGS. 33, 34, and 35, the portion of the groove 3121 that connects the chambers 4121 is formed in a straight line shape that connects at the shortest distance, but is not limited thereto. FIG. 36 is a plan view of an example of a nucleic acid detection device 3001 that shows another shape of the groove 3121 that connects the chambers 4121 to each other. In the example shown in FIG. 36, the portion of the groove portion 3121 that connects the chambers 4121 is configured to have a shape (for example, a curved shape) in which the flow path distance is longer than the linear shape. Therefore, the groove part 3121 shown in FIG. 36 can improve the independence of each chamber 4121 compared with the structure of the groove part 3121 which connects each chamber 4121 in the linear shape shown in FIG. Efficient nucleic acid amplification can be performed. Here, regarding the sixteenth embodiment, the configuration of the groove 3121 connecting the chambers 4121 has been described. However, the configuration shown in FIG. 36 is applied to the fifteenth embodiment as well. The effect of can be obtained.

  Specific detection means of the amplification product obtained locally includes detection means of a hybridization signal known per se, for example, detection and / or measurement of fluorescence intensity using a fluorescent label, or current using an intercalator This can be done using methods of detecting and / or measuring the response, and is not limited.

  Next, detection of a hybridization signal by the nucleic acid detection device 3001 will be described. FIG. 37A is a plan view of an example of the nucleic acid detection device 3001. FIG. 37B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. Note that the nucleic acid detection device 3001 shown in FIG. 37 is the same as FIG. 33 except that the support 3011 includes the probe-immobilized region 3111. The probe immobilization region 3111 is not particularly limited. For example, the probe immobilization region 3111 is disposed on the support 3011 at a position facing the chamber 4121 (primer immobilization region 3021). The probe immobilization region 3111 is a region where an electrode for detecting a hybridization signal is provided.

  In the probe immobilization region 3111, a plurality of probe nucleic acids including a complementary sequence of a desired sequence to be detected are immobilized. The nucleic acid detection device 3001 can obtain a hybridization signal in the probe immobilization region 3111 after performing an amplification reaction in the primer immobilization region 3021.

  In the case where the support 3011 is a current detection type sensor that particularly uses an intercalator, amplification inhibition occurs due to an eluate from the sensor protective film accompanying heating during the amplification reaction. However, since the nucleic acid detection device 3001 according to the sixteenth embodiment has a configuration in which the groove 3121 other than the amplification / detection region (chamber 4121) is narrowed (narrow in width), it is in contact with the sensor provided in the probe immobilization region 3111. Since the liquid area can be reduced, elution of amplification inhibitors can be effectively suppressed.

<Example 3 (part 2)>
Example 3-2
Nucleic acid detection with the nucleic acid detection device 3001 according to the sixteenth embodiment described above is performed, for example, as follows. A reaction solution containing a nucleic acid to be inspected is introduced into a groove 31211 formed in the nucleic acid detection device 3001 with an instrument such as a pipette. When the reaction solution is introduced and the template nucleic acid is present, the template nucleic acid is amplified by the corresponding free-diffused primer set 3031 to generate an amplification product.

  FIG. 38A is a plan view of an example of the nucleic acid detection device 3001 according to Example 3-2. FIG. 38B is a cross-sectional view of the nucleic acid detection device 3001 along the line XX in FIG. FIG. 38 (c) is a graph showing the results of the nucleic acid amplification reaction according to Example 3-2. FIG. 38C shows an amplification reaction in the regions A, B, and D (each chamber 4121) of the nucleic acid detection device 3001 shown in FIGS. 38A and 38B, and the amplification product corresponding to the probe nucleic acid. The results obtained when the target sequence complementary to the sequence is included are shown. As shown in FIG. 38C, the current values obtained for regions A, B, and D are larger than those of NC. On the other hand, the detection signals obtained for the regions C and E are approximately the same as the NC. This revealed that the template in the template solution could be reliably detected. Note that the detection signals are also obtained particularly in the adjacent regions A and B. Therefore, the nucleic acid detection device 3001 according to the thirteenth embodiment had no interference between adjacent regions, and was able to perform independently from nucleic acid amplification reaction to detection. As described above, according to Example 3-2, using the nucleic acid detection device 3001 according to the thirteenth embodiment, a plurality of types of target nucleic acids are amplified simultaneously and independently until detection. It was shown that it can.

  According to the above-described embodiment, it is difficult to maintain the primer set in the amplification region of the nucleic acid detection device, the primer set flows out, the amplification reaction is inhibited by the movement of the primer set and the amplification product, and the eluate from the protective film At least one problem among the inhibition of the amplification reaction can be solved. Therefore, a nucleic acid detection device that simultaneously and independently amplifies a plurality of types of target nucleic acids using a plurality of types of primer sets, and a nucleic acid detection apparatus using the same, perform efficient amplification reaction and detection simultaneously and independently. It can be realized.

  It is possible to combine the shape of the chamber 4121 according to the twelfth embodiment and the shape of the chamber 4121 according to the sixteenth embodiment.

5. Use of Protective Film As a further embodiment, the array-type primer probe chip may be provided as a nucleic acid detection device for performing nucleic acid amplification to detection in the same device.

  The array-type primer probe chip may be provided as a nucleic acid detection device that reduces inhibition of the nucleic acid amplification reaction.

  One of devices for detecting nucleic acids is a DNA chip. A DNA chip is a device in which a plurality of nucleic acid probes are immobilized on a substrate, and is characterized in that a large number of nucleic acid sequences can be detected at one time.

  There are various forms of the immobilization region of the nucleic acid probe, and there is a method in which the nucleic acid probe is immobilized on a sensor such as an electrode, a detection signal from the sensor is extracted by wiring, and is detected from the outside.

  In the case of such a form, regions other than the sensor portion and the outside contact portion are covered with a film called a protective film (passivation film). This is an application of semiconductor technology, and the protective film protects the obtained detection signal from noise from the wiring portion, contamination, and the like.

  On the other hand, a device called μ-TAS capable of sequentially performing a plurality of reactions involving a plurality of reagents in one device has been actively researched and developed. Such a device includes a reagent holding region, a reaction region, a sensor region, and the like, and includes a flow path that connects them. By applying this, a detection apparatus for detecting a nucleic acid has also been developed. When nucleic acid detection is performed, it is necessary to perform a plurality of reactions using a plurality of reagents. The plurality of reactions include a nucleic acid extraction reaction, a nucleic acid purification reaction, a nucleic acid amplification reaction, a nucleic acid hybridization reaction, and the presence / absence detection of hybridization. Among these reactions, nucleic acid amplification reactions include PCR, LAMP, and ICAN methods. All of these reactions are not only greatly affected by the amplification temperature and reagent composition, but also when impurities are mixed, amplification inhibition occurs. Since it has the feature of being easy to handle, the material and cleanliness of the reaction vessel are very important.

  Nucleic acid detection devices such as the above-mentioned DNA chip have various configurations. However, when a plurality of reactions are performed in separate reaction vessels as described above, the loss of reagents and test time is large. Therefore, a nucleic acid detection device capable of performing a nucleic acid amplification reaction and a nucleic acid hybridization reaction in the same reaction vessel has been developed.

  However, amplification inhibition occurs in the nucleic acid amplification reaction due to contamination with impurities. It has been clarified that the elution component from the protective film of the nucleic acid detection device strongly inhibits nucleic acid amplification, resulting in a decrease in sensitivity. Therefore, the nucleic acid detection device is required to reduce the amount of impurities eluted from the protective film and improve the sensitivity.

  According to the embodiment, when the array-type primer probe chip is provided as a nucleic acid detection device, the nucleic acid detection device includes a substrate, a sensor unit, a wiring, and a protection unit. The sensor unit is for detecting a nucleic acid formed on the substrate. The wiring is formed on the substrate and connected to the sensor. The protective film is formed on the substrate. The nucleic acid detection device detects a nucleic acid amplification product by the sensor unit after performing a nucleic acid amplification reaction in a chamber in which the sensor unit and a nucleic acid sample react. The protective film includes one or more openings that expose a lower layer portion including a part of the substrate in a liquid contact region of the nucleic acid sample on the substrate.

<Seventeenth embodiment>
FIG. 39 is a diagram showing a production procedure as an example of the nucleic acid detection device (DNA chip) 5100 according to the seventeenth embodiment. The nucleic acid detection device 5100 is configured by stacking each component in the order of (a) to (e) of FIG. FIG. 40 is a cross-sectional view in the stacking direction illustrating a schematic configuration as an example of the nucleic acid detection device 5100 according to the seventeenth embodiment. FIG. 41 is a diagram showing a schematic configuration as an example of a nucleic acid detection device 5100 according to the seventeenth embodiment.

  The nucleic acid detection device 5100 includes a substrate 5010, a sensor unit 5011, a pad 5012, a wiring 5013, and a protective film 5014. The substrate 5010 is a thin plate member as shown in FIG. The substrate 5010 is made of glass, silicon, polycarbonate, polypropylene, polyethylene, polyimide, ABS, metal, or the like, but is not particularly limited.

  The sensor unit 5011 is formed on the surface of the substrate 5010 as shown in FIG. The sensor unit 5011 is an electrode made of a conductive member. A plurality of sensor units 5011 are provided on one end side of the substrate 5010. In the sensor unit 5011, various nucleic acid probes for detecting a target nucleic acid are immobilized, and the target nucleic acid is detected. One sensor unit 5011 includes one or more sensors.

  The pad portion 5012 is formed on the surface of the substrate 5010 as shown in FIG. The pad portion 5012 is made of a conductive member. A plurality of pad portions 5012 are provided on the other end side of the substrate 5010. The pad unit 5012 is for transmitting a detection signal of the sensor unit 5011 to a detection device (not shown) via a wiring 5013 described later.

  The wiring 5013 is formed on the surface of the substrate 5010 as shown in FIG. The wiring 5013 is made of a conductive member. A wiring 5013 connects the sensor unit 5011 and the pad unit 5012. The wiring 5013 may be a three-dimensional wiring having a multilayer structure using a through hole or the like. The wiring 5013 is for taking out a detection signal from each sensor unit 5011 and sending it to the pad unit 5012. The protective film 5014 is formed on the surface of the substrate 5010 as shown in FIG.

  The protective film 5014 is a protective film made of an organic material. The protective film 5014 is made of, for example, a highly hydrophobic material. Generally, the protective film is classified into an organic protective film and an inorganic protective film. Inorganic protective films are known to have low impurity elution, but are expensive. Therefore, the protective film 5014 used in the fourteenth embodiment is an organic protective film. The shape of the protective film 5014 on the surface of the substrate 5010 will be described later. The protective film 5014 is used to prevent noise from being mixed into the detection signal from the wiring 5013 and the like and leakage of the signal to other wiring, and to protect the nucleic acid detection device 5100 from contamination and the like. According to the above-described procedure, the nucleic acid detection device 5100 is configured by laminating each component as shown in FIG. The nucleic acid detection device 5100 according to the seventeenth embodiment performs a nucleic acid amplification reaction in a flow path type reaction unit (chamber) 5201 (described later) for allowing the sensor unit 5011 and the nucleic acid sample to react with each other. Used to detect amplification products. In the seventeenth embodiment, the outermost surface on the protective film 5014 side in the stacking direction of the nucleic acid detection device 5100 is referred to as the surface of the nucleic acid detection device 5100.

  The surface of the nucleic acid detection device 5100 configured as described above can be roughly divided into a sensor region 5020, a wiring region 5021, a pad region 5022, and a reaction region 5023 as shown in FIG.

  The sensor region 5020 is a region where the sensor unit 5011 is formed. The wiring region 5021 is a region where the wiring 5013 is formed. The pad region 5022 is a region where the pad portion 5012 is formed. The reaction region 5023 is a region where a nucleic acid amplification reaction, a nucleic acid hybridization reaction, and the like are performed.

  Here, the reaction region 5023 will be described. As shown in FIG. 40B, the nucleic acid detection device 5100 is arranged so that a reaction portion defining member 5200, which will be described later, faces the protective film 5014 when used in a nucleic acid amplification reaction, a nucleic acid hybridization reaction, or the like. Is done. The sensor unit 5011 and the vicinity of the sensor unit 5011 are opposed to a later-described flow channel type reaction unit 5201 provided in the reaction unit defining member 5200. In the seventeenth embodiment, the reaction region 5023 refers to a region facing the flow channel reaction unit 5201 on the surface of the nucleic acid detection device 5100. Accordingly, the reaction region 5023 includes the sensor region 5020. The reaction region 5023 includes a wiring 5013 in the vicinity of the sensor unit 5011. Note that the reaction region 5023 is also a liquid contact region where a reaction solution (nucleic acid sample) for performing a nucleic acid amplification reaction on the substrate 5010 is in contact with the reaction region 5023. The reaction region 5023 on the surface of the nucleic acid detection device 5100 is defined by the shape of the flow channel reaction unit 5201.

  Next, the shape of the protective film 5014 on the surface of the nucleic acid detection device 5100 will be described. As shown in FIGS. 40 and 41, the protective film 5014 covers the wiring 5013 in the wiring region 5021 so that the wiring 5013 is not exposed. As shown in FIG. 41, the protective film 5014 is formed on the substrate so that in the pad region 5022, at least a part of each pad portion 5012 is exposed so that each pad portion 5012 contacts a detection device (not shown). 5010 is provided. That is, the protective film 5014 covers the surface of the nucleic acid detection device 5100 except for at least a part of each pad portion 5012 in a region other than the reaction region 5023.

  Next, the shape of the protective film 5014 in the reaction region 5023 will be described. FIG. 42 is an enlarged view near the reaction region 5023 on the surface of the nucleic acid detection device 5100 according to the seventeenth embodiment. The protective film 5014 covers the wiring 5013 so that the wiring 5013 is not exposed in the reaction region 5023. On the other hand, the protective film 5014 is not provided in the sensor unit 5011 in the reaction region 5023. The protective film 5014 includes one or more openings that expose the substrate 5010 or the sensor unit 5011 at least in a portion other than the wiring 5013 in the reaction region 5023. The protective film 5014 is not provided in the reaction region 5023 other than the minimum necessary portion. Note that the protective film 5014 may be provided over the substrate 5010 or in the vicinity of a boundary portion between the wiring 5013 and the substrate 5010.

  As described above, the protective film 5014 according to the seventeenth embodiment includes one or more openings that expose the lower layer portion (the substrate 5010 or the sensor portion 5011) including a part of the substrate 5010 in the reaction region 5023. The protective film 5014 may be configured to cover the surface of the nucleic acid detection device 5100 with a single film having one or more openings. In addition, the protective film 5014 may be configured to cover the surface of the nucleic acid detection device 5100 with a plurality of films and include one or more openings formed by a combination of the plurality of films. The configuration of the protective film 5014 in the reaction region 5023 as described above can significantly reduce the influence of amplification inhibition caused by impurities eluted from the protective film 5014 when an amplification reaction is performed in the reaction region 5023. it can. Usually, the detection of nucleic acid comprises a nucleic acid extraction reaction, a nucleic acid purification reaction, a nucleic acid amplification reaction, a nucleic acid hybridization reaction, detection of the presence or absence of hybridization, and the like. The nucleic acid detection device 5100 according to the fourteenth embodiment includes a nucleic acid high reaction. Not only the hybridization reaction but also the nucleic acid amplification reaction can be performed in the same reaction region 5023. Note that the protective film 5014 covers the wiring 5013 even in the reaction region 5023 because noise is mixed from the wiring 5013 if the wiring 5013 is not covered with the protective film 5014 as described above.

  FIG. 43 shows a nucleic acid detection device built-in cassette (liquid feeding cassette) 1 that can perform nucleic acid amplification reaction to nucleic acid detection in the same reaction vessel using the nucleic acid detection device 5100 according to the fourteenth embodiment. It is a figure which shows schematic structure used as an example. Note that in FIG. 43, the wiring 5013 and the protective film 5014 formed over the substrate 5010 are not illustrated for the sake of simplicity. FIG. 43 is a view of the nucleic acid detection device 5100 in which the reaction part defining member (reaction container) 5200 is disposed to face the reaction part defining member 5200 as viewed from the reaction part defining member 5200 side. FIG. 43 shows an example in which each sensor unit 5011 includes two sensors.

  As shown in FIG. 43, the nucleic acid detection device built-in cassette 5001 includes the nucleic acid detection device 5100, the reaction part defining member 5200, the first cassette 5300, and the second cassette 5400 described above.

  As shown in FIG. 44, the reaction part defining member 5200 includes a flow path type reaction part 5201. The flow path type reaction unit 5201 is a groove (flow path) provided on the surface in contact with the surface of the nucleic acid detection device 5100. In the flow path type reaction unit 5201, various solutions for performing from the nucleic acid amplification reaction to the nucleic acid detection in the same reaction vessel are injected from the sample injection port 5201a and discharged from the sample outlet 5201b. The flow channel reaction unit 5201 is provided in a meandering flow channel shape, but is not limited thereto. The flow path type reaction unit 5201 may be provided in a linear shape, a circular shape, or a square shape. The reaction part defining member 5200 may be a flat plate type or a tube type.

