JP6026196B2 - Multi-nucleic acid reaction device, method for producing the same, and method for amplifying or detecting nucleic acid using the same - Google Patents

Description

  The present invention relates to a multi-nucleic acid reaction device, a method for producing the same, and a method for amplifying or detecting a nucleic acid using the same.

  Currently, with the advancement of genetic testing technology, genetic testing is being carried out in various situations such as clinical sites and hygiene management facilities. In these genetic tests, it may be useful for the first time by detecting a plurality of target genes and quantifying the amount of each gene contained in a sample. For example, for the purpose of identifying pathogens in the clinical field, a plurality of types of bacteria and viruses suspected of infection from each symptom or each bacteria and virus are detected, and their types are further determined as necessary. They are used for determining diseases, grasping disease states, determining the effects of drug treatments, and the like. Therefore, a technique for detecting a plurality of target genes is very important.

  Conventionally, when a plurality of target genes are detected, each sample nucleic acid is first amplified in a certain reaction solution, and then the amplified product is detected in a further reaction apparatus. Amplification is selected from either a method of preparing a reaction container for amplifying each target gene or a method of performing a multi-nucleic acid amplification reaction by putting reagents for detecting all target genes in one reaction container. Is done. The detection is generally performed by using an amplification product such as a DNA chip or electrophoresis.

  In such a 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 the test increase as the number of target genes increases. To do. 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. Such a situation is the same in the detection process. Furthermore, since the number of reaction containers and amplification and detection need to be performed in different reaction containers, there is a possibility that a sample will 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.

  The problem to be solved by the present invention is to provide a multi-nucleic acid reaction device capable of performing a plurality of amplification reactions in one reaction field, a method for producing the same, and a method for amplifying or detecting a nucleic acid using the same. .

  According to an embodiment, a multi-nucleic acid reactor is provided. The multi-nucleic acid reaction tool is a support configured to support a reaction field in a liquid phase; when the reaction field is formed by the liquid phase, the multi-nucleic acid reaction tool is provided on at least one surface of the support that is in contact with the reaction field. A plurality of primer immobilization regions arranged independently of each other; a plurality of amplification reagent immobilization regions arranged at or near the same position as each of the plurality of primer immobilization regions; types of the plurality of primer immobilization regions A plurality of types of primer sets that are releasably immobilized every time; and are releasably immobilized for each composition in the amplification reagent immobilization region so as to enable an amplification reaction for each of the plurality of types of primer sets. An amplification reagent having a plurality of types of compositions is provided.

The figure which shows the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the mode at the time of use of the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the example of the multinucleic acid reaction tool which concerns on embodiment, and the detection signal obtained by it. The figure which shows the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the chip | tip raw material of embodiment. The figure which shows the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the mode at the time of use of the multi-nucleic acid reaction tool which concerns on embodiment. The figure which shows the mode at the time of use of the multi-nucleic acid reaction tool which concerns on embodiment.

  Hereinafter, embodiments of the present invention will be described in detail.

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 reaction tool and method for detecting target nucleic acid using the same First, a multi-nucleic acid reaction tool for detecting a target nucleic acid will be described.

<First Embodiment>
An example of the multi-nucleic acid reaction tool will be described with reference to FIGS. 1 (a) and (b). This multi-nucleic acid reaction tool is an example of a multi-nucleic acid amplification reaction tool for multi-amplifying a plurality of types of target nucleic acids. Therefore, for a plurality of types of template nucleic acids to be amplified, a partial sequence of each of the template nucleic acids is set as a target sequence, and a primer set for amplifying the target sequence is designed. A multi-nucleic acid amplification reaction tool is provided with such a primer set.

  FIG. 1A is a perspective view of an example of a multi-nucleic acid amplification reaction tool. A multi-nucleic acid amplification 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. FIG. 1B is an enlarged schematic view of the primer immobilization region 4. As shown there, one kind of primer set 6 and amplification reagent 20 are immobilized in one primer immobilization region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 and amplification reagents 20 are fixed for each type.

  Plural types of primer sets 6 and amplification reagents 20 are prepared for amplifying a plurality of target nucleic acids. One primer set 6 and an amplification reagent 20 for amplifying a specific target nucleic acid are immobilized in one primer immobilization 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 amplification reagent 20 may include components necessary for amplification of the target sequence by the primer set 6 immobilized on each primer immobilization region 4. The amplification reagent 20 is, for example, a DNA synthesizing enzyme such as a polymerase, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, and reverse transcription when simultaneously performing reverse transcription. It may be an enzyme, a substrate necessary for the enzyme, an additive that affects amplification efficiency, a pH adjuster, a salt that affects amplification, a substance that affects enzyme activity, and combinations thereof.

  Here, the amplification reagent 20 including components necessary for amplification of the target sequence by the primer set 6 immobilized on each primer immobilization region 4 is selected in consideration of the following.

  When amplification reactions are performed using different primer sets for a plurality of types of target sequences in one reaction space, that is, in each reaction region included in one reaction field, the amplification reaction for each primer set is optimized. The difference in the appropriate conditions and the reaction efficiency based thereon is a problem. For example, in the case of the multi-nucleic acid reaction tool shown in FIG. 1, one space is a space defined inside the container-type support 2 and defined by the reaction solution to be contained therein. That is, in the embodiment shown in FIG. 1, in the case of a multi-nucleic acid reaction tool, the inside of the container-type support 2 functions as a reaction part. Plural kinds of primer sets are immobilized in one space, and they amplify each target nucleic acid independently of each other. Therefore, the optimum conditions for the amplification reaction using a plurality of types of primer sets contained in one space are different. If the optimum conditions for each primer set are not taken into account, a part of the amplification reaction to be performed by one space or a part thereof may not be obtained. Therefore, in order to ensure that all amplification reactions to be performed by one space are performed to the desired extent, each primer set is immobilized, released, diffused, and the desired reaction in each reaction region defined. An amplification reaction needs to be performed with efficiency. For that purpose, it is necessary to adjust so that the amplification reaction performed by each primer set is performed to a desired extent. For this purpose, an amplification reagent having a composition configured to adjust amplification efficiency may be immobilized in the same region as and / or in the vicinity of the region where each primer set is immobilized. Thereby, the amplification efficiency of the amplification reaction occurring in each reaction region can be adjusted.

  The desired degree of amplification reaction means, for example, that each amplification reaction performed by a plurality of primer sets included in one reaction field is performed with substantially the same amplification efficiency, and a plurality of amplification reactions included in one reaction field. Amplification reaction is performed under optimal conditions using a primer set, and amplification efficiency is adjusted so that each amplification reaction performed by a plurality of primer sets included in one reaction field is changed at a desired ratio. It may be that. In addition, an amplification reaction is performed under optimum conditions by a plurality of primer sets contained in one reaction field, that is, a plurality of primer sets contained in one reaction field amplify a target nucleic acid under optimum conditions. It may be that the amplification efficiency that would be obtained in some cases is achieved.

  In order to achieve such a desired degree of amplification reaction, the type and amount of the amplification reagent to be immobilized corresponding to each primer set and / or target sequence is selected, and the same position as the primer immobilization region and / or Alternatively, it may be fixed in the vicinity thereof. In parallel with the release and diffusion of the primer set, the immobilized amplification reagent is released and diffused, so that when a reaction solution is added to one reaction part, it is desired in each of the plurality of reaction regions. Reaction conditions are achieved, and each amplification reaction can be achieved under such reaction conditions.

  The reaction conditions adjusted by immobilization of the amplification reagent may be conditions such as the type of nucleic acid, the reaction temperature, the type and / or concentration of a substance necessary for the reaction. For example, when DNA and RNA are amplified in one reaction field, for example, a polymerase is fixed to a primer set for amplifying DNA and reverse transcriptase is used for a primer set for amplifying RNA. And the polymerase may be immobilized. Also, different amplification enzymes may be used depending on the respective sequences, in which case, depending on the type of enzyme, the type and / or amount of salt and buffer necessary for the enzyme to be activated, etc. These substances may be immobilized together with the enzyme. Such selection and determination of the composition of the amplification reagent may be performed according to the type and / or sequence of the target nucleic acid to be amplified and the composition, type and / or sequence of the primer set used. Such selection and determination can be made prior to the production of the multi-nucleic acid reactor, how much amplification efficiency can be obtained under specific conditions for the desired target nucleic acid and / or primer set, or under what conditions It may also include performing a preliminary test to clarify whether the desired amplification efficiency can be achieved.

  By immobilizing amplification reagents for such adjustments corresponding to primer sets, multi-amplification in which multiple amplification reactions are performed in one space can be adjusted in advance for any target nucleic acid and / or primer set. An amplification reaction can be performed under the reaction conditions.

  Additives that affect amplification efficiency include, for example, ammonium sulfate, bovine serum albumin, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), formamide, gelatin, glycerol, PEG, SDS, spermidine, Triton X-100, Urea, BSA, Tween 20, and RT-mate may be used. The pH adjuster may be, for example, ADA, PEPES, POPSO, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, HEPPSO, EPPS, Tricine, Bicine, and Tris-HCl. The salt may be, for example, sodium chloride, sodium citrate, potassium chloride, sodium acetate, sodium glutamate, potassium glutamate and potassium acetate. Substances that affect enzyme activity may be magnesium chloride, magnesium sulfate, magnesium acetate, dithiothreitol (DTT), manganese chloride, and the like. The reagents used may be used alone or in combination of two or more of these substances. However, the substance used as the amplification reagent 20 is not limited to these.

  Furthermore, in order to retain the activity of DNA synthase and reverse transcriptase, a protein stabilizer may be immobilized as an amplification reagent during drying. The presence of the stabilizing agent stabilizes a protein such as an amplification enzyme, and the amplification efficiency is higher than that in a reaction region where no stabilizing agent is present. Examples of such stabilizers include, but are not limited to, for example, proteins such as sugars, nonionic surfactants, salts, sugar alcohols, polymers, amino acids, substrates, osmolytes and BSA. It may be present, and trehalose is preferable.

  Furthermore, in order to achieve almost the same amplification efficiency for all of the immobilized primer sets, for example, a primer set in which the amplification reaction proceeds with an outstanding amplification efficiency compared to other primer sets is inhibited. The agent may be immobilized.

  The amplification inhibitor to be immobilized may be any amplification inhibitor known per se, such as heparin, ethanol, urea, DMSO, DMF, formamide, SDS, salts, saccharides, surfactants and dyes. It may be.

  The primer set 6 and the amplification reagent 20 are fixed to the immobilization region 3 so as to be released in contact with a liquid phase for providing a reaction field. For example, when a plurality of primer sets are immobilized independently of each other, reaction regions defined by the release and diffusion of each primer set are formed and maintained independently of each other.

  Immobilization of the primer set 6 and the amplification reagent 20 to the primer immobilization region 4 is performed by, for example, dropping a solution containing one set of the primer set 6 and the amplification reagent 20 into one primer immobilization region 4 and then drying it. Can be achieved. Similarly, for the other primer immobilization regions 4, a solution containing the desired primer set 6 and amplification reagent 20 is dropped and dried, and a desired number of primer sets 6 and amplification reagent 20 are added. What is necessary is just to fix to the support body 2. As a result, the primer set 6 and the amplification reagent 20 are immobilized on all the primer immobilization regions 4 that are independently arranged on one inner bottom surface 3 of the support 2. However, the primer set 6 and the amplification reagent 20 may be immobilized on the primer immobilization region 4 in such a manner that they 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 case of a method of dropping a solution containing the primer set 6 and the amplification reagent 20, the solvent of the solution containing the primer set 6 and the amplification reagent 20 may be, for example, water, a buffer solution or an organic solvent. It is not limited to.

  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 the reaction regions included in one reaction field are arranged at intervals that do not interfere with the amplification that starts and / or proceeds for each primer set 6 and amplification reagent 20. It is.

  The liquid phase for providing the reaction field may be any liquid phase that allows the amplification reaction to proceed after the immobilized primer 6 and amplification reagent 20 are released. For example, for the desired amplification. It may be a necessary reaction solution.

  The support in the form of a container 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 may be any material that does not itself participate in the reaction, and any material that can perform the amplification reaction there may be used. For example, it may be arbitrarily selected from silicon, glass, resin and metal. Further, any commercially available container may be used as the support in the container form.

  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, the ceiling surface provided by any covering, or the like may be disposed.

  In the above description, an example in which the primer set 6 and the amplification reagent 20 are immobilized simultaneously has been shown. However, when the amplification reagent 20 is immobilized on the support 2, the amplification reagent 20 may be immobilized before the primer set 6 or at the same time as the primer set 6. It may be after the set 6 is fixed.

  When the amplification reagent 20 is immobilized on the support 2 simultaneously with the fixation of the primer set 6, the amplification reagent 20 may be dissolved in the solution in which the primer set 6 is dissolved. Alternatively, first, the amplification reagent 20 may be prepared as a solution and mixed with a solution in which the separately prepared primer set 6 is dissolved. What is necessary is just to fix the solution containing the obtained primer set 6 and amplification reagent 20 by dripping and drying.

  Further, in the above description, the example in which the primer set 6 and the amplification reagent 20 are immobilized on the primer immobilization region 4 has been described. However, the primer set 6 is immobilized on the primer immobilization region 4 and the amplification reagent. 20 may be immobilized in the vicinity of the primer immobilization region 4. That is, the amplification reagent 20 may be immobilized in the same region as the primer immobilization region 4 or may be immobilized in the vicinity of the primer immobilization region 4. The vicinity of the primer immobilization region 4 may be within a range in which amplification of a specific target nucleic acid is independently performed by the primer set 6 released and diffused from the primer immobilization region 4. When the amplification reagent 20 is immobilized at a position different from the primer immobilization region 4, the region where the amplification reagent 20 is immobilized may be arranged as the amplification reagent immobilization region.

  For example, the vicinity of the primer immobilization region 4 may be 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 more, preferably 100 μm -10 mm.

  Further, when the primer set 6 and the amplification reagent 20 are immobilized, a thickener may be used together. By using a thickener, the primer set 6 and the amplification reagent 20 can be stably immobilized. When a thickener is used in immobilizing the primer set 6 and the amplification reagent 20, the thickener is further dissolved in a solution containing the primer set 6 and the amplification reagent 20, and the resulting liquid is spotted on the support 2. And may be dried. Alternatively, the thickener may be further dissolved in a solution containing each of the primer set 6 and the amplification reagent 20, and the obtained liquid may be spotted on the support 2 and dried. Alternatively, the thickener may be further dissolved in either the solution containing the primer set 6 or the solution containing the amplification reagent 20, and the resulting liquid may be spotted on the support 2 and dried. Alternatively, after the primer set 6 and the amplification reagent 20 are immobilized, a thickener may be spotted so as to cover the immobilized primer set 6 and the amplification reagent 20 and dried.

  Thickeners are, for example, thickeners derived from sap, thickeners derived from seeds such as beans, thickeners derived from seaweed, thickeners derived from fruits, leaves and rhizomes, microorganisms, etc. These thickeners, as well as other thickener stabilizers, may be used alone or in combination of two or more selected from those thickeners.