  The nucleic acid amplification primer may be mixed with the nucleic acid sample and then injected into the flow channel reaction unit 5201, or may be held in advance in any part of the flow channel reaction unit 5201. In the latter case, the plurality of primer sets prepared for each amplification target may be held in different locations of the flow path type reaction unit 5201, or may be held all at one location. The method for holding the plurality of primer sets in the flow path type reaction unit 5201 is not limited. For example, the plurality of primer sets may be dried and held by a technique such as heat or vacuum drying, or may be held in a liquid state. Further, the plurality of primer sets may be frozen and held. The plurality of primer sets may be held on a holding carrier such as a membrane.

  In addition, although the flow path type reaction part 5201 is prescribed | regulated by forming in the reaction part prescription | regulation member 5020, it is not specifically limited. The flow path type reaction unit 5201 may be formed by etching the substrate 5010 or the like.

  The first cassette 5300 and the second cassette 5400 are outer frames that sandwich the nucleic acid detection device 5100 and the reaction part defining member 5200. The first cassette 5300 and the second cassette 5400 are made of, for example, a hard material. The cassette 5001 with a built-in nucleic acid detection device has a cassette structure in which the nucleic acid detection device 5100 and the reaction part defining member 5200, which are separate components, are sandwiched between the first cassette 5300 and the second cassette 5400. There is no particular limitation. The reaction part defining member 5200 may be configured integrally with the second cassette 5400. The cassette 5001 with a built-in nucleic acid detection device is a container type in which a reaction part defining member 5200, a first cassette 5300, and a second cassette 5400 are integrally formed, and the nucleic acid detection device 5100 is inserted into the container type. It may be configured.

  Next, as a comparative example, unlike the seventeenth embodiment, a nucleic acid detection device in which a part other than the sensor part (including not only the wiring but also the substrate) is covered with an organic protective film in the reaction region will be described. . The nucleic acid hybridization reaction is performed in the reaction region where the sensor part is formed. Therefore, usually, in the reaction region, the part other than the sensor part is covered with a protective film. However, it is generally known that the protective film has a slight eluate (impurities). In general, impurities do not affect the nucleic acid hybridization reaction. However, the nucleic acid amplification reaction is a very delicate reaction, and if an impurity is contained, amplification inhibition easily occurs. Therefore, when the nucleic acid amplification reaction is performed in the same reaction region as the nucleic acid hybridization reaction, the above-described amplification inhibition occurs due to the eluate from the protective film present in the reaction region.

  According to the seventeenth embodiment, by reducing the area occupied by the protective film 5014 in the reaction region 5023, the amount of impurities eluted from the protective film 5014 can be greatly reduced, and inhibition of the nucleic acid amplification reaction can be reduced. As a result, the sensitivity of the nucleic acid detection device 5100 is improved.

<Eighteenth embodiment>
The eighteenth embodiment will be described in detail with reference to the drawings. Note that the same components as those described in the seventeenth embodiment are denoted by the same reference numerals and description thereof is omitted. FIG. 45 is an enlarged view near the reaction region 5023 in the nucleic acid detection device 5100 according to the eighteenth embodiment. The eighteenth embodiment differs from the seventeenth embodiment in the shape of the protective film 5014 in the reaction region 5023.

  As in the seventeenth embodiment, the protective film 5014 covers the wiring 5013 so that the wiring 5013 is not exposed in the reaction region 5023. Further, the protective film 5014 covers the outer peripheral portion of the sensor portion 5011 so that the outer peripheral portion of the sensor portion 5011 (the boundary portion between the sensor portion 5011 and the substrate 5010) is not exposed in the reaction region 5023. In other words, the protective film 5014 includes an opening that exposes a substantially central portion of the sensor unit 5011. That is, the protective film 5014 includes one or more openings that expose the substrate 5010 or the sensor unit 5011 in at least a part other than the outer periphery of the wiring 5013 and the sensor unit 5011 in the reaction region 5023. Note that the protective film 5014 may be provided over the substrate 5010 or in the vicinity of a boundary portion between the wiring 5013 and the substrate 5010.

  As described above, the protective film 5014 according to the eighteenth embodiment includes one or more openings that expose the lower layer part (the substrate 5010 or the sensor part 5011) including a part of the substrate 5010 in the reaction region 5023.

  The protective film 5014 is provided on the outer peripheral portion of the sensor unit 5011 for the following reason. The detection signal from the sensor unit 5011 is proportional to the exposed area of the sensor unit 5011 (the wetted area not covered with the protective film 5014). Therefore, it is desirable that the exposed area of each sensor unit 5011 be constant. By providing the protective film 5014 having an opening in a portion facing the substantially central portion of each sensor unit 5011 on the substrate 5010, the area of each sensor unit 5011 can be defined strictly constant.

  According to the eighteenth embodiment, the same effect as in the seventeenth embodiment can be obtained. Furthermore, according to the eighteenth embodiment, the exposed area of each sensor unit 5011 (when each sensor unit 5011 is composed of a plurality of sensors, the sum of the exposed areas of the plurality of sensors) is constant. Therefore, the sensitivity of the nucleic acid detection device 5100 is further improved.

<Nineteenth embodiment>
The nineteenth embodiment will be described in detail with reference to the drawings. Note that the same components as those described in the seventeenth embodiment are denoted by the same reference numerals and description thereof is omitted. FIG. 46 is an enlarged view near the reaction region 5023 in the nucleic acid detection device 5100 according to the nineteenth embodiment. The nineteenth embodiment differs from the fourteenth embodiment in the shape of the protective film 5014 in the reaction region 5023.

  The protective film 5014 covers the wiring 5013 so that the wiring 5013 is not exposed in the reaction region 5023. Further, the protective film 5014 has a partition shape that covers the substrate 5010 in the boundary region between the sensor units 5011 in the reaction region 5023. Here, the boundary region between the sensor units 5011 is a substantially central portion between the sensor units 5011 in the reaction region 5023. The protective film 5014 provided in the boundary region between the sensor units 5011 is provided in the reaction region 5023 in the vicinity of an opening (exposed substrate 5010) provided in the vicinity of the sensor unit 5011 and another adjacent sensor unit 5011. The substrate 5010 is covered so as to divide (divide) the opening (exposed substrate 5010). Note that the shape of the protective film 5010 that covers the substrate 5010 in the boundary region between the sensor units 5011 is not particularly limited. On the other hand, the protective film 5014 is not provided in the sensor unit 5011 in the reaction region 5023. That is, the protective film 5014 includes one or more openings that expose the substrate 5010 or the sensor unit 5011 in the reaction region 5023 at least in a portion other than the boundary region between the wiring 5013 and each sensor unit 5011. Note that the protective film 5014 may be provided over the substrate 5010 or in the vicinity of a boundary portion between the wiring 5013 and the substrate 5010. The protective film 5014 may be provided so as to cover the outer peripheral portion of the sensor unit 5011 as in the fifteenth embodiment.

  As described above, the protective film 5014 according to the sixteenth embodiment includes one or more openings that expose the lower layer portion (the substrate 5010 or the sensor portion 5011) including a part of the substrate 5010 in the reaction region 5023.

  The reason why the protective film 5014 is provided in the boundary region between the sensor units 5011 is as follows. Various nucleic acid probes for detecting a target nucleic acid are immobilized on each sensor unit 5011. At the time of manufacture, a liquid containing each nucleic acid probe is dropped on each sensor unit 5011. However, when the substrate 5010 is highly hydrophilic, the nucleic acid probe solution dropped on a certain sensor unit 5011 is dropped on an adjacent sensor unit 5011 unless the protective film 5014 is provided in the boundary region between the sensor units 5011. Mix in contact with another prepared nucleic acid probe solution. In general, an organic protective film has low hydrophilicity. Therefore, the protective film 5014 provided in the boundary region between the sensor units 5011 prevents the nucleic acid probe solution dropped on one sensor unit 5011 from coming into contact with another nucleic acid probe solution dropped on the adjacent sensor unit 5011. be able to.

  According to the nineteenth embodiment, the same effects as in the seventeenth embodiment or the eighteenth embodiment can be obtained. Furthermore, according to the nineteenth embodiment, the nucleic acid probe can be accurately and easily fixed to each sensor unit 5014 at the time of manufacturing the nucleic acid detection device 5100.

<20th Embodiment>
The twentieth embodiment will be described in detail with reference to the drawings. Note that the same components as those described in the seventeenth embodiment are denoted by the same reference numerals and description thereof is omitted. FIG. 47 is an enlarged view of the vicinity of the reaction region 5023 in the nucleic acid detection device 5100 according to the twentieth embodiment. The twentieth embodiment differs from the sixteenth embodiment in the shape of the protective film 5014 in the boundary region between the sensor units 5011.

  The protective film 5014 covers the wiring 5013 so that the wiring 5013 is not exposed in the reaction region 5023. On the other hand, the protective film 5014 is not provided in the sensor unit 5011 in the reaction region 5023. In addition, the protective film 5014 has a partition shape that covers the substrate 5010 in the boundary region between the sensor units 5011 in the reaction region 5023 as in the sixteenth embodiment. The protective film 5014 provided in the boundary region includes an opening provided in the vicinity of the sensor unit 5011 (exposed substrate 5010) and an opening provided in the vicinity of another adjacent sensor unit 5011 (exposed substrate) in the reaction region 5023. 5010) is covered (divided) so as to cover the substrate 5010.

  The protective film 5014 is provided in a shape surrounding the sensor unit 5011 in the boundary region between the sensor units 5011. As an example, the protective film 5014 includes an opening having a convex arc shape in the vicinity of at least one adjacent sensor unit 5011 in the vicinity of the sensor unit 5011. Accordingly, the substrate 5010 is exposed in the vicinity of the sensor portion 5011, but the periphery thereof is further covered with the protective film 5014.

  That is, the protective film 5014 includes one or more openings that expose the substrate 5010 or the sensor unit 5011 in the reaction region 5023 at least in a portion other than the wiring 5013 and the boundary region. Note that the protective film 5014 may be provided over the substrate 5010 or in the vicinity of a boundary portion between the wiring 5013 and the substrate 5010. The protective film 5014 may be provided so as to cover the outer peripheral portion of the sensor unit 5011 as in the fifteenth embodiment.

  As described above, the protective film 5014 according to the twentieth embodiment includes one or more openings that expose the lower layer part (the substrate 5010 or the sensor part 5011) including a part of the substrate 5010 in the reaction region 5023.

  The protective film 5014 is provided in the boundary region between the sensor units 5011 so as to surround the sensor unit 5011. This prevents the liquid containing the nucleic acid probe dropped on the sensor unit 5011 from spreading more than necessary. Because.

  According to the twentieth embodiment, the same effects as those of the seventeenth embodiment or the eighteenth embodiment can be obtained. Furthermore, according to the twentieth embodiment, it is possible to accurately and easily fix the nucleic acid probe to each sensor unit when the nucleic acid detection device 5100 is manufactured.

<Example 4>
Example 4-1
In Example 4-1, a nucleic acid amplification reaction actually using the nucleic acid detection device 5100 according to the nineteenth embodiment described above will be described. FIG. 48 shows a comparison of the results of the nucleic acid amplification reaction using the nucleic acid detection device 5100 according to the nineteenth embodiment and the results of the nucleic acid amplification reaction using the nucleic acid detection device of the comparative example. The nucleic acid detection device of the comparative example has a configuration in which a portion other than the sensor 11 (including not only the wiring but also the substrate) is covered with an organic protective film in the reaction region. FIG. 48 shows the amplification time on the horizontal axis and the amount of amplified nucleic acid on the vertical axis. In Example 4-1, an amplification reaction reagent is injected into each reaction region of the nucleic acid detection device 5100 according to the nineteenth embodiment and the nucleic acid detection device of the comparative example, and amplification is performed for 40 minutes and 60 minutes after the injection. The amount of amplified nucleic acid at the time of the quantification was quantified. In the nucleic acid detection device of the comparative example, the amplification amount is low in 40 minutes, and 60 minutes is not sufficient. On the other hand, in the nucleic acid detection device 5100 according to the sixteenth embodiment, a sufficient amount of amplification is obtained at 40 minutes. Since the amplification is saturated at this point, the amount of amplified nucleic acid does not increase in 60 minutes.

  From the results shown in FIG. 48, it has been clarified that the configuration of the nucleic acid detection device 5100 according to the nineteenth embodiment has greatly reduced amplification inhibition. Note that the nucleic acid detection device 5100 according to the seventeenth, eighteenth and twentieth embodiments has the same configuration as the nucleic acid detection device 5100 according to the nineteenth embodiment, and therefore the same characteristics as described above can be obtained.

Example 4-2
In Example 4-2, a usage example of nucleic acid detection using the nucleic acid detection device built-in cassette 5001 according to the nineteenth embodiment described above will be specifically described. FIG. 49 is an enlarged view of the vicinity of the reaction region 5023 in the nucleic acid detection device 5100 according to the nineteenth embodiment. In Example 4-2, as shown in FIG. 49, the sensor unit 5011 is composed of a set of two sensors. The nucleic acid detection device 5100 detects a different nucleic acid for each sensor unit 5011 (for each set of two sensors). As described in the nineteenth embodiment, the protective film 5014 in the reaction region 5023 is formed only in the boundary region between the portion covering the wiring 5013 and each sensor unit 5011. The substrate 5010 (glass is used in Example 4-2) is exposed at other portions in the reaction region 5023.

(1) Preparation of Nucleic Acid Detection Device Built-in Cassette 5001 1-1. Preparation of Nucleic Acid Detection Device 5100 Five types of nucleic acid probes (sequences A to E) shown in Table 20 below are applied to each electrode (hereinafter, corresponding to a sensor constituting each sensor unit 5011) on the nucleic acid detection device 5100. Was fixed. Immobilization was performed by dropping a solution containing each nucleic acid probe onto each electrode set, and then washing and removing excess nucleic acid probes. In addition, the following 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th electrode groups constitute separate sensor units 5011. .

1) For negative control ... 1 and 2 electrodes 2) For gene-A detection ... 3, 4 electrode 3) For gene-B detection ... 5th and 6th electrode 4) For gene-C detection ... 7,8 electrode 5) Gene-D detection ... 9,10 electrode.

1-2. Assembly of Nucleic Acid Detection Device Built-In Cassette 5001 On the sensor unit 5011 of the nucleic acid detection device 5100, a reaction part defining member 5200 capable of forming a reaction region 5023 was attached. A sample injection port is formed in the reaction portion defining member 5200 and is fixed to the nucleic acid detection device 5100 so that the reaction solution does not leak. In the reaction part defining member 5200, a primer set as shown in Table 27 below was held in a dried state. The amplification primer is designed for amplification by the LAMP method.

(2) Detection of nucleic acid 2-1. Preparation of Template Solution As shown in Table 22 below, a template solution was prepared by mixing three types of templates of genes A, B, and D and an amplification reagent.

  The template solution contained Bst DNA polymerase and reaction mix, and was added with distilled water (ie, DW) so that the total amount was 50 μL with the template solution described later. An amplification reaction by the LAMP method is caused by the primer DNA (set A), and an amplification reaction by the LAMP method is caused by the template A detected by hybridization with the probe DNA (A) and the primer DNA (set B), A template B detected by hybridization with the probe DNA (B) and an amplification reaction by the LAMP method caused by the primer DNA (set D) and detected by hybridization with the probe DNA (D) And including.

Templates A, B, and D are synthetic oligo DNAs having the base sequences shown in Table 29 below.

2-2. Addition of Template Solution 50 μL of the template solution prepared in 2-1 was added to the reaction region.

2-3. Nucleic acid reaction Under the conditions shown below, the reaction region was heated or cooled to carry out various reactions.

Nucleic acid amplification reaction: 64 ° C., 60 minutes Hybridization reaction: 50 ° C., 10 minutes Washing reaction: 30 ° C., 5 minutes Current detection reagent (Hoechst 33258) reaction: 25 ° C., 3 minutes 2-4. Detection of Nucleic Acid The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecules specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The above series of reactions was carried out using an automatic DNA testing apparatus described in SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008.

(3) Results FIG. 50 is a graph showing the results obtained from each electrode. In the electrodes 3, 4, 5, 6, 9, and 10 on which the probes A, B, and D corresponding to the genes A, B, and D to which the template was added were immobilized, larger current values were obtained than in the negative control. On the other hand, the electrodes 7 and 8 on which the probe C corresponding to the gene C to which no template was added were immobilized had current values comparable to those of the negative control. From the results shown in FIG. 50, it was revealed that the gene to which the template was added could be reliably detected by using the nucleic acid detection device 5100 according to the nineteenth embodiment.

Example 4-3
In Example 4-3, a usage example of nucleic acid detection actually using the nucleic acid detection device built-in cassette 5001 according to the twentieth embodiment described above will be specifically described. FIG. 51 is an enlarged view near the reaction region 5023 in the nucleic acid detection device 5100 according to the twentieth embodiment. In Example 4-3, as shown in FIG. 51, the sensor unit 5011 is composed of a set of two sensors. The nucleic acid detection device 5100 detects a different nucleic acid for each sensor unit 5011 (for each set of two sensors). As described in the twentieth embodiment, the protective film 5014 in the reaction region 5023 includes a portion covering the wiring 5013, a boundary region between each sensor unit 5011, and two sensors constituting each sensor unit 5011. It is formed only on the outer periphery. The substrate 5010 (glass is used in Example 4-3) is exposed at other portions in the reaction region 5023. The materials and detection conditions used for detection, excluding the nucleic acid detection device 5100, are the same as in Example 4-1. In addition, the following 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th electrode groups constitute separate sensor units 5011. .

result
FIG. 52 is a graph showing the results obtained from each electrode. In the electrodes 3, 4, 5, 6, 9, and 10 on which the probes A, B, and D corresponding to the genes A, B, and D to which the template was added were immobilized, larger current values were obtained than in the negative control. On the other hand, the electrodes 7 and 8 on which the probe C corresponding to the gene C to which no template was added were immobilized had a current value comparable to that of the negative control. From the results shown in FIG. 52, it was revealed that the gene to which the template was added could be reliably detected by using the nucleic acid detection device 5100 according to the twentieth embodiment.

6). Prevention of inhibition of nucleic acid reaction In a further aspect, the array-type primer probe chip may be provided as a nucleic acid reaction device that does not inhibit the nucleic acid reaction.

  As described above, a technique for detecting a plurality of target genes is very important. However, it has become clear that some nucleic acid amplification devices and / or nucleic acid detection devices that have been developed in such situations inhibit the nucleic acid reaction. The reaction between the primer and the target nucleic acid and the reaction between the probe nucleic acid and the target nucleic acid are nucleic acid reactions that occur between the nucleic acids. In the reaction field, it is necessary to carry out the reaction that should originally occur without being inhibited.

  By using this embodiment, the array-type primer probe chip may be provided as a nucleic acid reaction tool that does not inhibit the nucleic acid reaction. For example, such a nucleic acid reaction tool may be a nucleic acid amplification reaction tool, a nucleic acid detection reaction tool, or a nucleic acid amplification detection reaction tool. Specifically, an array type probe chip, an array type primer chip, and an array type primer probe chip may be used.

  In such a nucleic acid reaction tool, a protective film is formed on the surface of a member constituting the reaction field. Here, the “reaction field” refers to a field where a nucleic acid reaction takes place.

  Protective film is polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, Acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicon resin, polyphenylene oxide and polysulfone, and glass, quartz glass, alumina, It is composed of at least one selected from the group consisting of sapphire, forsterite, silicon carbide and metal oxide.

  Examples of preferred protective films are novolak resins, epoxy resins, polyolefin resins, and silicon resins, and resin compositions containing these may also be used. Further, when a novolac resin is used, it is preferable that no photosensitive material is contained.

  The protective film may be formed so as to be in contact with the reaction field of the member constituting the reaction field of the nucleic acid reaction tool. The protective film may be formed using a technique known per se according to the type of the protective film material. Or a protective film should just be applied in order to prevent the substance which becomes a problem from releasing in the reaction field from the member which may inhibit reaction. In that case, at least a part of the member in contact with the reaction field may be covered with the protective film.

  A “primer set” is a collection of primers necessary for amplifying one target nucleic acid. For example, in the case of a primer set for PCR amplification, one primer set may include one kind of forward primer and one kind of reverse primer for amplifying one target nucleic acid. For example, in the case of a primer set for LAMP amplification, one primer set may include an FIP primer and a BIP primer for amplifying at least one target nucleic acid, and an F3 primer, a B3 primer, and an LP primer as necessary. That is, an LF primer and / or an LB primer may be included. When an amplification reaction is performed in one reaction tool, the amplification performed there is generally one specific amplification. Therefore, in the case of a reaction tool for LAMP amplification reaction, the plurality of types of primer sets included in one reaction tool may be different primer sets for amplifying target sequences having different base sequences. Alternatively, the plurality of types of primer sets included in one reaction tool may be primer sets having combinations of different primers for amplifying a specific target sequence.

  Below, the example of a nucleic acid reaction tool is described. The examples shown individually describe an array type probe chip, an array type primer chip and an array type primer probe chip as a nucleic acid reaction tool.

<Twenty-first embodiment>
Array type probe chip A schematic diagram of an embodiment of the array type probe chip 6001 is shown in FIG. The array type probe chip 6001 includes an electrode 6012 on a base 6011. A signal extraction unit 6013 for extracting electrical information of the electrode 6012 is disposed at a position corresponding to each electrode 6012. The electrode 6012 and the signal extraction unit 6013 are connected by a lead 6014. Probe nucleic acid 6015 is immobilized on the surface of electrode 6012. The surface of the base 6011 excluding the surface of the electrode 6012 and the surface of the lead 6014 are covered with a protective film.

  The formation of the protective film may be performed on a desired portion by coating, or after forming the protective film on the whole using techniques such as patterning, masking and etching, the desired size and A protective film having a shape may be formed. At least the lead 6014 may be covered to prevent contact between the lead 6014 and the reaction solution. Or you may form a protective film in the area | region which contacts a reaction liquid at least.

The base 6011 can be manufactured from, for example, glass, sapphire, ceramic, resin, rubber, elastomer, SiO ₂ , SiN, or Al ₂ O _3, but is not limited thereto. Preferably, it is made of any material known per se that is electrically and chemically inert.

  The electrode 6012 may be manufactured by a known means. The electrode is not particularly limited, but gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium, tungsten, and other metals or alloys thereof, or graphite, glassy carbon As well as these oxides or compounds.

  Immobilization of the probe nucleic acid 6015 to the electrode 6012 may be performed by chemical bonding, or may be performed by spotting and drying the probe nucleic acid solution.

  A hybridization signal generated by hybridization of probe nucleic acid 6015 and sample nucleic acid (not shown) is transmitted by electrode 6012 and extracted from signal extraction unit 6013.

  The array-type probe chip base shown in FIG. 53 includes ten electrodes 6012, but is not limited thereto, and the number of electrodes arranged on one base can be arbitrarily changed. Also, the arrangement pattern of the electrodes is not limited to that shown in the figure, and those skilled in the art can appropriately change the design as necessary. The base 6011 may be provided with a reference electrode and a counter electrode as necessary.

  The substrate 6022 may be a tube, a well, a chamber, a channel, a cup, a dish, and a plate including a plurality of them, for example, a multiwell plate, a plate, a sphere, a rod, and a part thereof. .

  In the case where the substrate 6011 is a shape having a container shape, for example, a tube, a well, a chamber, a channel, a cup and a dish, and a plate having a plurality of them, for example, a multiwell plate, A reaction field may be formed. Further, the array-type probe chip may be provided with a lid. The lid may be configured to cover at least a region where the probe nucleic acid 6015 is immobilized on the container-shaped or plate-shaped substrate 6022. The lid may be plate-shaped. A recess such as a groove may be formed in a part of the plate-like lid. A reaction field may be formed in a space between the concave portion of the lid and the base 6011.

  When the substrate 6011 has a plate shape, a spherical shape, a rod shape, or a part thereof, a reaction field may be formed by immersing the substrate 6011 in a further container containing a reaction solution. A reaction field may be formed by placing the reaction solution on the region where the probe nucleic acid is immobilized.

<Twenty-second embodiment>
Array-type primer chip An example of an array-type primer chip, which is a container-type multi-nucleic acid reaction tool, will be described with reference to FIGS. 54 (a), (b) and (c).

  FIG. 54A is a perspective view of an example of an array type primer chip. An array-type primer chip 6021 described in FIG. 54A includes a container 6022 having a protective film 6020 formed on the inner wall thereof. A plurality of primer-immobilized regions 6024 that are independent of each other are disposed on the inner bottom surface 6023 of the substrate 6022 on which the protective film 6020 is formed. FIG. 54B is a schematic diagram in which a portion of the primer immobilization region 6024 is enlarged. As shown therein, one kind of primer set 6025 is fixed to one immobilization region 6024. In each of the plurality of primer immobilization regions 6024, a plurality of primer sets 6025 are fixed for each set.

  Plural types of primer sets 6025 are prepared according to the type of target nucleic acid to be amplified. One primer set 6025 for amplifying one specific target nucleic acid is fixed to one primer fixing region 6024. For example, for PCR amplification, one primer set includes a forward primer and a reverse primer necessary for amplifying one specific target nucleic acid. In addition, for LAMP amplification, one primer set includes FIP primer, BIP primer, and F3 primer, B3 primer, and LP primer as necessary to amplify one specific target nucleic acid. It is.

  Primer set 6025 is fixed to primer immobilization region 6024 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 6025 to the primer immobilization region 6024 can be achieved, for example, by dropping a solution containing one set of primer sets onto one primer immobilization region 6024 and then drying. Further, similarly, a solution containing a desired primer set 6025 may be dropped and dried for the other primer immobilization regions 6024, and a desired number of primer sets may be fixed to the substrate 6022. As a result, the primer set 6025 is fixed to all the primer fixing regions 6024 that are independently arranged on one surface of the substrate 6022. However, the primer set 6025 may be immobilized on the primer immobilization region 6024 so as to be released in contact with a liquid phase for providing a reaction field. Therefore, any immobilization method known per se capable of such immobilization may be used. In the method of dropping a solution containing the primer set 6025, the solution containing the primer set 6025 may be, for example, water, a buffer solution or an organic solvent.

  The plurality of primer immobilization regions 6024 arranged on the substrate 6022 may be arranged independently of each other. Independently arranged means arranged at intervals that do not prevent amplification initiated and / or advanced for each primer set in the reaction field. For example, adjacent primer immobilization regions 6024 may be arranged in contact with each other, may be arranged in the vicinity of each other with a slight distance, or fixed in a commonly used detection device such as a so-called DNA chip. The probe nucleic acids may be arranged at the same distance from each other at the same distance.

  For example, the distance between adjacent primer immobilization regions 6024 may be 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or more, preferably 100 μm to 10 mm It may be.

  The liquid phase for providing the reaction field only needs to be a liquid phase that allows the amplification reaction to proceed after the fixed primer set is released, for example, a reaction liquid necessary for the desired amplification. It's okay.

  The nucleic acid reaction tool shown in FIGS. 54A and 54B is an example in which the substrate 6022 has a container shape. The container-shaped substrate 6022 may be, for example, a tube, a well, a chamber, a flow path, a cup and a dish, and a plate including a plurality of them, such as a multiwell plate. The base material may be any material. The base material 6022 may use the same base material as the above-described array type probe chip. Further, the base 6022 may have a plate shape. Furthermore, the array-type primer chip may be provided with a lid. The lid may be configured to cover at least the region where the primer 6025 is immobilized on the container-shaped or plate-shaped substrate 6022. The lid may be plate-shaped. A recess such as a groove may be formed in a part of the plate-like lid. A reaction field may be formed in a space between the concave portion of the lid and the base body 6022.

  In the multi-nucleic acid reaction device, a primer set 6025 may be fixed to a primer fixing region 6024 arranged on at least one surface 6023 of a plate-like substrate 6022 as shown in FIG. 54 (c). In this case, the reaction field may be formed by placing the reaction solution on at least the region where the primer set 6025 of the substrate 6022 is immobilized. In this case, a concave portion and / or a convex portion may be formed on the surface 6023 of the base 6022, or a flow path may be formed by the concave portion and / or the convex portion. The primer immobilization region 6024 and the primer set 6025 may be disposed in the concave portion of the substrate 6022, or may be disposed in a region surrounded by a plurality of convex portions. Further, the reaction field may be formed by disposing the substrate 6022 in a container including the reaction container. In this case, the substrate 6022 may have a plate shape, a spherical shape, a rod shape, or a shape made of a part thereof.

  For forming the protective film, any technique known per se may be used.

  In FIGS. 54A and 54B, an example in which the immobilization region 6024 is disposed only on the inner bottom surface of the base 6022 is shown, but the present invention is not limited to this. It suffices to be disposed on at least a part of the inside of the base body 6022, and may be disposed on any or all of the inner bottom surface and the inner side surface and the ceiling surface formed by the lid.

  FIG. 55 shows a nucleic acid amplification reaction using a container-type array primer chip 6031 similar to that in FIG. FIG. 55A shows an array type primer chip 6031 before the reaction. A plurality of primer sets 6034 are respectively fixed to a plurality of immobilization regions 6033 arranged on the inner bottom surface 25 of the base body 6032 having a protective film formed on the inner surface. FIG. 55B shows a state in which the reaction solution 6036 is added to the array type primer chip 6031 and accommodated therein.

  The reaction solution 6036 may contain components necessary for a desired amplification reaction. Although not limited to these, for example, when an enzyme such as polymerase or a primer is used as a starting point to form a new polynucleotide chain, a substrate such as oxynucleoside triphosphate, or when reverse transcription is performed simultaneously. In addition, a buffer such as reverse transcriptase and a substrate necessary for the enzyme may be further contained to maintain an appropriate amplification environment.

  As shown in FIG. 55 (b), the array type primer chip 6031 after the addition of the reaction solution 6036 is fixed to the inner bottom surface on which the protective film is formed, as schematically shown in FIG. 55 (c). The primer set is released and gradually diffuses. The free and diffused area is schematically represented by area 6037. The primer set 6034 that is released and diffuses encounters other components necessary for amplification such as template nucleic acid, polymerase, and substrate existing in the vicinity thereof, and an amplification reaction is started. A plurality of primer sets 6034, which are immobilized independently for each type, can start and proceed with an amplification reaction for the template nucleic acid independently for each type. Thereby, amplification of a plurality of template sequences using a plurality of types of primer sets is achieved independently and simultaneously. Here, the “reaction field” is theoretically referred to as a region defined by the reaction solution 6036 in which the amplification reaction can proceed, that is, a region where the reaction solution exists. Also, a region of the reaction field where the amplification reaction actually starts and proceeds there is called a “reaction region”. If the amplification reaction actually proceeds only within the region 6037, the region 6037 is interpreted as the reaction region.

  In the above example, an example in which only the primer set is immobilized on the substrate is shown. However, the present invention is not limited to this, and other components necessary for amplification, for example, enzymes such as polymerase and reverse transcriptase, substrates, and substrates, under conditions where the primer set is fixed to each immobilization region for each type. And / or a buffer may be fixed to the substrate together with the primer. In that case, the substance to be fixed may be contained in a desired liquid medium together with the primer, and fixed by dropping, drying or the like in the same manner as described above. When an amplification reaction is performed in such an array-type primer chip, the composition of the reaction solution added thereto may be selected according to the immobilized component.

<Twenty-third embodiment>
Array Type Primer Probe Chip An example of a further embodiment is shown in FIG. This embodiment includes the above-described substrate, a probe nucleic acid immobilized on at least one surface of the substrate, and a primer immobilized releasably. This embodiment may be referred to as an array-type primer probe chip. An array-type primer probe chip is a multi-nucleic acid reaction device including a probe nucleic acid and a primer on one substrate. The configuration of the substrate may be the same as that of the container-type multi-nucleic acid reaction tool described above.

  FIG. 56 (a) is a perspective view of an example of an array type primer probe chip. An array-type primer probe chip 6041 shown in FIG. 56A includes a container-shaped substrate 6042. A protective film 6043 is formed on the inner wall of the base body 6042. On the protective film 6043 on the inner bottom surface 6044 of the substrate 6042, a plurality of primer immobilization regions 6045 that are independent from each other are arranged. A plurality of probe immobilization regions 6046 are arranged adjacent to the plurality of primer immobilization regions 6045 and corresponding to the respective primer immobilization regions 6045.

  FIG. 56B is an enlarged schematic view of the primer immobilization region 6045. As shown there, one kind of primer set 6047 is fixed to one primer fixing region 6045. In each of the plurality of primer immobilization regions 6045, a plurality of primer sets 6047 are fixed for each set.

  The primer set 6047 may be fixed in the same manner as the array type primer chip described above.

  FIG. 56 (c) is an enlarged view of the probe immobilization region 6046 disposed in the vicinity of the primer immobilization region 6045. A plurality of probe nucleic acids 6048 including a complementary sequence of a desired sequence to be detected are immobilized in the probe immobilization region 6046.

  The desired sequence to be detected may be the complementary sequence of probe nucleic acid 6048. The probe immobilization region 6046 is arranged such that a hybridization signal between the probe nucleic acid 6048 and its target sequence is detected independently between the plurality of probe immobilization regions 6046.

  For immobilizing the probe nucleic acid 6048 to the probe immobilization region 6046, any general technique for immobilizing the probe nucleic acid 6048 to the substrate surface in a so-called DNA chip known per se may be used. The primer set 6047 may be immobilized after the probe nucleic acid 6048 is immobilized, or the probe nucleic acid 6048 may be immobilized after the primer set 6047 is immobilized. Further, the primer set 6047 and the probe nucleic acid 6048 may be immobilized at the same time.

  For example, the distance between adjacent probe immobilization regions 6046 may be 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or more, preferably 100 μm to 10 mm It may be.