  The thickener derived from sap may be, for example, almond gum, Elemi resin, dammar resin, gum arabic, karaya gum, tragacanth gum, arabinogalactan, gati gum and peach resin. Thickeners derived from seeds such as beans include, for example, ama seed gum, gua gum enzymatic degradation product, tamarind seed gum, cassia gum, silium seed gum, tara gum, carob bean gum = locust bean gum, mackerel mugwort seed gum, triacanthus gum, gua gum And sesbania gum. The thickener derived from seaweed may be, for example, alginic acid, fucronori extract, far selelain, carrageenan and the like. Thickeners derived from fruits, leaves, rhizomes and the like may be, for example, aloe vera extract, kidachi aloe extract, pectin, okra extract, troro mallow and the like. Thickeners derived from microorganisms include, for example, Aeromonas gum, Enterobacter gum, Natto gum, Aureobasidium culture solution, curdlan, pullulan, Azotobacter vinelandie gum, xanthan gum, macrohomopsis gum, welan gum, gellan gum, lambzan gum, Erwinia It may be mitsuen cis gum, sclerogum, levan, Enterobacter simanas gum, dextran and the like. Other thickening stabilizers may be, for example, yeast cell membrane, chitin, oligoglucosamine, microfibrous cellulose, chitosan, microcrystalline cellulose and glucosamine. The thickener may be a thickener generally used as a food or drink additive. Such thickeners may be, for example, agar, soy polysaccharides, nata de coco, starch, konjac potato extract, gelatin and the like. Preferred imaging agents include, but are not limited to, agar such as agarose and / or gelatin, polyethylene glycol and the like.

  When the amplification reagent 20 is fixed before or after the primer set 6 is fixed, the amplification reagent 20 is dropped, sprayed, printed, applied by brush, or the support is immersed in the amplification reagent 20, and then the primer is fixed by drying or the like. What is necessary is just to fix | immobilize on the surface of the support body 2 containing the formation area | region 4. FIG.

  As a method for drying the solution containing the primer set and the amplification reagent, 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.

  By providing the primer set 6 and the amplification reagent 20 to one reaction unit for immobilization, a decrease in sensitivity in the multi-amplification reaction, restriction on the type of amplification, and the like are suppressed. Conventionally, reagents other than the primer set had to be performed at a uniform concentration. However, since it is possible to immobilize a necessary amount of the necessary reagent for each primer set and / or target nucleic acid, Not only can the characteristics of the amplification reaction be improved, but also costs such as production costs and / or running costs can be reduced.

<Second Embodiment>
(1) Multi-nucleic acid reaction tool An example of a multi-nucleic acid reaction tool will be described with reference to FIGS. 2 (a), (b) and (c). This multi-nucleic acid reaction tool 1 detects hybridization of a plurality of target sequences and probe nucleic acids complementary to the respective target sequences by detecting a hybridization signal generated by the hybridization, and based on the detection, the target sequence For quantifying a template nucleic acid having a target sequence comprising For this purpose, for a plurality of types of template nucleic acids to be detected, a part of each sequence is set as a target sequence, and a primer set for amplifying the target sequence is designed. Furthermore, a part of the target sequence is set as a target sequence, and a probe nucleic acid having a sequence complementary to the target sequence is designed.

  FIG. 2A is a perspective view of an example of a multi-nucleic acid reaction tool. A multi-nucleic acid reaction tool 1 shown in FIG. 2A 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 in the vicinity of the plurality of primer immobilization regions 4 and corresponding to the respective primer immobilization regions 4. A plurality of primer sets 6 immobilized on the primer immobilization regions 4 arranged independently of each other are subjected to amplification reaction by each primer set 6 independently. For example, when a plurality of primer sets 6 are immobilized independently of each other, reaction regions defined by the release and diffusion of each primer set are formed and maintained independently of each other. Further, the amplification reagent 20 is immobilized together with the primer set 6 in the primer immobilization region 4.

  FIG. 2B 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, and an amplification reagent 20 (not shown) is fixed. 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 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 and the amplification reagent 20 are 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. The primer set 6 and the amplification reagent 20 are fixed to the primer immobilization region 4 by, for example, dropping a solution containing one set of primer set and the amplification reagent 20 into one primer immobilization region 4 and then drying the solution. It is possible to achieve. Further, similarly, a solution containing a desired primer set 6 and amplification reagent 20 is dropped and dried for each of the other primer-immobilized regions 4, and a desired number of primer sets 6 and amplification reagents 20 are placed on the support 2. What is necessary is to fix to. As a result, the primer set 6 and the amplification reagent 20 are immobilized on all the primer immobilization regions 4 that are independently arranged on one surface of the support 2. However, the primer set 6 and the amplification reagent 20 may be immobilized on the primer immobilization region 4 in such a manner that they 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 case of a method of dropping a solution containing the primer set 6 and the amplification reagent 20, the solvent of 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 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 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 may be any material that does not itself participate in the reaction, and any material that can perform the amplification reaction there may be used. For example, it may be arbitrarily selected from silicon, glass, resin and metal. Further, any commercially available container may be used as the support in the container form.

  FIG. 2 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 the primer immobilization region 4 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 any covering.

  FIG. 2C is an enlarged view of the probe immobilization region 5 disposed 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 support surface. Good. 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. A plurality of probe immobilization regions 5 may be arranged for one primer immobilization region 4.

(2) Nucleic Acid Amplification Detection Method Using Multi-Nucleic Acid Reactor FIG. 3 shows a reaction field after a nucleic acid amplification reaction performed using the same multi-nucleic acid reactor 1 as in the second embodiment. It is a schematic diagram. 3 (a-1) and 3 (b-1) show the multi-nucleic acid reaction tool 1 before the reaction. A probe immobilization region 5 is disposed in the vicinity of the plurality of primer immobilization regions 4 disposed on the inner bottom surface 3 of the support 2. A plurality of primer sets 6 and amplification reagents 20 are respectively fixed to the plurality of primer immobilization regions 4. Corresponding to each primer immobilization region 4, a plurality of probe nucleic acids 7 are immobilized for each desired type in the probe immobilization region 5 arranged in the vicinity thereof.

  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. 3 (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 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 sample to the reaction solution 8 in advance before adding the reaction solution 8 to the multi-nucleic acid reaction device 1, and after adding the reaction solution 8 to the multi-nucleic acid reaction device 1. Alternatively, the sample may be added to the multi-nucleic acid reaction device 1 before the reaction solution 8 is added to the multi-nucleic acid reaction device 1.

  As shown in FIGS. 3 (a-2) and (b-2), in the multi-nucleic acid reaction tool 1 after the addition of the reaction solution 8, schematically shown in FIGS. 3 (a-3) and (b-3). As shown, the primer set 6 fixed to the inner bottom surface 3 is released and gradually diffuses. 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 then the 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. Here, the “reaction field” is theoretically referred to as 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 9, the region 9 is interpreted as the reaction region 10. The diagram of FIG. 3A-3 is a schematic diagram when an amplification reaction is caused by the primer set 6 fixed to all the primer fixing regions 4. FIG. FIG. 3 (b-3) shows a part of all the primer-immobilized regions fixed to the inner bottom surface 3 by the fixed primer set 6, and FIG. 3 (b-3) shows that only three regions are amplified. It is a schematic diagram when it occurs.