  Further, for example, the distance between the probe immobilization region 6046 and the primer immobilization region 6045 is 0 μm to 0.1 μm, 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or The above may be sufficient, Preferably, it may be 100 micrometers-10 mm.

  For example, when the distance between the probe immobilization region 6046 and the primer immobilization region 6045 is 0 μm, the probe immobilization region 6046 and the primer immobilization region 6045 may be understood to be at the same position on the substrate surface. . Further, the probe immobilization region 6046 may be included in the primer immobilization region 6045, and the primer immobilization region 6045 may be included in the probe immobilization region 6046.

  FIG. 57 is a schematic diagram showing the state of the reaction field after the nucleic acid amplification reaction performed using the array-type primer probe chip 6041. FIGS. 57 (a-1) and (b-1) show an array-type primer probe chip 6041 before the reaction. A protective film 6043 is formed on the inner wall of the base body 6042. A plurality of primer sets 6047 are respectively fixed to a plurality of primer fixing regions 6045 arranged on the protective film 6043 on the inner bottom surface 6044 of the base body 6042. Corresponding to each primer immobilization region 6045, a probe immobilization region 6046 is arranged in the vicinity thereof. A plurality of probe nucleic acids 6048 are immobilized for each type in the probe immobilization region 6046.

  The reaction solution 6050 is added to the array type primer probe chip 6041 and the state in which it is accommodated is shown in FIGS. 57 (a-2) and (b-2).

  The reaction solution 6050 may contain components necessary for a desired amplification reaction. Although not limited to these, for example, when an enzyme such as polymerase or a primer is used as a starting point to form a new polynucleotide chain, a substrate such as oxynucleoside triphosphate, or when reverse transcription is performed simultaneously. In addition, a buffer such as reverse transcriptase and a substrate required therefor, and salts for maintaining an appropriate amplification environment may be included.

  The sample may be added to the reaction field by adding the reaction solution 6050 to the array-type primer probe chip 6041 in advance before adding the reaction solution 6050 to the array-type primer probe chip 6041. Or may be performed by adding a sample to the array-type primer probe chip 6041 before adding the reaction solution 6050 to the array-type primer probe chip 6041.

  As shown in FIGS. 57 (a-2) and (b-2), the array type primer probe chip 6041 after the addition of the reaction solution 6050 is schematically shown in FIGS. 57 (a-3) and (b-3). As shown, the primer set 6047 immobilized on the protective film 6043 on the bottom surface 6044 is released and gradually diffuses. The free and diffused region is schematically indicated by region 6051. The released and diffusing primer meets other components necessary for amplification such as template nucleic acid, polymerase and substrate present in the vicinity. Thereafter, the amplification reaction is started. A plurality of primer sets 6047, which are independently fixed for each type, can start and proceed with an amplification reaction for the template nucleic acid independently for each type. Thereby, amplification for a plurality of template sequences using a plurality of types of primer sets 6047 is achieved independently and simultaneously. Here, the “reaction field” is theoretically referred to as a region defined by the reaction solution 6050 in which the amplification reaction can proceed, that is, a region where the reaction solution exists. Also, a region of the reaction field where the amplification reaction actually starts and proceeds there is called a “reaction region”. If the amplification reaction actually proceeds only within the region 6051, the region 6051 is interpreted as the reaction region. The diagram of FIG. 57 (a-3) is a schematic diagram when an amplification reaction is caused by the primer set 6047 fixed to all the primer fixing regions 6045. FIG. 57 (b-3) shows a part of all the primer-immobilized regions 6045 formed on the protective film 6043 on the bottom 6044 by the fixed primer set, and three regions in FIG. 57 (b-3). It is a schematic diagram when amplification occurs only in FIG.

  When the nucleic acid containing the target sequence is present in the amplification product amplified in the region 6051, the probe nucleic acid 6048 hybridizes with the nucleic acid. The probe nucleic acid 6048 immobilized on the probe immobilization region 6046 is immobilized so as to hybridize only with the amplification product in the corresponding primer immobilization region 6045. That is, the probe nucleic acid 6048 immobilized on one probe immobilization region 6046 is kept at a distance so as to hybridize only with the amplification product in the corresponding primer immobilization region 6045, and each probe immobilization region 6046 and primer immobilization are maintained. The conversion area 6045 is arranged.

  Detection of hybridization between the probe nucleic acid 6048 and its target sequence may be performed by a known hybridization signal detection means. For example, a fluorescent material may be previously imparted to the primer, or a fluorescent material may be imparted to a substrate such as oxynucleoside triphosphate. The presence / absence and amount of hybridization may be determined using the fluorescence intensity from these fluorescent substances as an index. Alternatively, the hybridization signal may be detected by electrochemical means.

  The detection of hybridization may be performed after the inside of the array type primer probe chip 6041 is cleaned, or may be performed without cleaning. When detecting by electrochemical means, the hybridizing signal may be detected using an intercalator. In this case, for example, an intercalator may be included in the reaction solution 6050 in advance, and may be added before the hybridization reaction starts, during the hybridization reaction, or after the hybridization reaction. In any of these cases, the detection may be performed after washing the inside of the array type primer probe chip 6041, or the detection may be performed without washing. The start of the hybridization reaction, the determination during the reaction, and the determination after the reaction may be performed according to the reaction conditions such as the sequence of the primer, the probe nucleic acid and the template nucleic acid, the reaction temperature, and may be determined by preliminary experiments.

  The length of the primer is not limited to this, but about 5 bases or more, about 6 bases or more, about 7 bases or more, about 8 bases or more, about 9 bases or more, about 10 bases or more, about 15 bases More than about 20 bases, about 25 bases or more, about 30 bases or more, about 35 bases or more, about 40 bases or more, about 45 bases or more, about 55 bases or more, about 80 bases or less, about 75 bases or less About 70 bases or less, about 65 bases or less, about 60 bases or less, about 55 bases or less, about 50 bases or less, about 45 bases or less, about 40 bases or less, about 35 bases or less, about 30 bases or less, about 25 bases or less , About 20 bases or less, about 25 bases or less, or about 20 bases or less, or a combination of any of these lower and upper limits. For example, examples of preferable base length may be about 10 bases to about 60 bases, about 13 to 40 bases, about 10 to 30 bases, and the like. In addition, the lengths of the primers simultaneously fixed to one immobilization region may be the same for all primers, may be different for all primers, or some of the primers have the same length. Alternatively, some primers may have different lengths. Moreover, you may differ for every primer set. In addition, the primer sets fixed to one immobilization region may have different lengths for each type, and all the primer sets fixed to one immobilization region may have the same length.

  The length of the probe nucleic acid is, for example, 3 bases to 10 bases, 10 bases to 20 bases, 20 bases to 30 bases, 30 bases to 40 bases, 40 bases to 50 bases, 50 bases to 60 bases, preferably 10 bases to It may be 50 bases. The probe nucleic acid contains a sequence complementary to the target sequence to be detected. The probe nucleic acid may contain a further sequence such as a spacer sequence in addition to the complementary sequence of the target sequence.

  The length of the target sequence may be, for example, 10 bases to 100 bases, 100 bases to 200 bases, bases 200 to 300 bases, 300 bases to 400 bases, preferably 100 bases to 300 bases.

  The length of the target sequence is, for example, 3 bases to 10 bases, 10 bases to 20 bases, 20 bases to 30 bases, 30 bases to 40 bases, 40 bases to 50 bases, 50 bases to 60 bases, preferably 10 bases to It may be 50 bases.

  The type of primer set immobilized on one primer immobilization region may be one type for amplifying one type of target nucleic acid, or multiple types for amplifying two or more types of target nucleic acids. May be.

  The type of probe nucleic acid group immobilized on one probe immobilization region may be one type for hybridizing with one type of target sequence, or multiple types for amplifying two or more types of target nucleic acids. It may be. Further, it may be a probe nucleic acid having a common target sequence portion and a sequence different from other target sequences.

  The lower limit of the number of primer immobilization regions arranged on one array type primer probe chip is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 175 or more, 200 or more, 300 or more, 400 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, and the upper limit is 10,000 or less, It may be 5000 or less, 2500 or less, 2000 or less, 1500 or less, 1000 or less, 500 or less, 250 or less, 200 or less, or 150 or less, or a range in which any of these upper and lower limits is combined.

  The number of primer-immobilized regions and probe-immobilized regions arranged on one array-type primer probe chip may be the same or different. That is, the same number of probe immobilization regions may be arranged so as to correspond to all the primer immobilization regions, and the number of primer immobilization regions may be larger than the number of probe immobilization regions. May be smaller than the number of probe immobilization regions. Further, a positive control and / or a negative control for confirming the amplification reaction state or for confirming the state of the hybridization reaction may be included. Such positive control and / or negative control may be provided for the primer set and / or probe nucleic acid, respectively.

  In the above example, an example in which only the primer set is immobilized on the substrate is shown. However, the present invention is not limited to this, and other components necessary for amplification, for example, enzymes such as polymerase and reverse transcriptase, substrates, and substrates, under conditions where the primer set is fixed to each immobilization region for each type. And / or a buffer may be fixed to the substrate together with the primer. In that case, the substance to be fixed may be contained in a desired liquid medium together with the primer, and fixed by dropping, drying or the like in the same manner as described above. In such an array-type primer probe chip, when an amplification reaction is performed, the composition of the reaction solution added thereto may be selected according to the immobilized component.

<Twenty-fourth embodiment>
The array-type primer probe chip of the 24th embodiment will be described with reference to FIGS.

(1) Chip Material First, an example of the structure and manufacturing method of an array type primer probe chip that detects a hybridizing signal by electrochemical detection will be described with reference to FIGS. 58 (a) and 58 (b). 58A is a plan view of the chip material, and FIG. 58B is a cross-sectional view taken along line BB of the chip material of FIG. 58A.

  The chip material 111 includes, for example, four electrodes 113a to 113d arranged on a rectangular substrate 112 along the longitudinal direction thereof. Each of the electrodes 113a to 113d has a structure in which a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order. Each of the electrodes 113a to 113d has a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118. An insulating protective film 6118 is coated on the substrate 112 including the electrodes 113a to 113d. The circular window 120 is opened in the insulating protective film 6118 corresponding to the large rectangular portion 116. The rectangular window 121 is opened at the insulating protective film 6118 portion corresponding to the small rectangular portion 117. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113a functions as the first working electrode 122a. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113b functions as the second working electrode 122b. The large rectangular portion exposed from the circular window 120 of the electrode 113 c functions as the counter electrode 123. The large rectangular portion 116 exposed from the circular window 120 of the electrode 113d functions as the reference electrode 124. The small rectangular portion 117 exposed from the rectangular window 121 of the electrodes 113a to 113d functions as a prober contact portion.

  Such a chip material 111 can be manufactured by the following method.

  First, a first metal thin film and a second metal thin film are deposited on the substrate 112 in this order by, for example, a sputtering method or a vacuum evaporation method. Subsequently, these metal thin films are sequentially selectively etched using, for example, a resist pattern as a mask, and a first metal thin film pattern 114 and a second metal thin film pattern 115 are stacked in this order, for example, four electrodes 113a to 113a. 113 d is formed along the longitudinal direction of the substrate 112. These electrodes 113 a to 113 d have a shape in which a large rectangular portion 116 and a small rectangular portion 117 are connected by a thin line 118.

  Next, a protective film 6118 is deposited on the substrate 112 including the electrodes 113a to 113d by, for example, a sputtering method or a CVD method. Subsequently, the protective film 6118 portion corresponding to the large rectangular portion 116 of each electrode 113a to 113d and the protective film 6118 portion corresponding to the small rectangular portion 117 are selectively etched using the resist pattern as a mask, so that the large rectangular portion 116 is formed. The circular window 120 is opened in the insulating protective film 6118 portion corresponding to, and the rectangular window 121 is opened in the insulating protective film 6118 portion corresponding to the small rectangular portion 117. Thereby, the above-described chip material 111 is produced.

  The substrate 112 is made of glass or resin such as Pyrex (registered trademark) glass, for example.

  The first metal thin film functions as a base metal film for bringing the second metal thin film into close contact with the substrate 112, and is made of, for example, Ti. The second metal thin film is made of, for example, Au.

  Examples of etching when patterning the first and second metal thin films include plasma etching using an etching gas or reactive ion etching.

  The insulating protective film 6118 is made of polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, poly Acrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicon resin, polyphenylene oxide and polysulfone, glass, quartz It may be selected from the group consisting of glass, alumina, sapphire, forsterite, silicon carbide and metal oxide. For example, a metal oxide film such as a silicon oxide film or a metal nitride film such as a silicon nitride film may be used.

  Examples of etching when patterning the insulating protective film 6118 include plasma etching using an etching gas or reactive ion etching.

(2) Array-type Primer Probe Chip Next, an example of the configuration and production method of the array-type primer probe chip 1 in which the primer set and the probe nucleic acid are fixed to the chip material 111 produced in the above (1) is shown in FIG. ) And FIG. 59 (b). 59A is a plan view of the array-type primer probe chip 1, and FIG. 59B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 51A.

  The first working electrode 122a of the electrode 113a formed on the chip material 111 is defined as a first probe immobilization region 201a, and the first probe immobilization region 201a includes a first sequence that includes a complementary sequence of the first target sequence. The probe nucleic acid 202a is fixed. A plurality of the first probe nucleic acids 202a to be immobilized are immobilized as one probe nucleic acid group. Similarly, the second working electrode 122b of the electrode 113b is used as a second probe immobilization region 201b, and a complementary sequence of a second target sequence different from the first target sequence is provided in the second probe immobilization region 201b. The contained second probe nucleic acid 202b is fixed.

  An example of a method for immobilizing the probe nucleic acids 202a and 202b to the probe immobilization region 201 is to introduce a thiol group at the 3 ′ end into the first and second probe nucleic acids 202a and 202b for the chip material 111 having a gold electrode. Including methods.

  Next, the first primer immobilization region 203a is disposed in the vicinity of the first working electrode 122a, and the second primer immobilization region 203b is disposed in the vicinity of the second working electrode 122b. The first primer set 204a is fixed on the first primer fixing region 203a, and the second primer set 204b is fixed on the second primer fixing region 203b. Thereby, the array type primer probe chip 1 is produced.

  The first primer set 204a has a sequence designed to amplify the first target sequence, and the second primer immobilization region 204b is a second target sequence comprising a sequence different from the first target sequence. Has a sequence designed to amplify.

  The first and second primer sets 204a and 204b are fixed to the first and second primer fixing regions 203a and 203b, respectively. For example, the primer set is included in a liquid such as water, a buffer solution, or an organic solvent. For example, in the case of room temperature, it is allowed to stand for 10 minutes until it is dropped and then dried under an appropriate temperature condition such as room temperature.

(3) Array-type primer probe chip at the time of use The usage method of the array-type primer probe chip 1 produced in said (2) is demonstrated referring FIG. 60 and FIG.

  60 (a) is a plan view of the array-type primer probe chip 1 in use, and FIG. 60 (b) is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 60 (a). It is.

  When the array type primer probe chip 1 of the present embodiment is used, the first working electrode 122a, the second working electrode 122b, the counter electrode 123, the reference electrode 124, and the first primer formed on the electrodes 113a to 113d, respectively. The reaction solution is maintained so that the immobilization region 203a and the second primer immobilization region 203b are included in the same one reaction field. Therefore, the covering 301 is mounted on the array type primer probe chip 1 before using the array type primer probe chip 1. The covering 301 is formed using a material such as a silicon resin such as silicon rubber and / or a resin such as a fluororesin. The desired shape of the covering 301 is formed by using the above-mentioned materials by any of the resin molding methods known per se, such as extrusion molding, injection molding or stamping and / or bonding with an adhesive. Is done. After the covering 301 is mounted, the reaction solution 302 containing the template nucleic acid 303 is added to the space formed by the array type primer probe chip 1 and the covering 301.

  In the array-type primer probe chip 1 to which the covering 301 is attached, the small rectangular portions 117 exposed from the rectangular windows 121 of the electrodes 113a to 113d are exposed.

  Examples of attaching the cover 301 to the array-type primer probe chip 1 include, for example, pressure bonding and bonding with an adhesive.

  Next, the reaction solution 302 is added after the covering 301 is attached to the array type primer probe chip 1.

  The method of adding a liquid to the space formed by the array-type primer probe chip 1 and the cover 301 may be, for example, by previously providing an opening in a part of the cover 301 and adding from the opening Alternatively, it may be added by inserting into a part of the covering 301 using an injector having a sharp tip such as a needle.

  The reaction solution 302 is used when a sample, an amplification reagent, for example, an enzyme such as a polymerase, a primer, and a substrate such as oxynucleoside triphosphate, which are necessary for forming a new polynucleotide chain, and reverse transcription simultaneously. Includes a reverse transcriptase and its necessary substrates, as well as buffers such as salts to maintain an appropriate amplification environment and an intercalator that recognizes a double stranded nucleic acid such as Hoechst 33258 and generates a signal. including. When the template nucleic acid containing the target sequence to be amplified by the primer set immobilized on the specific primer immobilization region is present in the sample to be examined, the primer immobilization region and the corresponding probe immobilization region are included. An amplification product is formed in the reaction field. This is schematically shown in FIG.

  FIG. 61 (a) schematically shows a state where an amplification product is formed in the reaction field 302. FIG. 61 (a) is a plan view of the array-type primer probe chip in use, and FIG. 61 (b) is a cross-sectional view taken along line BB of the array-type primer probe chip in FIG. 61 (a). . As described above, the sample added in FIG. 61 contained a nucleic acid containing a sequence to which the second primer set 204b can bind, so that as shown in FIGS. 61 (a) and 61 (b) The second primer set 204 b in the reaction field 302 provided by the reaction solution 302 is released and diffused in the region 401. Furthermore, an amplification reaction occurs after the second primer set 204b meets the template nucleic acid 303. As a result, an amplification product is formed. The amplification product by the second primer set 204b diffuses around the second primer immobilization region 203b and reaches the second probe immobilization region 201b. When the reached amplification product includes the target sequence, the second probe nucleic acid 202b and the amplification product are hybridized to form a double-stranded nucleic acid. The intercalator contained in the reaction solution 302 is combined with this double-stranded nucleic acid to generate a hybrid signal.

  The hybrid signal is generated by, for example, bringing a prober into contact with the small rectangular portion 117 exposed from each rectangular window 121 of the electrodes 113a to 113d and measuring the current response of an intercalator such as Hoechst 33258.

  By using an array-type primer probe chip that utilizes electrochemical detection, the target nucleic acid contained in the amplification product can be detected after the target nucleic acid contained in the sample has been amplified more easily and in a short time. Is possible.

<25th Embodiment>
Detection Method When nucleic acid is detected using an array type probe chip and an array type primer probe chip, the detection may be performed as follows.

(A) Current detection method A method for electrochemically detecting a double-stranded nucleic acid will be described. In this method, a double-stranded recognizer that specifically recognizes a double-stranded nucleic acid is used. Examples of double strand recognizers include, for example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator, polyintercalator, etc. Not. Furthermore, these substances can be modified with an electrochemically active metal complex such as ferrocene or viologen.

  The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.

  During or after the hybridization reaction, a double-stranded recognizer is added to the reaction solution. When a double-stranded nucleic acid is generated by hybridization, a double-stranded recognizer binds to this. Therefore, for example, by applying a potential higher than the potential at which the double strand recognizer reacts electrochemically, the reaction current value derived from the double strand recognizer can be measured. At this time, the potential may be applied at a constant speed, or a constant potential may be applied by applying a pulse. During measurement, the current and voltage may be controlled using a device such as a potentiostat, a digital multimeter, and a function generator. For example, known electrochemical detection means described in JP-A-10-146183 is preferably used.

(B) Fluorescence detection method A method for detecting double-stranded nucleic acid by fluorescence will be described. At least one primer included in the primer set is previously labeled with a fluorescently active substance. Alternatively, detection is performed using a secondary probe nucleic acid labeled with a fluorescently active substance. Alternatively, multiple labels may be used. Fluorologically active substances include, but are not limited to, fluorescent dyes such as FITC, Cy3, Cy5, or rhodamine. The fluorescent substance is detected using, for example, a fluorescence detector. A labeled detection sequence or a secondary probe nucleic acid is detected using an appropriate detection device according to the type of label.

<Example 5>
Example 5-1
In order to examine the influence of the protective film on the nucleic acid reaction, an amplification reaction was performed in an amplification chip having a flow path as a reaction field without immobilizing primers. Thereafter, detection was performed with an array type probe chip.

(1) Production of Chip Material A wafer-sized Pyrex glass was used as a plate-like substrate (that is, a substrate). A protective film material was applied to the surface with a spin coater. As the protective film material, a negative resist (epoxy), a positive resist (novolak, polyolefin), and a material not containing a photosensitizer (novolak) were used.

  After applying each material, the substrate was placed in a drying oven and prebaked at 150 ° C. to dry the film.

  Subsequently, in the case of a negative photoresist material, exposure was performed at 400 mJ with a contact type exposure machine, and then development processing was performed. In the case of a positive resist material, only the development process was performed without performing the exposure process. The materials other than the photoresist were not exposed or developed.

  Post-baking was performed at 160 ° C. in a drying oven to completely cure the film. After curing, a chemical dry etching (CDE) treatment was performed for 2 minutes to form a chip material.

(2) Amplifying chip Silicon rubber having a channel formed in advance was attached to the chip material. This was used as an amplification chip.

(3) Preparation of LAMP reaction solution and LAMP reaction Table 30 shows the base sequences of the primers used.

Table 31 shows the composition of the LAMP reaction solution.

Table 32 shows the composition of Reaction Mixture used in the LAMP reaction solution.

Table 33 shows the base sequence of the template contained in the LAMP reaction solution.

  50 μL of LAMP reaction solution was injected into the channel of the amplification chip, set in a generator with a Peltier temperature set to 63 ° C., and subjected to LAMP reaction for 1 hour.

<Preparation of DNA chip for LAMP amplification product detection>
(4) Array type probe chip FIG. 58 shows a schematic diagram of a chip material for an array type primer probe chip. Titanium and gold thin films were formed on the Pyrex glass surface by sputtering. Thereafter, titanium and gold electrodes were formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and a circular window and a rectangular window were opened in the insulating film by an etching process to expose the working electrode, the counter electrode, the reference electrode, and the prober contact portion. This was used as a chip material for an array type primer probe chip.

(5) Preparation of array-type probe chip Probe nucleic acids (3′-end SH-labeled synthetic oligos) shown in Table 34 were synthesized.

  A probe DNA solution containing 3 μM of probe DNA was prepared, and 100 nL of this solution was spotted on the working electrode. After drying at 40 ° C. and washing with ultrapure water, the ultrapure water remaining on the surface of the working electrode was removed, and the probe DNA was immobilized on the working electrode of the chip material.

  For details of the DNA chip and the DNA chip measuring apparatus, refer to the following document (SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008).

<Detection of LAMP amplification product>
In order to test the influence of each protective film on the amplification reaction, the LAMP amplification product amplified in the amplification chip was detected with an array type probe chip.

The results are shown in Table 35.

  As shown in Table 35, when novolak resin (containing naphthoquinone photosensitizer) was used as a protective film, the hybridization signal was negative. When novolac resin, epoxy resin, polyolefin resin and silicon resin were used as the protective film, the hybridization signal was positive.

  Accordingly, it has been clarified that novolak resin (containing naphthoquinone photosensitizer) affects nucleic acid reactions such as amplification reactions. On the other hand, it became clear that novolak resin, epoxy resin, polyolefin resin, and silicon resin are less likely to affect nucleic acid reactions such as amplification reactions.

  A novolak resin (containing a naphthoquinone photosensitizer) is an example of a positive resist that is generally used in the manufacture of devices intended to perform a nucleic acid reaction. Such materials have been shown to affect nucleic acid reactions in this study. In a device for performing a nucleic acid reaction, it is necessary to avoid the use of a material that affects the nucleic acid reaction.

  From the above results, it was shown that novolak resin, epoxy resin, polyolefin resin, and silicon resin are materials that are preferably used in devices intended to perform nucleic acid reactions.

<Example 5-2>
An array-type primer probe chip for electrochemical detection, comprising: a primer set fixed to a primer-immobilized region; and a probe DNA as a probe nucleic acid fixed to a probe-immobilized region near the primer-immobilized region. An example of making and using is described. The probe immobilization region was composed of electrodes and was used as a sensor for detecting a current response generated depending on the presence of hybridization.

(1) Production of chip material A schematic diagram of a chip material for an array type primer probe chip is shown in FIG. Titanium and gold thin films were formed on the Pyrex glass surface by sputtering. Thereafter, titanium and gold electrodes were formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and a circular window and a rectangular window were opened in the insulating film by an etching process to expose the working electrode, the counter electrode, the reference electrode, and the prober contact portion. This was used as a chip material for an array type primer probe chip.

(2) Preparation of array type primer probe chip First, probe DNA was fixed on the working electrode. Table 36 shows the base sequence of the probe DNA used.

  A probe DNA solution containing 3 μM each of probe DNA (A) and probe DNA (B) was prepared, and 100 nL of this solution was spotted on the working electrode. After drying at 40 ° C. and washing with ultrapure water, the ultrapure water remaining on the surface of the working electrode was removed, and the probe DNA was immobilized on the working electrode of the chip material.

Next, primer DNA used as a primer set was prepared. The primer DNA to be used is a primer set for amplification by a loop-mediated isal amplification (LAMP) method. Table 37 shows the base sequence of the primer DNA used.

  For the primer DNA (set A), 200 μM FIP, BIP, F3, B3, and LPF were prepared, respectively, and 0.1 μL, 0.1 μL, 0.0125 μL, 0.00125 μL, and 0.05 μL were respectively added. 275 μL of the solution was used to immobilize the working electrode, which is a primer immobilization region in the vicinity of the corresponding probe DNA (A). Specifically, 0.275 μL of each prepared solution was spotted on the corresponding working electrode to which the probe DNA (A) was fixed, and dried at 63 ° C. for 5 minutes. Thereby, an array type primer probe chip was obtained.

(3) Preparation of LAMP reaction solution Table 38 shows the composition of the LAMP reaction solution.

Compositions (1) to (4) include Bst DNA polymerase and reaction mix in common, and distilled water (ie, DW) is added so that the total amount is 50 μL when combined with the template solution described below. It was used. The composition (1) includes a template B that is detected by hybridization with the probe DNA (B) in which an amplification reaction by the LAMP method is caused by the primer DNA (set B). The composition (2) does not contain a template, and the composition (3) contains a template A that undergoes an amplification reaction by the LAMP method with the primer DNA (set A) and is detected by hybridization with the probe DNA (A). As the composition (1), one containing both template A and template B was used. Templates A and B are synthetic oligo DNAs having the base sequences shown in Table 39.

(4) LAMP amplification reaction on array type primer probe chip and detection of target nucleic acid by probe DNA As shown schematically in FIG. 60, so as to include an electrode as a probe immobilization region and a primer DNA immobilization region, Silicon rubber molded as a covering for forming the reaction vessel was mounted on the array-type primer probe chip, and the LAMP reaction solution was poured into the reaction vessel through a hole previously provided in the silicon rubber, and then the lid was capped. This was set | placed on the plate set to 63 degreeC, and LAMP reaction was performed for 60 minutes. As outlined in FIG. 61, for a particular primer, if it can bind and the template containing the target sequence to be amplified is included in the LAMP reaction solution, that primer is immobilized. The LAMP reaction proceeds locally at the site, and the resulting LAMP product hybridizes with the nearby probe DNA.

  After the LAMP reaction for 60 minutes, a hybridization reaction was performed at 45 ° C. for 10 minutes, and washing was performed at 45 ° C. for 10 minutes. Thereafter, the washing solution was removed and a 75 μM Hoechst 33258 solution was injected. The potential was swept across each probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecules specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The above series of reactions was carried out using an automatic DNA testing apparatus described in the following literature (SICE Journal of Control, Measurement, and System Integration, Vol. 1, No. 3, pp. 266-270, 2008).

(5) Detection results Table 40 shows the detection results.

  When the LAMP reaction solution composition (1) containing the template B is added, the LAMP reaction with the primer DNA (set B) immobilized in the vicinity of the working electrode 2 proceeds, and the resulting LAMP product becomes the probe DNA (B). As a result, a current value of 70 nA was obtained.

  On the other hand, with the primer DNA (set A) immobilized in the vicinity of the working electrode 2, the LAMP reaction did not proceed and no current value was obtained.

  From these two numerical values, it was possible to determine that the template B was contained in the LAMP reaction solution.

  In addition, when the LAMP reaction solution composition (2) containing no template is added, the LAMP amplification reaction using the primer DNA (set A) and the primer DNA (set B) immobilized in the vicinity of the working electrodes 1 and 2 is The current value was not obtained.

  From the above results, it was shown that the template contained in the LAMP reaction solution can be detected and the sequence thereof can be identified using the array-type primer probe chip described in this example.

7). Device Suitable for Fully Automatic Processing As a further embodiment, a nucleic acid detection cassette and a nucleic acid detection apparatus suitable for fully automatic processing of detection of a target nucleic acid following a pretreatment step are provided.

  Conventionally, as a system for detecting a nucleic acid, a system in which each device such as a nucleic acid extraction device, a nucleic acid amplification device, a hybridization device, a nucleic acid detection device, and a data analysis device is individually used is known. In such a system, the adjustment of the sample other than that realized by these apparatuses or the movement of the sample between the apparatuses requires manual labor.

  In nucleic acid amplification, there is a problem that if a sample before amplification is mixed with a very small amount of other nucleic acid, the nucleic acid is also amplified in a large amount to cause false detection. It is known that nucleic acid molecules are stable even in a dry state, adsorb to various substances, and may float in the air. Therefore, in order to prevent erroneous detection, a strict management system is required such that no sample after amplification is brought into the place where nucleic acid extraction is performed.

  In recent years, fully automatic nucleic acid detection apparatuses have been developed that automatically perform steps from nucleic acid extraction, amplification to hybridization, detection to data analysis. However, the existing fully-automatic nucleic acid detectors are not intended to take measures against contamination of nucleic acid molecules that are not subject to detection as described above, and many of them are large, so they are intended for research use. Yes. In addition, products that have been dealt with by adopting a sealed structure have been put on the market, but because the number of parts such as cassettes is large and the structure is complicated, it is difficult to downsize, and these consumables are expensive. Detection costs increase.

  When a closed structure is used in general fully automatic nucleic acid detection, multiple containers for loading nucleic acid samples and chemical solutions are formed, and a flow path is formed for each container, and each valve is equipped with a control. Since the number of parts such as a cassette is large and the structure is complicated, the cassette is expensive. In addition, the nucleic acid detection apparatus also requires complicated control in accordance with the structure of the cassette, so the structure is complicated, and it is difficult to downsize and expensive.

  According to a further embodiment, a small sealed nucleic acid detection cassette and a nucleic acid detection apparatus suitable for consistently and automatically processing from nucleic acid amplification to detection of a target nucleic acid are provided.

<Twenty-sixth embodiment>
FIG. 62 is an exploded perspective view showing a schematic configuration as an example of a nucleic acid detection (nucleic acid extraction) cassette 7022 according to this embodiment. The nucleic acid detection cassette 7022 mainly includes three components: a flow path packing 7001, an upper plate 7002, and a lower plate 7003. FIG. 63 is a perspective view showing a schematic configuration as an example of the nucleic acid detection cassette 7022 in which the flow path packing 7001, the upper plate 7002, and the lower plate 7003 shown in FIG. 62 are combined. FIG. 63A is a perspective view seen from the front surface side of the nucleic acid detection cassette 7022. In this embodiment, the outer surface of the upper plate 7002 in the stacking direction of the flow path packing 7001, the upper plate 7002, and the lower plate 7003 is the surface of the nucleic acid detection cassette 7022. FIG. 63B is a perspective view of the nucleic acid detection cassette 7022 viewed from the back side. In this embodiment, the outer surface of the lower plate 7003 in the stacking direction of the flow path packing 7001, the upper plate 7002, and the lower plate 7003 is set as the back surface of the nucleic acid detection cassette 7022.

  The flow path packing 7001 includes a sample syringe 7004, a cleaning syringe 7005, an insertion agent syringe 7006, a flow path 7007, a nucleic acid detection flow path 7008, a waste liquid flow path 7009, and a waste liquid syringe 7010. The flow path packing 7001 is a thin plate having a front surface (first surface) and a back surface (second surface) opposite to the front surface. In the flow path packing 7001, the sample syringe 7004, the cleaning syringe 7005, the insertion syringe 7006, the flow path 7007, the nucleic acid detection flow path 7008, the waste liquid flow path 7009, and the waste liquid syringe 7010 are integrally configured (integrated molding) as one part. Yes. Therefore, the flow path packing 7001 can reduce the number of parts. The flow path packing 7001 is made of a soft material such as silicone or elastomer, for example. In addition, since an elastomer is a material denser than silicone, it can prevent evaporation of a liquid more.

  A specimen syringe 7004 is a container having an opening for loading (injecting) a liquid specimen (also referred to as a nucleic acid to be detected, a specimen sample, or a nucleic acid sample) on the surface, and a thin film part that can be easily deformed on the back surface. Shape. Therefore, the sample syringe 7004 can be easily deformed and crushed by external pressurization to the thin film portion side. On the other hand, the sample syringe 7004 expands on the thin film portion side by being filled with a liquid in a crushed state, for example. The sample syringe 7004 can store a sample.

  The cleaning syringe 7005 has a container shape including an opening for loading a cleaning liquid on the front surface and a thin film portion that can be easily deformed on the back surface. Therefore, like the syringe syringe 7004, the cleaning syringe 7005 can be easily deformed and crushed by external pressure applied to the thin film portion side. On the other hand, the cleaning syringe 7005 is inflated on the thin film portion side by being filled with a liquid in a crushed state, for example. The cleaning syringe 7005 can store a cleaning solution for performing cleaning after hybridization.