  When the nucleic acid containing the target sequence is present in the amplification product amplified in the region 9, the probe-immobilized region 5 hybridizes with the nucleic acid. The probe nucleic acid 7 immobilized on the probe immobilization region 5 is immobilized so as to hybridize only with the amplification product in the corresponding primer immobilization region 4. That is, the probe nucleic acid 7 immobilized on one probe immobilization region 5 is maintained at a distance so as to hybridize only with the amplification product in the corresponding primer immobilization region 4, and each probe immobilization region 5 and primer immobilization are maintained. The conversion area 4 is arranged.

  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 inside of the multi-nucleic acid reaction tool 1 is washed, or may be carried out 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 78 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 the inside of the multi-nucleic acid reaction tool 1 is washed, 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 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 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 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. 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-immobilized regions 4 arranged in one array type 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 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 It may be 10,000 or less, 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 controls and / or negative controls may be provided for primer sets and / or probe nucleic acids.

  In the above example, only the primer set 6 is immobilized on the primer immobilization region 4. However, the present invention is not limited to this, and other components necessary for amplification, for example, an enzyme such as a polymerase or a reverse transcriptase, a substrate, 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 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 preferable to use the board | substrate which has a flow path as the support body 71 like 4th Embodiment mentioned later.

<Third Embodiment>
Next, the principle of a multi-nucleic acid reaction tool for detecting a plurality of target nucleic acids will be described.

  One example of the multi-nucleic acid reaction tool will be described with reference to FIG.

  The multi-nucleic acid reaction tool 1 illustrated in FIG. 4A is a chip-type multi-nucleic acid reaction tool, that is, an array-type primer probe chip 1. This array type primer probe chip 1 detects a hybridization between one target sequence and a probe nucleic acid by detecting a hybridization signal generated by the hybridization, and based on that, a template having a target sequence including the target sequence It is for quantifying nucleic acids. For this purpose, a partial sequence of the template nucleic acid to be quantified is set as a target sequence, and a primer set for amplifying the target sequence is designed. Furthermore, a part of the target sequence is set as a target sequence, and a probe nucleic acid having a sequence complementary to the target sequence is designed.

  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. Corresponding to each primer immobilization region 4, a probe immobilization region 5 is arranged in the vicinity thereof.

  In the primer immobilization region 4, one kind of primer set 6 and an amplification reagent 20 designed so that an amplification reaction using each primer set is performed under the optimum conditions for one primer immobilization region 4 are immobilized. Is done. In order to quantify a specific target sequence, a primer set 6 for amplifying a specific target sequence including the specific target sequence is used.

  Five types of primer sets 6 necessary for amplifying different template nucleic acids are immobilized on the five primer immobilization regions 4 included in the array type primer probe chip 1 shown in FIG.

  The composition and amount of the amplification reagent 20 that is immobilized so that the amplification reaction is performed under the optimum conditions for each primer set is different for each primer set. When the liquid mixture for immobilizing each amplification reagent 20 is indicated as composition A, B, C, D and E, respectively, composition A, composition B, composition C, composition D and composition E depend on each primer set. The composition is necessary to make the amplification reaction performed appropriately.

  A plurality of probe nucleic acids 7 including sequences complementary to five types of target sequences are immobilized on the probe nucleic acid 7 immobilized on the probe immobilization region 5. That is, five types of probe nucleic acids 7 necessary for detecting different target sequences are immobilized on the five probe immobilization regions 5 included in the array-type primer probe chip 1 of FIG. .

  The surface 3 of the array-type primer probe chip 1 is brought into contact with the sample so that all the primer-immobilized regions 4 and all the probe-immobilized regions 5 of the array-type primer-probe chip 1 are included. The soybean is detected.

  For example, the amplification reaction and hybridization may be detected by placing the array-type primer probe chip 1 on a flat surface and placing the sample on the surface 3 of the array-type primer probe chip 1. Or you may arrange | position in the container which can maintain this array type primer probe chip 1, for example. A reaction field is formed by adding a reaction solution into the container.

  For example, except that the amplification reagent 20 is not immobilized, an array-type primer probe chip for comparison, which is designed in the same manner as the above-described array-type primer probe chip 1, that is, designed to obtain optimum conditions. In the case of a non-arrayed primer probe chip, since the amplification efficiency differs, a detection signal as shown in FIG. 4B is obtained. That is, the hybridization signals detected by each probe are disjoint.

  However, in the case of the array type primer probe chip of the embodiment designed so as to obtain the optimum condition as described above, it is possible to obtain hybridization signals having substantially the same magnitude as shown in FIG. It becomes possible. In this example, a current value is used as an example of the hybrid signal.

  Optimal conditions for each primer and / or target nucleic acid may be selected by conducting a preliminary test prior to manufacturing the array-type primer probe chip. In addition, the examination of the kind and amount of the amplification reagent for obtaining such optimum conditions may also be selected by conducting a preliminary test prior to manufacturing the array type primer probe chip.

  By designing in this way, it becomes possible to detect a plurality of types of nucleic acids with high accuracy using an array type primer probe chip.

  The selection of the amplification reagent to achieve the indicated condition may change the amount of the amplification reagent to be immobilized, and may change the configuration of the amplification reagent to be immobilized (that is, the combination of components used). The kind of amplification reagent to be immobilized may be changed, or an amplification inhibitor may be added to the amplification reagent to be immobilized. An amplification accelerator may be added to the amplification reagent to be immobilized. However, the amount of the amplification inhibitor to be immobilized needs to be an amount that does not completely suppress the amplification.

  By immobilizing the amplification inhibitor in different amounts, a difference occurs in amplification efficiency in each primer immobilization region. Thereby, similar amplification efficiencies may be achieved for all primer sets.

<Fourth Embodiment>
An example of the multi-nucleic acid reaction tool 1 for quantifying a template nucleic acid having a plurality of target sequences will be described with reference to FIG. This multi-nucleic acid reaction tool 1 is for detecting the hybridization of a plurality of target sequences and probe nucleic acids complementary to them by detecting a hybridization signal generated by the hybridization. Therefore, for a plurality of template nucleic acids to be detected, a part of each sequence is set as a target sequence, and a primer set for amplifying the target sequence is designed. Furthermore, a part of each target sequence is set as a target sequence, and a probe nucleic acid having a complementary sequence of each target sequence is designed.

  FIG. 5A is a perspective view of an example of a multi-nucleic acid reaction tool. A multi-nucleic acid reaction tool 1 shown in FIG. 5A 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 in the vicinity of the plurality of primer immobilization regions 4 and corresponding to the respective primer immobilization regions 4.

  FIG. 5B is an enlarged schematic view of the primer immobilization region 4. As shown there, one kind of primer set 6 is immobilized in one primer immobilization region 4. In each of the plurality of primer immobilization regions 4, a plurality of primer sets 6 are immobilized for each type. Although not shown, at the same time as each primer set 6, amplification reagents are immobilized so that optimum amplification conditions are achieved for each primer set.

  Plural types of primer sets 6 are prepared for amplifying a plurality of target nucleic acids. Further, the same primer set 6 is immobilized on three primer immobilization regions in order to obtain three hybridization signals for each target nucleic acid (referred to as “one series”). In addition, the amplification reagent 20 is immobilized simultaneously with the primer set 6. The same plurality of amplification reagent immobilization regions 4 are set. Probe nucleic acids 7 having the same sequence are immobilized on the probe immobilization region 5 corresponding to one series of primer immobilization regions.

  For primer sets 6 of different series, amplification reagents selected so that the amplification reaction by each series is performed under optimum conditions are immobilized. The selection of the amplification reagent so that the amplification reaction is performed under the optimum conditions can be performed by changing the type, concentration and / or configuration of the immobilized amplification reagent as described in the above embodiment. May be done.