  The insertion syringe 7006 is provided with an opening for loading a liquid insertion agent (chemical solution used for nucleic acid detection) for the oxidation-reduction reaction at the time of current detection on the surface, and can be easily deformed on the back surface. It is a container shape provided with a thin film part. Therefore, as with the sample syringe 7004, the intercalating syringe 7006 can be easily deformed and crushed by external pressure applied to the thin film portion side. On the other hand, the insertion agent syringe 7006 expands on the side of the thin film portion by being filled with a liquid in a crushed state, for example. The insertion agent syringe 7006 can store the insertion agent.

  The channel 7007 connects the sample syringe 7004, the washing syringe 7005, the intercalating syringe 7006, and the nucleic acid detection channel 7008. The flow path 7007 further includes flow paths 7071, 7072, and 7073 that are branched independently. The channel 7007 is connected to the sample syringe 7004, the cleaning syringe 7005, and the intercalating agent syringe 7006 via the channels 7071, 7072, and 7073. The channel 7007 is also connected to the nucleic acid detection channel 7008. Therefore, the channel 7007 is a channel for sending (flowing) the liquid stored in the sample syringe 7004, the cleaning syringe 7005, and the intercalating syringe 7006 to the nucleic acid detection channel 7008. In addition, check valves 7011a, 7011b, and 7011c are provided at connection portions between the sample syringe 7004, the cleaning syringe 7005, the insertion agent syringe 7006, and the flow path 7007, respectively. Each of the check valves 7011a, 7011b, and 7011c has a function of preventing inflow of liquid from the flow path 7007 to the sample syringe 7004, the cleaning syringe 7005, and the insertion agent syringe 7006. Further, each of the check valves 7011a, 7011b, and 7011c also has a function of preventing a significant outflow of each liquid other than during the liquid feeding from the sample syringe 7004, the cleaning syringe 7005, and the insertion agent syringe 7006.

  The nucleic acid detection channel 7008 is, for example, a groove provided on the back surface of the channel packing 7001. The nucleic acid detection channel 7008 has a liquid inflow side connected to the channel 7007 and an outflow side connected to a waste liquid channel 7009. The nucleic acid detection channel 7008 is a channel (region) for processing from nucleic acid extraction, nucleic acid amplification, hybridization, and nucleic acid detection.

  The waste liquid flow path 7009 connects the nucleic acid detection flow path 7008 and the waste liquid syringe 7010. The waste liquid flow path 7009 is a flow path for sending the liquid (waste liquid) flowing out from the nucleic acid detection flow path 7008 to the waste liquid syringe 7010.

  The waste liquid syringe 7010 is formed in a bag shape having a thin film portion that can be easily deformed on the back surface of the flow path packing 7001. Accordingly, the waste liquid syringe 7010 can be easily deformed and crushed by external pressure applied to the thin film side. The waste liquid syringe 7010 is configured in a normal state (before the waste liquid flows in) in a state in which the thin film is previously folded and crushed (shrinked). When the waste liquid flows in, the waste liquid syringe 7010 expands from the state in which the thin film side is crushed. Therefore, the waste liquid syringe 7010 can store the liquid flowing out from the nucleic acid detection flow path 7008.

  According to the configuration of the flow path packing 7001 described above, the sample syringe 7004, the cleaning syringe 7005, and the insertion syringe 7006 are connected to the waste liquid syringe 7010 via the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009. Yes.

  The upper plate 7002 includes injection ports 7012a, 7012b, and 7012c, a nucleic acid detection port 7014, and a positioning hole 7015. The upper plate 7002 is made of a hard material such as plastic, glass, or metal. The upper plate 7002 has a thin shape. The upper plate 7002 is opposed (opposed) to the surface of the flow path packing 7001 and is in close contact with the surface of the flow path packing 7001. That is, the upper plate 7002 is used for sealing (sealing) the flow path packing 7001.

  Each of the injection ports 7012a, 7012b, and 7012c is provided at a position facing the sample syringe 7004 (the opening), the cleaning syringe 7005 (the opening), and the insertion agent syringe 7006 (the opening). Each of the inlets 7012a, 7012b, and 7012c is an opening for loading a sample syringe 7004, a cleaning syringe 7005, and an insertion agent syringe 7006 with liquid after the nucleic acid detection cassette 7022 is assembled. The inlets 7012a, 7012b, and 7012c are sealed using a cap seal 7013 after the liquid is loaded into the sample syringe 7004, the cleaning syringe 7005, and the insertion agent syringe 7006.

The nucleic acid detection port 7014 is provided at a position facing the substrate 7020a provided on the lower plate 7003 without being opposed to the flow path packing 7001 after the nucleic acid detection cassette 7022 is assembled. The substrate 7020a is connected to a nucleic acid detection unit 7020 described later, and is used to transmit a signal detected by the nucleic acid detection unit 7020 to a nucleic acid detection substrate 7024 described later. The nucleic acid detection port 7014 is an opening for inserting the nucleic acid detection substrate 7024 when detecting nucleic acid.
The positioning hole 7015 is an opening used for positioning (alignment) of the nucleic acid detection cassette 7022 as will be described later.

  The lower plate 7003 includes a specimen liquid supply hole 7016, a cleaning liquid supply hole 7017, an insertion agent liquid supply hole 7018, a waste liquid recess 7019, a nucleic acid detection unit 7020, and a temperature adjustment hole 7021. Similarly to the upper plate 7002, the lower plate 7003 is made of a hard material such as plastic, glass, or metal. The lower plate 7003 faces the back surface of the flow path packing 7001 and is in close contact with the back surface of the flow path packing 7001. That is, the lower plate 7003 has a thin shape. The lower plate 7003 is used together with the upper plate 7002 to seal the flow path packing 7001.

  The sample feeding hole 7016 is provided in the lower plate 7003 at a position facing the sample syringe 7004. The sample liquid feeding hole 7016 is an opening that is secured so as not to prevent the expansion of the sample syringe 7004 toward the lower plate 7003 even when the sample syringe 7004 is fully stored. Therefore, as shown in FIG. 63, on the back side of the nucleic acid detection cassette 7022, the bottom portion (thin film portion side) of the sample syringe 7004 is exposed from the lower plate 7003 through the sample liquid supply hole 7016.

  The cleaning liquid feeding hole 7017 is provided on the lower plate 7003 at a position facing the cleaning syringe 7005. The cleaning liquid feeding hole 7017 is an opening that is secured so as not to prevent expansion of the cleaning syringe 7005 toward the lower plate 7003 even when the specimen is stored in the cleaning syringe 7005 full. Therefore, as shown in FIG. 63, on the back side of the nucleic acid detection cassette 7022, the bottom portion (thin film portion side) of the cleaning syringe 7005 is exposed from the lower plate 7003 through the cleaning liquid feeding hole 7017.

  The insertion agent feeding hole 7018 is provided at a position facing the insertion agent syringe 7006 in the lower plate 7003. The insertion agent feeding hole 7018 is an opening that is secured so as not to hinder the expansion of the insertion agent syringe 7006 toward the lower plate 7003 even when the specimen is fully stored in the insertion agent syringe 7006. Therefore, as shown in FIG. 63, on the back side of the nucleic acid detection cassette 7022, the bottom portion (thin film side) of the insertion agent syringe 7006 is exposed from the lower plate 7003 through the insertion agent feeding hole 7018.

  The waste liquid recess 7019 is provided on the surface of the lower plate 7003 facing the surface of the flow path packing 7001 and at a position facing the waste liquid syringe 7010. The waste liquid recess 7019 is a recess (gap) that is secured so as not to hinder the expansion of the waste liquid syringe 7010 toward the lower plate 7003 even when the waste liquid 7010 is fully stored in the waste liquid syringe 7010.

  The nucleic acid detection unit 7020 is provided on the surface of the lower plate 7003 facing the back surface of the flow path packing 7001 and at a position facing the nucleic acid detection flow path 7009. The nucleic acid detection unit 7020 detects a target nucleic acid. The nucleic acid detection unit 7020 is a sensor region where a nucleic acid probe is immobilized.

  The temperature adjustment hole 7021 is a surface corresponding to the back surface of the nucleic acid detection cassette 7022, and is provided at a position facing the nucleic acid detection unit 7020. That is, the nucleic acid detection unit 7020 is exposed from the lower plate 7003 through the temperature adjustment hole 7021. The temperature adjustment hole 7021 is an opening for performing high-precision heating and cooling directly on the nucleic acid detection unit 7020 during nucleic acid detection.

  As described above, the three parts of the flow path packing 7001, the upper plate 7002, and the lower plate 7003 are combined so that the flow path packing 7001 is sandwiched by the upper plate 7002 and the lower plate 7003. Since the flow path packing 7001 is pressurized by the upper plate 7002 and the lower plate 7003, hermeticity is maintained. That is, the nucleic acid detection cassette 7022 is a sealing device that ensures the sealing of the flow path packing 7001. The nucleic acid detection cassette 7022 can prevent nucleic acid from leaking outside due to the sealing property of the flow path packing 7001. For joining the upper plate 7002 and the lower plate 7003, various methods such as adhesion, welding, and screwing can be adopted, and the method is not limited.

  FIG. 64 is a perspective view showing a schematic configuration as an example of a nucleic acid detection device 7100 that uses the nucleic acid detection cassette 7022 according to the present embodiment. In the present embodiment, the nucleic acid detection cassette 7022 and the nucleic acid detection device 7100 are described as separate components, but the nucleic acid detection device 7100 may include the nucleic acid detection cassette 7022.

  The nucleic acid detection device 7100 includes a cassette stand 7023, a nucleic acid detection substrate 7024, positioning pins 7025, a nucleic acid detection substrate front / rear mechanism 7026, a heating / cooling device 7027, a heating / cooling device front / rear mechanism 7028, a specimen feeding rod 7029, a washing feeding rod 7030, An insertion agent feeding rod 7031, a rod front-rear mechanism (moving mechanism) 7032, and springs 7033a, 7033b, 7033c are provided.

  The cassette stand 7023 is provided near the center of the nucleic acid detection device 7100. The cassette stand 7023 holds the nucleic acid detection cassette 7022. The cassette stand 7023 is a slot into which the nucleic acid detection cassette 7022 can be inserted and removed, for example.

  The nucleic acid detection substrate 7024 is provided at a position facing the nucleic acid detection port 7014 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The nucleic acid detection substrate 7024 has a size that can be inserted into and removed from the nucleic acid detection port 7014. The nucleic acid detection substrate 7024 is a substrate for performing nucleic acid detection by acquiring a signal detected by the nucleic acid detection unit 7020 and determining the presence or absence of a target nucleic acid.

  The positioning pin 7025 is provided at a position facing the positioning hole 7015 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The positioning pin 7025 positions the nucleic acid detection cassette 7022 with respect to the nucleic acid detection device 7100.

  The nucleic acid detection substrate front-rear mechanism 7026 includes a nucleic acid detection substrate 7024 and positioning pins 7025. The nucleic acid detection substrate front-rear mechanism 7026 can move in the front-rear direction simultaneously with the nucleic acid detection substrate 7024 and the positioning pins 7025 as one body. In the present embodiment, the direction toward or away from the nucleic acid detection cassette 7022 (or cassette stand 7023) is defined as the front-rear direction. The nucleic acid detection substrate front-rear mechanism 7026 fits the nucleic acid detection substrate 7024 and the positioning pins 7025 into the back surface of the nucleic acid detection cassette 7022. The nucleic acid detection substrate 7024 is in contact with the substrate of the lower plate 7003 through the nucleic acid detection port 7014. The positioning pin 7025 is inserted into the positioning hole 7015, thereby positioning the nucleic acid detection cassette 7022 with respect to the nucleic acid detection device 7100.

  The heating / cooling device 7027 is provided at a position facing the temperature adjustment hole 7021 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The heating / cooling device 7027 has a size that can be inserted into and removed from the temperature adjustment hole 7021. The heating / cooling device 7027 controls the nucleic acid detection unit 7020 (the corresponding nucleic acid detection flow path 7008) to an optimum temperature.

  The heating / cooling device front / rear mechanism 7028 is equipped with a heating / cooling device 7027. That is, the heating / cooling device 7027 and the heating / cooling device front / rear mechanism 7028 are provided on the opposite side of the nucleic acid detection substrate front / rear mechanism 7026 with the cassette stand 7023 interposed therebetween. The heating / cooling device front / rear mechanism 7028 can move the heating / cooling device 7027 in the front / rear direction. The nucleic acid detection substrate front-rear mechanism 7026 fits the heating / cooling device 7027 into the back surface of the nucleic acid detection cassette 7022. The heating / cooling device 7027 is brought into contact with the nucleic acid detection unit 7020 by fitting into the temperature adjustment hole 7021.

  The sample feeding rod 7029 is provided at a position facing the sample feeding hole 7016 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The sample feeding rod 7029 has a function of applying pressure to the thin film portion of the sample syringe 7004 through the sample feeding hole 7016 and sending the sample in the sample syringe 7004 to the flow path 7007. The sample feeding rod 7029 is provided with a surface orthogonal to the front-rear direction at the tip portion facing the nucleic acid detection cassette 7022. The surface of the tip portion of the sample liquid feeding rod 7029 has substantially the same shape as the shape of the sample liquid feeding hole 7016. The width in the front-rear direction of the distal end portion of the sample feeding rod 7029 is the distance from the back surface of the nucleic acid detection cassette 7022 to the opening of the sample syringe 7004 (the surface of the upper plate 7002 that contacts this) in the front and back direction of the nucleic acid detection cassette 7022. Is almost the same. Therefore, the sample feeding rod 7029 can completely crush the thin film portion of the sample syringe 7004 and can send all the sample in the sample syringe 7004 to the flow path 7007.

  The washing liquid feeding rod 7030 is provided at a position facing the washing liquid feeding hole 7017 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The cleaning liquid feeding rod 7030 has a function of pressurizing the thin film portion of the cleaning syringe 7005 through the cleaning liquid supply hole 7017 and sending the specimen in the cleaning syringe 7005 to the flow path 7007. The cleaning liquid feeding rod 7030 includes a surface orthogonal to the front-rear direction at the tip portion facing the nucleic acid detection cassette 7022. The surface of the distal end portion of the cleaning liquid feeding rod 7030 has substantially the same shape as the shape of the cleaning liquid feeding hole 7017. The width in the front-rear direction of the front end portion of the cleaning / feeding rod 7030 is the distance from the back surface of the nucleic acid detection cassette 7022 to the opening of the cleaning syringe 7005 (the surface of the upper plate 7002 that is in contact with the back surface). Is almost the same. Therefore, the cleaning liquid feeding rod 7030 can completely crush the thin film portion of the cleaning syringe 7005, and can send all the specimen in the cleaning syringe 7005 to the flow path 7007.

  The insertion agent feeding rod 7031 is provided at a position facing the insertion agent feeding hole 7018 when the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023. The intercalating agent feeding rod 7031 has a function of applying pressure to the thin film portion of the intercalating agent syringe 7006 via the intercalating agent feeding hole 7018 and feeding the specimen in the intercalating agent syringe 7006 to the flow path 7007. The insertion agent feeding rod 7031 is provided with a surface perpendicular to the front-rear direction at the tip portion facing the nucleic acid detection cassette 7022. The surface of the distal end portion of the insertion agent feeding rod 7031 has substantially the same shape as the shape of the cleaning solution feeding hole 7017. The width in the front-rear direction of the distal end portion of the insertion agent feeding rod 7031 is from the back surface of the nucleic acid detection cassette 7022 to the opening of the insertion agent syringe 7006 (the surface of the upper plate 7002 in contact therewith) in the front and back direction of the nucleic acid detection cassette 7022. The distance is approximately the same. Therefore, the insertion agent feeding rod 7031 can completely crush the thin film portion of the insertion agent syringe 7006, and can send out all of the insertion agent in the insertion agent syringe 7006 to the flow path 7007.

  The rod front / rear mechanism 7032 is equipped with a specimen liquid feeding rod 7029, a cleaning liquid feeding rod 7030, and an intercalating agent liquid feeding rod 7031. That is, the sample liquid feeding rod 7029, the washing liquid feeding rod 7030, the intercalating agent liquid feeding rod 7031, and the rod front / rear mechanism 7032 are provided on the opposite side to the nucleic acid detection substrate front / rear mechanism 7026 with the cassette stand 7023 interposed therebetween. The rod back-and-forth mechanism 7032 can move in the front-rear direction with respect to the nucleic acid detection cassette 7022 at the same time as the sample liquid-feeding rod 7029, the cleaning liquid-feeding rod 7030, and the insertion agent liquid-feeding rod 7031. The rod front-rear mechanism 7032 presses the specimen liquid feeding rod 7029, the cleaning liquid feeding rod 7030, and the intercalating agent liquid feeding rod 7031 against the back surface of the nucleic acid detection cassette 7022. The sample feeding rod 7029 comes into contact with the thin film portion of the sample syringe 7004 through the sample feeding hole 7016 and pressurizes it. Similarly, the cleaning liquid feeding rod 7030 comes into contact with the thin film portion of the cleaning syringe 7005 through the cleaning liquid feeding hole 7017 and pressurizes it. The insertion agent feeding rod 7031 contacts and pressurizes the thin film portion of the insertion agent syringe 7006 through the insertion agent feeding hole 7018.