  FIG. 5C is an enlarged view of the probe immobilization region 5 disposed in the vicinity of the amplification reagent 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. After the probe nucleic acid 7 is immobilized, the primer set 6 and the amplification reagent 20 may be immobilized, and after the primer set 6 and the amplification reagent are immobilized, the probe nucleic acid 7 may be immobilized. The primer set 6 and the amplification The immobilization of the reagent 20 and the immobilization of the probe nucleic acid 7 may be performed simultaneously.

  FIG. 5D schematically shows the types of the primer set 6, the amplification reagent 20, and the probe nucleic acid 7 that are immobilized. Three types of primer sets 6 of the first series, the second series, and the third series are immobilized on the multi-nucleic acid reaction tool 1. In the first series, in order to quantify the first target nucleic acid, the composition of the amplification reagent in which the same kind of primer set 6a is immobilized and the resulting amplification is performed under the optimum conditions A is designed. In the second series, in order to quantify the second target nucleic acid, the composition of the amplification reagent in which the same kind of primer set 6b is immobilized and the resulting amplification is performed under optimum conditions B is designed. In the third series, in order to quantify the third target nucleic acid, the composition of the amplification reagent in which the same kind of primer set 6c is immobilized and the resulting amplification is performed under optimum conditions C is designed.

  Probe immobilization regions 5 having different sequences for each series are arranged at positions corresponding to the primer immobilization regions 4 to which the first series, second series, and third series primer sets 6 are respectively immobilized. Is done. That is, the probe nucleic acid 7a for the first series has a sequence complementary to the first target sequence which is at least a partial sequence of the first target nucleic acid. The probe nucleic acid 7b for the second series has a sequence complementary to the second target sequence, which is the sequence of at least a part of the second target nucleic acid. The probe nucleic acid 7c for the third series has a sequence complementary to the third target sequence, which is at least a partial sequence of the third target nucleic acid.

  The primer set 6a for amplifying the first target nucleic acid is fixed to the primer fixing region 4 arranged in the same row as shown in FIG. 5 (d). In the probe immobilization region 5 corresponding to these, at least a part of the sequence contained in the amplification product generated by amplification of the first target nucleic acid is used as a target sequence (first target sequence), and complementary to the target sequence. The probe nucleic acid 7a containing the correct sequence is immobilized.

  The probe nucleic acids 7a immobilized on the probe immobilization region 5 corresponding to the first series of primer immobilization region 4 are all for detecting the same target sequence. Therefore, the probe nucleic acid 7a immobilized on the probe immobilization region 5 includes a sequence complementary to one target sequence (first target sequence).

  Similarly, a primer set 6b for amplifying the second target nucleic acid is immobilized on the plurality of primer immobilization regions 4 included in the second series. Therefore, the probe nucleic acid 7b immobilized on the probe immobilization region 5 includes a sequence complementary to one target sequence (second target sequence).

  Furthermore, a primer set 6c for amplifying the third target nucleic acid is immobilized on the plurality of primer immobilization regions 4 included in the third series. Accordingly, the probe nucleic acid 7c immobilized on the probe immobilization region 5 includes a sequence complementary to one target sequence (third target sequence).

  A method for detecting three types of template nucleic acids using such a multi-nucleic acid reaction tool 1 is performed as follows. A reaction solution containing a sample and any further amplification reagent is added to the multi-nucleic acid reaction tool 1. Thereafter, an amplification reaction is performed, and a hybridization reaction between the generated amplification product and the probe nucleic acid 7 is performed. Next, the generated hybridization signal is detected.

  In the above example, the number of template nucleic acids quantified by the multi-nucleic acid reaction tool 1 is three, and a configuration for obtaining three examples of hybridization signals has been shown. However, the number of primer immobilization regions and probe immobilization regions may be changed, and the number of template nucleic acids to be detected may be one or more, two or more, or three or more. As a result, a desired number of nucleic acids can be detected by one multi-nucleic acid reaction device.

  Such a multi-nucleic acid reaction tool makes it possible to detect a plurality of nucleic acids simply and with high accuracy.

<Fifth Embodiment>
A further example of the multi-nucleic acid reaction device will be described with reference to FIG. The multi-nucleic acid reaction tool 1 shown in FIG. 6 is an example using a support having a flow path. 6A is a plan view of the multi-nucleic acid reaction tool 1, and FIG. 6B is a cross-sectional view of the multi-nucleic acid reaction tool 1 cut along a line mm ′.

  The multi-nucleic acid reaction tool 1 has a flow channel 41 formed inside the support 2, a plurality of mutually independent primer-immobilized regions 50 arranged on the bottom surface 42 of the flow channel 41, and can be liberated for each type. A plurality of primer sets 6 and amplification reagents 51 immobilized on the probe, a probe immobilization region 5 arranged at the same position as the plurality of primer immobilization regions 50, and a probe nucleic acid 7 immobilized thereon.

  The multi-nucleic acid reaction tool 1 is for detecting a plurality of target nucleic acids. Accordingly, a plurality of primer sets 6 for amplifying at least a part of each of the plurality of target nucleic acids is prepared. Furthermore, amplification reagents having a plurality of types of compositions are designed and prepared for each primer set 6 so that the amplification reaction using these primer sets 6 is performed under optimum conditions. The design of the amplification reagent for performing the amplification reaction by each primer set 6 under optimum conditions is performed by a preliminary test for each type of primer set 6. In order to achieve the optimum conditions, for example, the presence / absence of use of an amplification reagent, the type, amount and combination of amplification reagents used are adjusted among a plurality of types of primer sets.

  The plurality of primer sets 6 are immobilized along the flow direction of the flow path 41 of the multi-nucleic acid reaction tool 1 for each type of target nucleic acid to be amplified.

  A plurality of types of probe nucleic acids 7 are prepared so as to hybridize with a plurality of types of amplification products formed by amplification with the primer set 6. The plurality of probe nucleic acids 7 are immobilized at the same positions as the corresponding primer immobilization regions 50 along the flow direction of the flow path 41 of the multi-nucleic acid reaction tool 1.

  As described above, in this embodiment, the probe nucleic acid 7, the primer set 6, and the other amplification reagents 51 are immobilized at the same position. In this case, the probe nucleic acid 7 remains immobilized on the support 2 from the addition of the reaction solution to the end of detection, and the primer set 6 and the amplification reagent 51 are released by contact with the added reaction solution. Need to be immobilized as follows. For this purpose, for example, the probe nucleic acid 7 may be first immobilized on the support 2 and then the primer set 6 and the amplification reagent 51 may be immobilized releasably.

  This multi-nucleic acid reaction tool is manufactured using, for example, a first substrate and a second substrate. First, a plurality of types of probe nucleic acids 7 are immobilized on a predetermined probe immobilization region 5 on the first substrate. Next, a plurality of primer sets 6 and amplification reagents 51 are releasably immobilized in the primer immobilization region 50. The primer set 6 and the amplification reagent 51 are immobilized for each kind of primer and each kind of composition.

  On the other hand, a recess 43 is formed in the second substrate so as to correspond to the shape of the desired flow path 41. The recess 43 can be formed by a method known per se according to the material of the substrate used. Further, the positions where the primer immobilization region 50 and the probe immobilization region 5 are arranged are within the flow path formed by the concave portion 43 formed on the second substrate and the surface of the second substrate facing the concave portion 43. To be included.

  Next, the first substrate and the second substrate are integrated. By integration, the flow path 41 is formed by the inner wall of the recess 43 and the surface of the first substrate. This channel 41 is a channel-shaped reaction part. The two substrates are integrated so that the concave portion 43 of the second substrate faces the first substrate side. Further, the through holes 72 and 73 may be provided in a portion corresponding to the end of the concave portion 43 of the second substrate. The through holes 72 and 73 may be used as an inlet / outlet for 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, the example in which the primer set, the amplification reagent, and the probe nucleic acid are immobilized on the channel bottom surface of the support having the channel is shown, but the arrangement and shape of the channel are not limited thereto. . The surface on which the probe set, amplification reagent, and probe nucleic acid are immobilized may be any surface that defines the flow path, and the probe set, amplification reagent, and probe nucleic acid are immobilized on all surfaces that define the flow path. It may be fixed to a plurality of surfaces. Furthermore, the primer set, amplification reagent, and probe nucleic acid may be immobilized in different regions. The primer set and the amplification reagent are immobilized in the same region, and only the probe nucleic acid is immobilized in different regions. May be.

  In addition, in the production of the multi-nucleic acid reaction tool, the first substrate on which the channel is formed by forming a recess or a groove in advance, with respect to the primer immobilization region disposed on a part of the wall surface of the channel A plurality of primer sets and amplification reagents may be immobilized independently. Thereafter, a multi-nucleic acid reaction tool may be manufactured by attaching a lid such as silicon rubber.

  With such a multi-nucleic acid reaction tool, an amplification reaction is performed for each target nucleic acid under optimum conditions for each target nucleic acid, and each reaction product is detected, thereby detecting a plurality of target nucleic acids simply and with high accuracy. It becomes possible to do.

<Sixth Embodiment>
An array-type primer probe chip according to a sixth embodiment will be described with reference to FIGS. This array type primer probe chip is for detecting two specific types of nucleic acids in a sample.

(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. 7 (a) and 7 (b). FIG. 7A is a plan view of the chip material, and FIG. 7B 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. 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 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 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 and the amplification reagent 20a are immobilized on the first primer immobilization region 203a, and the second primer set 204b and the amplification reagent 20b are immobilized on the second primer immobilization 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 composition of the amplification reagent 20a is designed so that the amplification reaction by the first primer set 204a is performed under optimum conditions. The composition of the amplification reagent 20b is designed so that the amplification reaction by the second primer set 204b is performed under optimum conditions.

  Immobilizing the first and second primer sets 204a and 204b and the amplification reagents 20a and 10b to the first and second primer immobilization regions 203a and 203b, respectively, can be performed using, for example, water, a buffer solution, or an organic solvent. In such a solvent, each primer set 204 and the corresponding amplification reagent 20 are added dropwise, and then, for example, a time until drying under an appropriate temperature condition such as room temperature, for example, 10 minutes in the case of room temperature. To do.

(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. 9 and FIG.

  9A is a plan view of the array-type primer probe chip 1 in use, and FIG. 9B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 9A. 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 an oxynucleoside which is necessary for forming a new polynucleotide chain starting from a sample and a reagent necessary for amplification other than the immobilized amplification reagent, for example, an enzyme such as a polymerase and a primer. When performing reverse transcription simultaneously with a substrate such as triphosphate, a reverse transcriptase and a substrate necessary therefor, a buffer such as salts for maintaining an appropriate amplification environment, and, for example, Hoechst 33258 Includes an intercalator that recognizes double-stranded nucleic acid and generates a signal. When a template nucleic acid containing a target sequence to be amplified by a primer set immobilized on a specific primer immobilization region is present in the sample to be examined, the primer immobilization region and the corresponding probe immobilization region An amplification product is formed in the reaction region containing This is schematically shown in FIG.

  FIG. 10A 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. 10A is a plan view of the array-type primer probe chip 1 in use, and FIG. 10B 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. 9 contained a nucleic acid containing a sequence that can be bound by the second primer set 204b, and as shown in FIGS. 10 (a) and 10 (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.

  This array-type primer probe chip is a device for amplifying and detecting two kinds of specific nucleic acids in a sample, and is provided with two electrodes for that purpose. However, by increasing the number of electrodes to increase the probe-immobilized region and the corresponding primer-immobilized region, it is possible to similarly amplify and detect two or more types of nucleic acids.

  According to such an embodiment, it becomes possible to amplify a plurality of nucleic acids simply and with high accuracy, and to detect an amplification product generated by the amplification.

<Method for producing multi-nucleic acid reaction device>
Moreover, the manufacturing method of a multi-nucleic acid reaction tool is provided as further embodiment. As one example, the manufacturing method includes:
Multiple types of target nucleic acids, multiple types of primer sets for amplifying the multiple types of target nucleic acids, respectively, and amplification of multiple types of compositions designed to perform amplification using the respective primer sets under optimum conditions Providing a reagent and a support configured to support a liquid phase reaction field;
In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently of each other on at least one surface of the support that is in contact with the reaction field,
Arranging a plurality of amplification reagent immobilization regions at or near the same position as each of the plurality of primer immobilization regions;
Immobilizing each primer set in each of the plurality of primer immobilization regions in a releasable manner; and immobilizing an amplification reagent in each of the plurality of amplification reagent immobilization regions.

By way of further example, a method for producing a multi-nucleic acid reaction device includes:
A plurality of types of target nucleic acids, a primer set for amplifying each of the plurality of types of target nucleic acids, and amplification reagents of a plurality of types designed to perform amplification with each of the primer sets under optimum conditions; A plurality of types of probe nucleic acids containing sequences complementary to at least a part of an amplification product generated by each of the target nucleic acid and the primer set, and a support configured to support a liquid phase reaction field To prepare,
In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently of each other on at least one surface of the support that is in contact with the reaction field,
Arranging a plurality of probe immobilization regions at or near the same position as each of the plurality of primer immobilization regions;
The primer set and amplification reagent can be releasably immobilized for each type in each of the plurality of primer immobilization regions, and the hybridization signal can be independently detected in each of the plurality of probe immobilization regions. Immobilize each probe nucleic acid.

Here, a probe nucleic acid containing a sequence complementary to at least a part of the specific target sequence is immobilized at the same position as or near the region where the primer set for amplifying the specific target sequence is immobilized. . That is, by hybridizing with the target sequence contained in the amplification product obtained using the target nucleic acid as a template at or near the position where the primer set for amplifying the target nucleic acid to be amplified is immobilized. A probe nucleic acid for detecting the target nucleic acid is immobilized. In other words, an amplification product generated by amplification in one reaction region is detected only when a probe nucleic acid capable of detecting it is present in the same reaction region. Therefore, the primer set and probe nucleic acid corresponding to each other are immobilized so that they can exist in the same reaction region.

  Such a production method provides a device that can achieve a plurality of amplification reactions in one reaction field and detect the resulting amplification product to quantify nucleic acids in a sample.

<Nucleic acid amplification method>
A method for amplifying a plurality of target nucleic acids using a multi-nucleic acid reactor as described above by way of example is also provided as a further embodiment. Such a method may include, for example, the following steps.

(A) a plurality of types of target nucleic acids, a plurality of types of primer sets for amplifying the plurality of types of target nucleic acids, respectively, and a plurality of types of primers designed to perform amplification with the respective primer sets under optimum conditions Providing an amplification reagent of composition and a support configured to support a liquid phase reaction field;
(B) In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently from each other on at least one surface of the support that is in contact with the reaction field. about,
(C) disposing a plurality of amplification reagent immobilization regions at the same position as or near each of the plurality of primer immobilization regions;
(D) immobilizing each of the plurality of primer immobilization regions so that the primer set is releasable for each type;
(E) immobilizing an amplification reagent in each of the plurality of amplification reagent immobilization regions;
(F) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(G) bringing a sample into the reaction field; and (h) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field.

  By such an amplification method, it becomes possible to amplify the nucleic acid in the sample simply and with high accuracy.