  Here, the mounting positional relationship of the specimen liquid feeding rod 7029, the cleaning liquid feeding rod 7030, and the insertion agent liquid feeding rod 7031 in the rod front-rear mechanism 7032 will be described. The mounting position relationship regarding the specimen liquid feeding rod 7029 and the cleaning liquid feeding rod 7030 is as follows. Before any of the specimen liquid feeding rod 7029, the washing liquid feeding rod 7030, and the intercalating agent liquid feeding rod 7031 is in contact with the nucleic acid detection cassette 7022, the tip part of the specimen liquid feeding rod 7029 is more than the tip part of the washing liquid feeding rod 7030. It is located near the nucleic acid detection cassette 7022 by a predetermined distance in the front-rear direction. Here, the predetermined distance is, for example, a distance greater than or equal to the width of the tip portion of the specimen liquid feeding rod 7029. That is, when the specimen liquid feeding rod 7029, the washing liquid feeding rod 7030, and the intercalating agent liquid feeding rod 7031 move toward the nucleic acid detection cassette 7022, the specimen liquid feeding rod 7029 completely crushes the thin film portion of the specimen syringe 7004. Before all the specimen in the specimen syringe 7004 is sent out to the flow path 7007, the cleaning liquid feeding rod 7030 comes into contact with the thin film portion of the cleaning syringe 7005 and starts sending the cleaning agent in the cleaning syringe 7005 to the flow path 7007. Absent.

  The mounting position relationship regarding the cleaning liquid feeding rod 7030 and the intercalating agent liquid feeding rod 7031 is as follows. Under the same conditions, the distal end portion of the cleaning liquid feeding rod 7030 is positioned closer to the nucleic acid detection cassette 7022 by a predetermined distance in the front-rear direction than the distal end portion of the insertion agent liquid feeding rod 7031. Here, the predetermined distance is, for example, a distance greater than or equal to the width of the tip portion of the cleaning liquid feeding rod 7030. That is, when the specimen feeding rod 7029, the washing feeding rod 7030, and the intercalating agent feeding rod 7031 move toward the nucleic acid detection cassette 7022, the washing feeding rod 7030 completely crushes the thin film portion of the washing syringe 7005. Before sending all the cleaning agent in the cleaning syringe 7005 to the flow path 7007, the insertion agent feeding rod 7031 comes into contact with the thin film portion of the insertion syringe 7006, and the insertion agent in the insertion syringe 7006 is transferred to the flow path 7007. It will not start sending out.

  From the relationship described above, the mounting position relationship regarding the sample liquid feeding rod 7029 and the intercalating agent liquid feeding rod 7031 is as follows. Under the same conditions, the tip portion of the sample feeding rod 7029 is positioned closer to the nucleic acid detection cassette 7022 by a predetermined distance in the front-rear direction than the tip portion of the intercalating agent feeding rod 7031.

  Each of the springs 7033a, 7033b, and 7033c is provided between the specimen liquid feeding rod 7029, the cleaning liquid feeding rod 7030, the insertion agent liquid feeding rod 7031, and the rod front and rear mechanism 7032. The springs 7033a, 7033b, and 7033c have elasticity that can expand and contract in the front-rear direction (the moving direction of the rod front-rear mechanism 7032).

Each of the springs 7033a, 7033b, and 7033c contracts in the front-rear direction due to contact of the sample feeding rod 7029, the washing feeding rod 7030, the intercalating agent feeding rod 7031, and the nucleic acid detection cassette 7022. Instead of the spring 7033, another elastic body may be used. Further, instead of the springs 7033a, 7033b, and 7033c, a mechanical configuration that contracts in the front-rear direction by contact of the sample feeding rod 7029, the washing feeding rod 7030, the intercalating agent feeding rod 7031 and the nucleic acid detection cassette 7022 may be used. Good.

  Next, a usage procedure (method) of the nucleic acid detection cassette 7022 and the nucleic acid detection device 7100 according to the present embodiment and an example of a nucleic acid detection procedure using these will be described. In addition, the procedure demonstrated below is an example and can be replaced | exchanged suitably.

  First, an example of a preparation procedure for the nucleic acid detection cassette 7022 will be described. As shown in FIG. 63, the sample syringe 7004 of the sealed nucleic acid detection cassette 7022 contains a sample, the washing syringe 7005 contains a washing liquid, and the insertion agent syringe 7006 contains an insertion agent via the inlets 7012a, 7012b, and 7012c. Loaded. The inlet 7012 is sealed using a cap seal 7013. The nucleic acid detection cassette 7022 is inserted into the cassette stand 7023 so that the portion provided with the sample syringe 7004, the washing syringe 7005, and the insertion agent syringe 7006 is on the upper side, and the upper plate 7002 is opposed to the nucleic acid detection substrate 7024.

  Next, an example of a procedure for using the nucleic acid detection device 7100 in which the nucleic acid detection cassette 7022 is inserted into the cassette stand 7023 will be described.

  First, the nucleic acid detection substrate front-rear mechanism 7026 is operated. The nucleic acid detection substrate front-rear mechanism 7026 advances the nucleic acid detection substrate 7024 and the positioning pins 7025 toward the nucleic acid detection cassette 7022. The nucleic acid detection substrate back-and-forth mechanism 7026 moves the nucleic acid detection substrate 7024 to a detection position (a position in contact with the substrate 7020a of the lower plate 7003). At the same time, the nucleic acid detection substrate front-rear mechanism 7026 moves the positioning pin 7025 to the positioning hole 7015 of the nucleic acid detection cassette 7022 and inserts it. The nucleic acid detection cassette 7022 is positioned with respect to the nucleic acid detection device 7100 by inserting the positioning pins 7025 into the positioning holes 7015.

  Next, the heating / cooling device front-rear mechanism 7028 is operated. The heating / cooling device front / rear mechanism 7028 advances the heating / cooling device 7027 toward the nucleic acid detection cassette 7022. The heating / cooling device front / rear mechanism 7028 moves the heating / cooling device 7027 to a position in contact with the nucleic acid detection unit 7020 through the temperature adjustment hole 7021.

  Next, the rod front-rear mechanism 7032 is operated. The rod front-rear mechanism 7032 advances the specimen liquid feeding rod 7029, the washing liquid feeding rod 7030, and the insertion agent rod 7031 toward the nucleic acid detection cassette 7022. The rod front / rear mechanism 7032 presses the sample feeding rod 7029 against the sample syringe 7004 through the sample feeding hole 7016. The sample syringe 7004 has a thin film structure and easily deforms as described above. Therefore, the sample is sent out to the nucleic acid detection flow path 7008 via the flow path 7007 by pressurization of the sample liquid feeding rod 7029. The rod front / rear mechanism 7032 advances the sample feeding rod 7029 until the sample syringe 7004 is completely crushed and all the samples are sent to the flow path 7007. The distal end portion of the cleaning liquid feeding rod 7030 and the distal end portion of the intercalating agent liquid feeding rod 7031 are respectively connected to the cleaning syringe 7005 and the intercalating agent syringe 7006 according to the positional relationship with the distal end portion of the specimen liquid feeding rod 7029 as described above. Not touching.

  At this time, the air in the nucleic acid detection flow path 7008 is pushed out by the specimen and flows into the waste liquid syringe 7010 via the waste liquid flow path 7009. The waste liquid syringe 7010 expands slightly due to an increase in internal pressure caused by the inflowing air. The waste liquid syringe 7010 ensures a capacity for the waste liquid to flow in due to expansion. Note that the volume of the sample syringe 7004 is substantially the same as the amount that fills the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009. Therefore, all the specimens sent from the specimen syringe 7004 to the flow path 7007 fill the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009, but do not flow to the waste liquid syringe 7010.

  Next, the heating / cooling device front-rear mechanism 7028 is operated. The heating / cooling device front / rear mechanism 7028 advances the heating / cooling device 7027 toward the nucleic acid detection cassette 7022. The heating / cooling device front / rear mechanism 7028 brings the heating / cooling device 7027 into contact with the nucleic acid detection unit 7020 through the temperature adjustment hole 7021. Next, the heating / cooling device 7027 is operated to heat the specimen. Since the amplification primer is fixed to the inner wall of the nucleic acid detection channel 7008 in advance, the primer is eluted into the sample by heating. In the nucleic acid detection channel 7008, nucleic acid amplification is performed, and at the same time, hybridization to the probe electrode fixed to the nucleic acid detection unit 7020 is performed.

  Next, the rod front-rear mechanism 7032 is operated again. The rod front-rear mechanism 7032 advances the specimen liquid feeding rod 7029, the washing liquid feeding rod 7030, and the insertion agent rod 7031 toward the nucleic acid detection cassette 7022. The rod front-rear mechanism 7032 presses the cleaning liquid supply rod 7030 against the cleaning syringe 7005 through the cleaning liquid supply hole 7017. Note that the sample feeding rod 7029 is configured to be freely stretchable by the spring 7033a as described above. Therefore, even if the rod back-and-forth mechanism 7032 further moves the sample feeding rod 7029 to the nucleic acid detection cassette 7022 side, the sample feeding rod 7029 does not destroy the nucleic acid detection cassette 7022 because the spring 7033 contracts.

  Further, as described above, the cleaning syringe 7005 has a thin film structure and easily deforms. Therefore, the cleaning liquid is sent out to the nucleic acid detection flow path 7008 through the flow path 7007 by pressurization of the cleaning liquid supply rod 7030. The cleaning liquid cleans the nucleic acid detection channel 7008. The rod front-rear mechanism 7032 advances the cleaning liquid feeding rod 7030 until the cleaning syringe 7005 is completely crushed and all specimens are sent to the flow path 7007. Note that the distal end portion of the intercalating agent feeding rod 7031 is not in contact with the intercalating agent syringe 7006 due to the positional relationship with the distal end portion of the cleaning fluid feeding rod 7030 as described above.

  The capacity of the cleaning syringe 7005 is substantially the same as the amount that fills the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009. Therefore, all of the cleaning liquid sent from the cleaning syringe 7005 to the flow path 7007 is replaced with the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009 instead of pushing the specimen into the waste liquid syringe 7010. The nucleic acid detection flow path 7008 and the waste liquid flow path 7009 are filled. That is, all of the specimen in the nucleic acid detection flow path 7008 flows into the waste liquid syringe 7010 through the waste liquid flow path 7009. Note that the cleaning liquid fills the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009, but does not flow out to the waste liquid syringe 7010. Since the waste liquid syringe 7010 is easily expanded, the waste liquid syringe 7010 is further expanded by the inflowing sample.

  Next, the rod front-rear mechanism 7032 is operated again. The rod front-rear mechanism 7032 advances the specimen liquid feeding rod 7029, the washing liquid feeding rod 7030, and the insertion agent rod 7031 toward the nucleic acid detection cassette 7022. The rod back-and-forth mechanism 7032 presses the insertion agent feeding rod 7031 against the insertion agent syringe 7006 through the insertion agent feeding hole 7018. Note that the cleaning liquid feeding rod 7030 is configured to be extendable and contractable by the spring 7033b as described above. Therefore, even if the rod front-rear mechanism 7032 moves the washing / feeding rod 7030 to the nucleic acid detection cassette 7022 side again, the washing / feeding rod 7030 does not destroy the nucleic acid detection cassette 7022 because the spring 7033b contracts. .

  Further, as described above, the insertion agent syringe 7006 has a thin film structure and easily deforms. Therefore, the insertion agent is sent out to the nucleic acid detection channel 7008 via the channel 7007 by pressurization of the insertion agent feeding rod 7031. In the nucleic acid detection flow path 7008, a nucleic acid detection reaction is performed by an intercalating agent. The rod front-rear mechanism 7032 advances the insertion agent feeding rod 7031 until the insertion agent syringe 7006 is completely crushed and all specimens are sent to the flow path 7007.

  Note that the volume of the insertion agent syringe 7006 is substantially the same as the amount that fills the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009. Therefore, all the intercalating agents sent from the intercalating syringe 7006 to the flow path 7007 are replaced with the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009 instead of pushing the cleaning liquid into the waste liquid syringe 7010. 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009 are filled. That is, all the cleaning liquid in the nucleic acid detection flow path 7008 flows into the waste liquid syringe 7010 through the waste liquid flow path 7009. The insertion agent fills the flow path 7007, the nucleic acid detection flow path 7008, and the waste liquid flow path 7009, but does not flow out to the waste liquid syringe 7010. Since the waste liquid syringe 7010 is easily expanded, the waste liquid syringe 7010 is further expanded by the flowing cleaning liquid.

  As described above, the rod front-rear mechanism 7032 sequentially crushes the sample syringe 7004, the cleaning syringe 7005, and the insertion agent syringe 7006 with the sample supply rod 7029, the cleaning solution supply rod 7030, and the insertion agent rod 7031 arranged in parallel. Then, the solution is sequentially sent to the nucleic acid detection channel 7008. The waste liquid discharged from the nucleic acid detection channel 7008 is sent to the waste liquid syringe 7010 as the waste liquid syringe 7010 spontaneously expands due to the pressure increase.

  Needless to say, when performing the nucleic acid detection reaction from the series of nucleic acid amplification described above, the nucleic acid detection unit 7020 is controlled to an optimum temperature using the heating / cooling device 7027. After completion of the nucleic acid detection reaction, nucleic acid detection is performed using the nucleic acid detection substrate 7024 to determine the presence or absence of the target nucleic acid.

  In the present embodiment, the example in which the spring 7033c is provided between the insertion agent feeding rod 7031 and the rod front-rear mechanism 7032 has been described. However, the spring 7033c may not be provided. This is because the rod front / rear mechanism 7032 does not further move the insertion agent feeding rod 7031 to the nucleic acid detection cassette 7022 side after all of the insertion agent in the insertion agent syringe 7006 has been sent out to the flow path 7007. In this embodiment, the example in which the nucleic acid detection cassette 7022 includes the cleaning syringe 7005 has been described. However, the nucleic acid detection cassette 7022 may not include the cleaning syringe 7005. This is because the washing solution is used to increase the detection accuracy of the target nucleic acid, and is not essential for the nucleic acid amplification to the nucleic acid detection reaction. In this case, the nucleic acid detection cassette 7022 does not need to include the check valve 7011b, the inlet 7012b, and the cleaning liquid feeding hole 7017, and the nucleic acid detection device 7100 does not need to include the cleaning liquid feeding rod 7030 and the spring 7033c.

  According to this embodiment, an inexpensive and compact sealed nucleic acid detection cassette 7022 and a nucleic acid detection apparatus 7100 using the same are automatically and consistently processed from nucleic acid amplification to target nucleic acid detection with a very simple configuration. can do.

  Although several embodiments of the present invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the scope of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are included in the invention described in the claims and the equivalents thereof.

  The following embodiments are also included.

[1] a support configured to support a liquid phase reaction field;
When the reaction field is formed by the liquid phase, a plurality of types that are releasably fixed to a plurality of primer-immobilized regions that are independent from each other on at least one surface of the support in contact with the reaction field. A plurality of primer sets configured to amplify each target sequence of
A complementary sequence of the target sequence, which is immobilized for each type so that the hybridization signal can be independently detected for each probe immobilization region at the same position as or near the probe immobilization region of the plurality of primer immobilization regions. At least one probe nucleic acid comprising:
An array-type primer probe chip comprising:

[2] The array-type primer probe chip according to [1], wherein the support has a container form or a flow path, and the inside of the container or the flow path is a reaction field.

[3] It is configured to amplify a plurality of types of target sequences that are releasably immobilized for each type in a substrate and a plurality of independent primer-immobilized regions on at least one surface of the substrate. A plurality of types of primer sets and a probe immobilization region at the same position as or near the plurality of primer immobilization regions are immobilized for each type so that hybridization signals can be independently detected for each probe immobilization region. An array type primer probe chip carrier comprising at least one kind of probe nucleic acid containing a complementary sequence of a target sequence.

[4] (a) At least one surface of a support configured to support one reaction field in a liquid phase, and contacts the reaction field when the reaction field is formed by the liquid phase. Fixing a plurality of types of primer sets for amplifying a plurality of types of target nucleic acids to a plurality of immobilization regions independent of each other in a releasable manner for each type;
(B) The probe-immobilized region at or near the plurality of primer-immobilized regions includes a complementary sequence of the target sequence for each type so that a hybridization signal can be detected independently for each probe-immobilized region. Immobilizing at least one probe nucleic acid;
(C) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(D) bringing a sample into the reaction field;
(E) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field;
(F) independently detecting the presence or amount of hybridization between the amplification product obtained by the amplification reaction and the probe nucleic acid for the amplification product obtained by each of the primer sets;
(G) detecting or measuring the presence of the target nucleic acid containing the target sequence based on the result of (f),
A target nucleic acid detection method comprising:

[5] A support configured to support a reaction field in a liquid phase, and independent of at least one surface of the support in contact with the reaction field when the reaction field is formed by the liquid phase. A plurality of primer immobilization regions arranged in a plurality of types, and a plurality of types of primer immobilization regions, each of which is configured to amplify a plurality of types of target sequences. A multi-nucleic acid reaction device comprising a primer set and a thickener releasably immobilized on the primer immobilization region.