<Nucleic acid detection method>
A method for amplifying a plurality of target nucleic acids using the multi-nucleic acid reaction tool as described above as an example and quantifying the nucleic acid using the hybridization signal as an index is also provided as a further embodiment. The method for quantifying nucleic acids may comprise the following steps;
(A) A plurality of types of target nucleic acids, a primer set for amplifying each of the plurality of types of target nucleic acids, and amplification of a plurality of types of compositions designed to perform amplification using each of the primer sets under optimum conditions Supports configured to support a liquid phase reaction field, and a plurality of types of probe nucleic acids containing a sequence complementary to at least a part of the amplification product generated by each of the target nucleic acid and the primer set. Preparing the body,
(B) In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently from each other on at least one surface of the support that is in contact with the reaction field. about,
(C) disposing a plurality of probe immobilization regions at the same position as or near each of the plurality of primer immobilization regions;
(D) immobilizing and immobilizing a primer set and an amplification reagent for each type in each of the plurality of primer immobilization regions;
(E) immobilizing a probe nucleic acid in each of the plurality of probe immobilization regions so that a hybridization signal can be independently detected;
(F) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(G) bringing a sample into the reaction field;
(H) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field;
(I) the presence or absence and / or amount of hybridization between the amplification product obtained by the amplification reaction and the probe nucleic acid is independently detected for the amplification product obtained by each of the primer sets; and (j) The presence / absence and / or amount of the target nucleic acid in the sample is determined based on the result of (i).

  Such a detection method makes it possible to amplify and detect nucleic acids in a sample easily and with high accuracy.

  The reaction solution may be a liquid necessary for the amplification reaction and subsequent hybridization in the multi-nucleic acid reaction device. The liquid necessary for the amplification reaction may be water when the amplification reaction can be performed only with the immobilized primer set and the immobilized amplification reagent. If necessary, the reaction solution contains reagents necessary for amplification, for example, enzymes such as polymerase, substrates such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, reverse transcription. In the case of carrying out simultaneously, a buffer such as reverse transcriptase and a substrate necessary therefor, and salts for maintaining an appropriate amplification environment may be included.

  The sample brought into the reaction field may be added to the reaction solution in advance, and then added to the reaction part of the multi-nucleic acid reaction tool. Alternatively, the sample may be added to the reaction unit before or after the reaction solution is added to the reaction unit of the multi-nucleic acid reaction tool.

  Moreover, you may adjust the temperature in a reaction part as needed, for example by heating or cooling the support body to which the said primer was fixed. Furthermore, you may perform operation which satisfy | fills the conditions for starting and / or maintaining an amplification reaction as needed.

  The detection of the amplification product is a method for detecting a hybridization signal known per se, for example, a method for detecting and / or measuring fluorescence intensity using a fluorescent label, or a method for detecting and / or measuring current response using an intercalator. May be used.

  As an example, according to the multi-nucleic acid reaction device shown in the embodiment, it is possible to detect a plurality of target sequences without being affected by a difference in sequences existing between a plurality of target nucleic acids to be amplified and / or between primer sets. Amplification can be performed independently for each of a plurality of reaction regions included in one reaction field. In addition, the presence or absence and / or 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 region where the amplification reaction is performed simultaneously with the amplification reaction 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 a problem is also solved by the embodiments disclosed herein.

  That is, when the amplification reaction is performed using the multi-nucleic acid reaction tool illustrated in the embodiment, the amplification reaction proceeds only in the vicinity of the immobilized primer set and amplification reagent, that is, in the respective reaction regions. Therefore, the amplification reactions in a plurality of existing reaction regions can proceed independently without interfering with each other in the same container and / or in the same solution. Furthermore, simultaneously with or following such an amplification reaction, the presence and / or amount of the target nucleic acid can be detected and / or measured in the same reaction vessel in which the amplification reaction was performed. Thereby, it is possible to perform a plurality of amplifications simply and with high accuracy, and it is possible to independently detect the amplification products generated by the respective reactions with high accuracy. Therefore, nucleic acid can be detected simply and with high accuracy.

[Example]
Example 1
Simultaneous detection of RNA and DNA (1) Preparation of chip material A chip material for a multi-nucleic acid amplification detection reaction tool as shown in FIG. 8 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 multinucleic acid amplification detection reaction tool Probe DNA was immobilized on the working electrode of the chip material produced as described above. Table 1 shows the base sequence of the probe DNA used.

  Two types of probe DNAs (A) and (B) were immobilized on the chip material prepared as described above. Probe DNA solutions each containing 3 μM of probe DNA were prepared. 100 nL of these solutions were 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 and an amplification reagent, DNA polymerase, were immobilized on the bottom surface of the groove of the covering, and in addition, reverse transcriptase was immobilized at a location where RNA was detected. The immobilized region of the primer set and the amplification reagent 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. Table 2 shows the base sequence of the primer DNA.

  For primer DNA (set A) and (set B), 200 μM FIP, BIP, F3, B3 and LPF, LPB were prepared. For 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, LPF, and LPB, respectively, and a mixed solution of 0.1 μL of DNA polymerase In Set B, 0.050 μL of reverse transcriptase was added. This aqueous solution was fixed to the primer on the bottom surface of the groove portion of the silicon rubber as a covering and the immobilization region necessary for nucleic acid amplification.

  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, a multi-nucleic acid amplification detection reaction tool 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 Table 3 shows the composition of the LAMP reaction liquid (1).

Table 4 shows the template sequences in the LAMP reaction solution (1).

(4) LAMP amplification reaction on multi-nucleic acid amplification detection reaction tool 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, 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 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 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 result A LAMP reaction solution (1) containing template A (DNA) and template B (RNA) was added, and as a result of measurement, a current value of 30 nA or more was obtained for probe A, probe B, and both probes. It was. From this result, it was shown that multi-amplification and detection in which DNA and RNA coexist were successful.

Example 2
Necessary reagents are additionally fixed for each target nucleic acid, and multiple detections are performed with different characteristics. First, a preliminary test was performed by reacting one target nucleic acid with one kind of primer set in a tube. In this example, amplification is almost the same for a target sequence and primer set combination that requires a high amplification temperature to enable an amplification reaction, and for a target sequence and primer set combination that allows an amplification reaction even at a low amplification temperature. In order to obtain an amplification reaction with efficiency, the composition of the amplification reagent was determined.

The results are shown in Table 5.

  This preliminary test revealed that the optimal concentrations of ammonium sulfate and betaine differed depending on the target sequence and primer set design. Based on this result, the composition of the amplification reagent was designed according to the sequence of the target nucleic acid and primer set.

(1) Production of chip material A chip material for a multi-nucleic acid amplification detection reaction tool as shown in FIG. 8 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 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 the same as that shown in Table 1.

  Four types of probe DNAs (C), (D), and (E) shown in SEQ ID NOs: 1 to 4 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 were 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 and DNA polymerase as an amplification reagent were fixed to the bottom surface of the groove of the covering. The immobilized region of the primer set and the amplification reagent 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 sequence of the primer DNA is the same as the sequence shown in Table 2.

  For the primer DNA (set C), (set D), and (set E), 200 μM FIP, BIP, F3, B3 and LPF, LPB were prepared. For 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, LPF, and LPB, respectively, and a mixed solution of 0.1 μL of DNA polymerase Set C adds 0.1 μL of 1 M magnesium sulfate, Set D adds 0.050 μL of reverse transcriptase, 0.14 μL of 3.5 M ammonium sulfate, and Set E adds 0.500 μL of 2 M potassium chloride (Condition 1). This aqueous solution was fixed to the primer on the bottom surface of the groove portion of the silicon rubber as a covering and the immobilization region necessary for nucleic acid amplification. As a control, an aqueous solution to which no amplification reagent other than the enzyme was added was prepared (Condition 2).