[6] The array-type primer probe chip according to [5], further including a covering attached to a surface of the support that supports the reaction field, wherein the covering includes at least the support. A groove portion formed in a portion corresponding to a region including all the primer-immobilized regions, and through-holes opened at one end and the other end of the groove portion, respectively, and the groove portion of the covering body and the support body A multi-nucleic acid reaction tool in which a reaction part is formed by a surface supporting the reaction part.

[7] The above [1] further comprising a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions, and a plurality of probe nucleic acids immobilized on the plurality of probe immobilization regions. Or the multi-nucleic acid reaction tool as described in [2].

[8] The multi-nucleic acid reaction device according to any one of [5] to [7], wherein the thickener is agar or gelatin.

[9] The multi-nucleic acid reaction tool according to any one of [1] to [8], wherein the thickener is fixed so as to cover the primer.

[10] The multi-nucleic acid reaction tool according to any one of [5] to [9], wherein the thickener is fixed to the primer-immobilized region together with the primer.

[11] A plurality of types of target sequences that are releasably immobilized for each type in a support and a plurality of independent primer-immobilized regions on at least one surface of the support are configured to be amplified respectively. A multi-nucleic acid reaction carrier comprising a plurality of primer sets and a thickener releasably immobilized in the primer immobilization region.

[12] The above [11] further comprising a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions, and a plurality of probe nucleic acids immobilized in the plurality of probe immobilization regions. The multi-nucleic acid reaction carrier according to 1.

[13] The multi-nucleic acid reaction carrier according to [11] or [12], wherein the thickener is agar or gelatin.

[14] The multi-nucleic acid reaction carrier according to any one of [11] to [13], wherein the thickener is immobilized so as to cover the primer.

[15] The multi-nucleic acid reaction carrier according to any one of [11] to [13], wherein the thickener is immobilized together with the primer in the primer immobilization region.

[16] A plate-shaped support, a cover fixed to one surface of the support, and having an axially extending groove that opens to the surface on the support, the groove of the cover, and the support A flow path constituted by the one surface, a first through hole opened at one end of the flow path, a second through hole opened at the other end of the flow path, and the inner wall of the flow path An array-type primer probe chip comprising a plurality of primer sets releasably fixed to the primer-immobilized region and a thickener releasably fixed to the primer-immobilized region. A primer set is independently fixed to a primer fixing region for each type, and one primer set includes a plurality of primers for amplifying one target nucleic acid.

[17] The above [16], further comprising: a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions; and a plurality of probe nucleic acids immobilized on the plurality of probe immobilization regions. A multi-nucleic acid reaction device according to 1.

[18] (a) a plate-like support, a covering that is fixed to one surface of the support and that extends in the axial direction on the surface on the support, the groove of the covering, and the support A flow path constituted by the one surface of the body, a first through hole opened at one end of the flow path, a second through hole opened at the other end of the flow path, and the flow A plurality of primer immobilization regions arranged independently from each other on the inner wall of the road, a plurality of primer sets releasably fixed to the plurality of primer immobilization regions, and a releasably fixed to the primer immobilization region Preparing a multi-nucleic acid reaction device comprising a thickener;
Here, the plurality of primer sets are independently fixed to the primer fixing region independently for each type, and one primer set includes a plurality of primers for amplifying one target nucleic acid,
(B) adding a reaction solution containing a target nucleic acid to the channel from the first opening;
(C) amplifying the target nucleic acid;
A multi-nucleic acid reaction method comprising:

[19] (a) A plate-like support, a covering that is fixed to one surface of the support and that extends in the axial direction on the surface on the support, the groove of the covering, and the support A flow path constituted by the one surface of the body surface, a first through hole opened at one end of the flow path, a second through hole opened at the other end of the flow path, and the flow Providing a multi-nucleic acid reaction device comprising a plurality of primer-immobilized regions arranged independently on the inner wall of the road and a plurality of primer sets releasably fixed to the plurality of primer-immobilized regions, respectively. ,
Here, the plurality of primer sets are independently fixed to the primer immobilization region for each type, and one primer set includes a plurality of primers for amplifying one target nucleic acid,
(B) adding a reaction liquid containing a target nucleic acid and a thickener to the channel from the first opening;
(C) amplifying the target nucleic acid;
A multi-nucleic acid reaction method comprising:

[20] The multi-nucleic acid reaction tool further includes a plurality of probe immobilization regions arranged in the vicinity of each of the plurality of primer immobilization regions, and a probe nucleic acid immobilized on the plurality of probe immobilization regions. Equipped,
(E) detecting a hybridization signal between the amplification product obtained in (c) and the probe nucleic acid;
The multi-nucleic acid reaction method according to [18] or [19], further comprising:

[21] The multinucleic acid reaction method according to [18] to [20], wherein the thickener is immobilized on the primer immobilization region after the primer set is immobilized on the primer immobilization region. .

[22] The multi-nucleic acid reaction method according to [18] to [21], wherein a mixture of the primer set and the thickener is immobilized on the primer immobilization region.

[23] The multi-nucleic acid reaction method according to any one of [18] to [22], wherein the reaction solution is added at a flow rate of 10 mm / second or more.

[24] comprising a first member having a groove on the first surface;
The groove includes a flow channel chamber for a nucleic acid sample to react,
The cross-sectional area of the flow channel chamber is larger than the cross-sectional area of the groove other than the flow channel chamber,
Nucleic acid detection device.

[25] The nucleic acid detection device according to [24], wherein a depth of the flow channel chamber is deeper than a depth of a region other than the flow channel chamber in the groove.

[26] The nucleic acid detection device according to [24], wherein a width of the flow channel chamber is wider than a width of a region other than the flow channel chamber in the groove.

[27] The cross-sectional area of the flow channel chamber and the cross-sectional area of the groove portion other than the flow channel chamber are cross-sectional areas based on a plane orthogonal to the first surface. Nucleic acid detection device.

[28] The nucleic acid detection device according to [24], wherein the plurality of flow channel chambers hold a plurality of types of primer sets configured to amplify a plurality of types of target sequences, respectively.

[29] The nucleic acid detection device according to [24], wherein the flow channel chamber holds the primer set on a wall surface.

[30] Any of the above [24] to [29], further comprising a second member that faces the first surface of the first member and includes a nucleic acid detection electrode at a position facing the groove. 2. A nucleic acid detection device according to item 1.

[31] A nucleic acid detection apparatus using the nucleic acid detection device according to any one of [24] to [30],
A nucleic acid detection apparatus for detecting a nucleic acid based on a current value from the electrode.

[32] a substrate;
A nucleic acid detection sensor part formed on the substrate;
Wiring formed on the substrate and connected to the sensor;
A protective film formed on the substrate;
With
In a nucleic acid detection device for detecting a nucleic acid amplification product by the sensor unit after performing a nucleic acid amplification reaction in a chamber for reacting the sensor unit and the nucleic acid sample,
The protective film includes one or more openings that expose a lower layer portion including a part of the substrate in a wetted region of the nucleic acid sample on the substrate.
Nucleic acid detection device.

[33] The nucleic acid detection device according to [32], wherein the sensor unit is an electrode.

[34] The nucleic acid detection device according to [32], wherein the protective film covers the wiring in the liquid contact area.

[35] The nucleic acid detection device according to [34], wherein the protective film covers an outer peripheral portion of the sensor unit.

[36] In the liquid contact region, the protective film covers the substrate so as to separate an opening provided in the vicinity of the sensor unit and an opening provided in the vicinity of another sensor unit adjacent to the sensor unit. The nucleic acid detection device according to [32].

[37] The nucleic acid detection device according to [32], wherein the sensor unit includes one or more sensors.

[38] A substrate, a plurality of first electrodes disposed independently of each other on at least one surface of the substrate, a probe nucleic acid immobilized on each of the plurality of first electrodes, and the plurality of first electrodes A plurality of detection signal extraction portions arranged corresponding to one electrode, a lead connecting the plurality of first electrodes and the detection signal extraction portion corresponding to the plurality of first electrodes, the lead surface, and the base body A protective film covering at least one exposed portion of the surface, the protective film comprising polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, Polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene Acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicon resin, polyphenylene oxide and polysulfone, and glass, quartz glass, alumina, A nucleic acid reaction tool which is at least one selected from the group consisting of sapphire, forsterite, silicon carbide and metal oxide.

[39] Further, a plurality of primer-immobilized regions arranged at or near the plurality of first electrodes on the at least one surface of the substrate, and each kind releasably in the primer-immobilized region. The nucleic acid reaction device according to [38], further comprising a plurality of primer sets fixed to the surface.

[40] The nucleic acid reaction tool according to [38] or [39], wherein the substrate has a plate-like shape.

[41] The nucleic acid reaction according to [40], wherein the second electrode is disposed independently of each other in a region different from a region where the first electrode is disposed on the at least one surface of the substrate. Ingredients.

[42] The method according to any one of [38] to [40], further comprising a covering attached to the base so as to cover the region including the probe nucleic acid immobilization region and the primer immobilization region. Nucleic acid reaction tool.

[43] comprising a base, a protective film covering the exposed portion of the at least one surface of the base, and a plurality of primer sets arranged independently on the protective film, the protective film comprising: Polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate , Polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicon resin, polyphenylene oxide and police Hong, as well as glass, quartz glass, alumina, sapphire, forsterite, nucleic acid reaction member is at least one selected from the group consisting of silicon carbide and metal oxides.

[44] The nucleic acid reaction tool according to any one of [38] to [43], wherein the protective film contains a novolac resin, an epoxy resin, a polyolefin resin, and a silicon resin.

[45] A nucleic acid detection channel, a first syringe for storing a nucleic acid sample, a second syringe for storing a chemical used for nucleic acid detection, a third syringe for storing a liquid flowing out of the nucleic acid detection channel, A first flow path that connects the first syringe, the second syringe, and the nucleic acid detection flow path; and a second flow path that connects the nucleic acid detection flow path and the third syringe; A flow path packing configured integrally;
A first plate made of a hard material and facing the first surface of the flow path packing;
A second plate made of a hard material, facing the second surface opposite to the first surface, and sealing the flow path packing together with the first plate;
A nucleic acid detection cassette comprising:

[46] The nucleic acid detection cassette according to [45], wherein the first syringe and the second syringe include a thin film part that can be easily deformed on the second surface.

[47] The lower plate includes a first opening at a position facing the first syringe, a second opening at a position facing the second syringe, and relative to the nucleic acid detection flow path. The nucleic acid detection cassette according to [46], further including a nucleic acid detection unit that detects a target nucleic acid at a position to be detected.

[48] The volume of the first syringe and the volume of the second syringe are substantially the same as the volume that fills the nucleic acid detection channel, the first channel, and the second channel, [47] The nucleic acid detection cassette according to [47].

[49] The first flow path includes a first check valve that prevents liquid from flowing into the first syringe, and a second check valve that prevents liquid from flowing into the second syringe. The nucleic acid detection cassette according to [48], further comprising a valve.

[50] A nucleic acid detection apparatus using the nucleic acid detection cassette according to any one of [47] to [49],
A stand for holding the nucleic acid detection cassette;
A first rod that pressurizes the first syringe through the first opening;
A second rod that pressurizes the second syringe through the second opening;
The first rod and the second rod are mounted so that the tip portion of the first rod is positioned closer to the nucleic acid detection cassette by a predetermined distance than the tip portion of the second rod, A moving mechanism capable of moving one rod and the second rod with respect to the nucleic acid detection cassette;
A nucleic acid detection apparatus comprising:

[51] The nucleic acid detection device according to [50], further including an elastic body having elasticity in a moving direction of the moving mechanism between the first rod, the second rod, and the moving mechanism.

Claims (15)

A support configured to support a liquid phase reaction field;
A plurality of primer-immobilized regions arranged independently of each other on at least one surface of the support in contact with the reaction field when the reaction field is formed by the liquid phase;
A plurality of types of primer sets that are releasably immobilized for each type in the plurality of primer immobilization regions, and are configured to amplify a plurality of types of target sequences, respectively.
A plurality of probe immobilization regions independently disposed at the same position or the vicinity thereof as the plurality of primer immobilization regions;
A plurality of types of probe nucleic acids including a complementary sequence of a target sequence, which is immobilized for each type, so that a hybridization signal can be independently detected for each probe-immobilized region in the probe-immobilized region;
A multi-nucleic acid reaction device comprising:   The multi-nucleic acid reaction tool according to claim 1, wherein the support has a container form or a flow path, and the inside of the container or the flow path is a reaction field.   The multi-nucleic acid reaction device according to claim 1, further comprising a thickener releasably immobilized on the primer immobilization region.   The multi-nucleic acid reaction tool according to claim 1, further comprising a covering body that is attached to the support body and forms a chamber for maintaining a reaction solution together with the support body.   The multi-nucleic acid reaction tool according to claim 1, wherein the support comprises a substrate. The support includes a groove portion on a first surface thereof,
The groove includes a flow channel chamber for a nucleic acid sample to react,
The cross-sectional area of the flow channel chamber is larger than the cross-sectional area of the groove other than the flow channel chamber,
The multi-nucleic acid reaction tool according to claim 5, which is a nucleic acid detection device.   The flow path type chamber has an injection part for adding a reaction solution upstream of the flow path, and a plurality of primer immobilization regions are arranged downstream of the injection part, corresponding to each primer immobilization area. The multi-nucleic acid reaction tool according to claim 6, wherein a plurality of probe immobilization regions are independently arranged in the vicinity of the downstream of each primer immobilization region. A sensor unit for nucleic acid detection formed on the support;
Wiring formed on the support and connected to the sensor;
A protective film formed on the support;
The multi-nucleic acid reaction device according to claim 5, further comprising:
The array-type primer probe chip is a nucleic acid detection device that detects a nucleic acid amplification product by the sensor unit after performing a nucleic acid amplification reaction in a chamber for the nucleic acid sample to react with the sensor unit,
The protective film includes one or more openings that expose a lower layer portion including a part of the substrate in a wetted region of the nucleic acid sample on the substrate.
Multi-nucleic acid reaction tool.   The multi-nucleic acid reaction tool according to claim 8, wherein the sensor unit is an electrode.   The multi-nucleic acid reaction tool according to claim 9, wherein the protective film covers the wiring in the liquid contact area.   The protective film is polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene. , Acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicon resin, polyphenylene oxide and polysulfone, and glass, quartz glass, alumina 9. At least one selected from the group consisting of sapphire, forsterite, silicon carbide and metal oxides Multi-nucleic acid reaction fixture of the mounting.   The multi-nucleic acid reaction device according to claim 8, wherein the protective film contains a novolac resin, an epoxy resin, a polyolefin resin, and a silicon resin. The support comprises a first plate made of a hard material;
A first syringe for storing a nucleic acid reaction channel; a second syringe for storing a chemical used for a nucleic acid reaction; a third syringe for storing a liquid flowing out of the nucleic acid reaction channel; A first flow path connecting the syringe, the second syringe, and the nucleic acid reaction flow path; and a second flow path connecting the nucleic acid reaction flow path and the third syringe; Flow path packing,
The array type primer probe chip according to claim 1, further comprising a second plate made of a hard material,
The first plate is opposed to the first surface of the flow path packing, and the second plate is opposed to the second surface opposite to the first surface, together with the first plate The multi-nucleic acid reaction tool according to claim 1, which is a nucleic acid reaction cassette that seals the flow path packing.   A substrate, a plurality of independent primer-immobilized regions on at least one surface of the substrate, and a plurality of types of target sequences that are releasably immobilized in the plurality of primer-immobilized regions for each type. A plurality of types of primer sets, a probe immobilization region at or near the same position as the plurality of primer immobilization regions, and a hybridization signal for each probe immobilization region in the probe immobilization region A multi-nucleic acid reaction device comprising at least one type of probe nucleic acid containing a complementary sequence of a target sequence, which is immobilized for each type so as to be independently detectable. (A) At least one surface of a support configured to support one reaction field in a liquid phase, and when the reaction field is formed by the liquid phase, the surfaces contacting the reaction field are mutually Immobilizing a plurality of types of primer sets for amplifying a plurality of types of target nucleic acids in each of a plurality of independent primer immobilization regions in a releasable manner for each type,
(B) The probe-immobilized region at or near the plurality of primer-immobilized regions includes a complementary sequence of the target sequence for each type so that a hybridization signal can be detected independently for each probe-immobilized region. Immobilizing at least one probe nucleic acid;
(C) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(D) bringing a sample into the reaction field;
(E) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field;
(F) independently detecting the presence or amount of hybridization between the amplification product obtained by the amplification reaction and the probe nucleic acid for the amplification product obtained by each of the primer sets;
(G) detecting or measuring the presence of the target nucleic acid containing the target sequence based on the result of (f),
A target nucleic acid detection method comprising:

Images