  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, a multi-nucleic acid amplification detection reaction tool 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 (2) is shown in Table 6.

The sequence of the template contained in the LAMP reaction solution (2) is shown in Table 7.

(4) LAMP amplification reaction on multi-nucleic acid amplification detection reaction tool 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, 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 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 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 result As a result of adding and measuring the LAMP reaction solution (2) containing the template C, template D, and template E, in condition 1, probe C, probe D, probe E, and all probes, 30 nA or more A current value was obtained. On the other hand, in condition 2 in which the amplification reagent was not additionally fixed for each target nucleic acid, D had a low current value, and C and E were negative because the current value was not detected. From this result, it is possible to succeed in multi-amplification by individually fixing the reagents necessary for amplification separately for each target nucleic acid, and in the case where the reagent is not additionally fixed, the current value is not detected or low detection can be performed in multi-detection. It has been shown.

Example 3
Betaine was added and multi-detection at the same temperature First, a preliminary test was performed by reacting one target nucleic acid with one kind of primer set in a tube. In this example, amplification is almost the same for a target sequence and primer set combination that requires a high amplification temperature to enable an amplification reaction, and for a target sequence and primer set combination that allows an amplification reaction even at a low amplification temperature. In order to obtain an amplification reaction with efficiency, the composition of the amplification reagent was determined.

  The results are shown in Table 5 above. As a result, it was shown that a higher betaine concentration is better for a target nucleic acid having a high optimum amplification temperature. That is, when a target nucleic acid having a low optimal amplification temperature requires 0.4 M betaine to achieve a specific amplification efficiency, a target nucleic acid having a high optimal amplification temperature is required to achieve the same amplification efficiency. So 1.4M betaine was needed. From this result, even when nucleic acids were detected, the addition of betaine improved the amplification characteristics, and it became possible to detect the types of nucleic acids that could not be detected before the improvement.

(1) Production of chip material A chip material for a multi-nucleic acid amplification detection reaction tool as shown in FIG. 8 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 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 was the same as the sequence shown in Table 1.

  Two types of probe DNAs (A) and (F) were immobilized on the chip material prepared as described above. Probe DNA solutions each containing 3 μM of probe DNA were prepared. 100 nL of these solutions were 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 and an amplification reagent, DNA polymerase, were immobilized on the bottom surface of the groove of the covering. The immobilized region of the primer set and the amplification reagent 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 sequence of the primer DNA was the same as that shown in Table 2.

  About primer DNA (set A) and (set F), 200 micromol FIP, BIP, F3, B3 and LPF, LPB were prepared, respectively. For 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, LPF, and LPB, respectively, and a mixed solution of 0.1 μL of DNA polymerase In Set C, 0.050 μL of 5M betaine was added.

This aqueous solution was fixed to the primer on the bottom surface of the groove portion of the silicon rubber as a covering and the immobilization region necessary for nucleic acid amplification.

  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, a multi-nucleic acid amplification detection reaction tool was obtained. Two through holes are opened at two ends of the groove of the silicon rubber that is the covering.

(3) Production of LAMP reaction solution Table 8 shows the composition of the LAMP reaction solution (3).

Table 8 shows the template sequences contained in the LAMP reaction solution (3).

(4) LAMP amplification reaction on multi-nucleic acid amplification detection reaction tool 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, 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 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 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 result As a result of adding and measuring LAMP reaction solution (3) containing template A and template F, a current value of 30 nA or more was obtained for probe A, probe F, and both probes under the conditions where betaine was added. Obtained. On the other hand, when betaine was not added, the current value of F was not detected, resulting in a negative result. From this result, it was shown that by immobilizing betaine, the target nucleic acid F that could not be amplified can be amplified and multiple detection can be performed.

Claims (10)

A support configured to support a liquid phase reaction field;
When the reaction field is formed by the liquid phase, a plurality of primer-immobilized regions arranged independently of each other on at least one surface of the support in contact with the one reaction field;
A plurality of amplification reagent immobilization regions disposed at the same position as and / or in the vicinity of each of the plurality of primer immobilization regions;
A plurality of types of primer sets fixed releasably for each type in the plurality of primer-immobilized regions; and
An amplification reagent group having a plurality of types of compositions each releasably immobilized in the amplification reagent immobilization region for each composition and configured to allow an amplification reaction for each of the plurality of types of primer sets;
A multi-nucleic acid reaction device comprising: A plurality of probe immobilization regions arranged at or near the same position as each of the primer immobilization regions, and at the same position as or near each of the amplification reagent immobilization regions;
Hybridization signals are independently and detectably immobilized on each of the plurality of probe immobilization regions, and are complementary to at least a part of an amplification product generated by the primer set, the target sequence, and the amplification reagent. A probe nucleic acid comprising a sequence;
The multi-nucleic acid reaction device according to claim 1, comprising: Each of the amplification reagent group, DNA synthase, reverse transcriptase, buffer, dNTPs, ammonium sulfate, Tween20, potassium chloride, magnesium sulfate, betaine, protein stabilizers and amplification inhibitors, as well as from the group consisting of combinations thereof The multi-nucleic acid reaction device according to claim 1 or 2, comprising at least one selected. It said support is a support having a container form or channels, multi nucleic reaction device according to any one of claims 1 to 3, wherein the container or the flow path is a reaction field. A plurality of types of target nucleic acids, a plurality of types of primer sets for amplifying the plurality of types of target nucleic acids, respectively, and a plurality of compositions each configured to allow an amplification reaction for each of the plurality of types of primer sets Providing a group of amplification reagents of different compositions and a support configured to support a liquid phase reaction field;
In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently of each other on at least one surface of the support that is in contact with the one reaction field. ,
Disposing a plurality of amplification reagent immobilization regions at the same position as and / or in the vicinity of each of the plurality of primer immobilization regions;
To each of the plurality of primer immobilization region, be releasable immobilize the primer set for each type, and each of the plurality of amplification reagent immobilization region, immobilizing the amplification reagent group for each composition A method for producing a multi-nucleic acid reaction device. A plurality of types of compositions comprising a plurality of types of target nucleic acids, a primer set for amplifying each of the plurality of types of target nucleic acids, and a composition each configured to allow an amplification reaction for each of the plurality of types of primer sets and amplification reagents group, configured to support a plurality kinds of probe nucleic acids, a reaction field of a liquid phase comprising a sequence complementary to at least a portion of each of said target nucleic acid and the primer set and the resulting amplification products Preparing a supported support,
In the support, when the reaction field is formed by the liquid phase, a plurality of primer immobilization regions are arranged independently of each other on at least one surface of the support that is in contact with the one reaction field. ,
Arranging a plurality of amplification reagent immobilization region in the same position and / or near the each of the plurality of primer immobilization region,
Wherein the same location or near the respective primer-immobilized regions, it and arranging a plurality of probe immobilization region in the same position or near the each of the amplification reagent immobilization region,
To each of the plurality of primer immobilization region, to respective fixed releasable to the primer set for each type,
The amplification reagent group can be releasably immobilized for each composition in each of the plurality of amplification reagent immobilization regions, and a hybridization signal can be independently detected in each of the plurality of probe immobilization regions. method for producing a multi-nucleic acid reaction tool comprising respectively immobilize the probe nucleic acid. (A) a plurality of types of target nucleic acids, a plurality of types of primer sets for amplifying the plurality of types of target nucleic acids, a group of amplification reagents of a plurality of types corresponding to each of the primer sets , and a liquid phase Providing a support configured to support the reaction field;
(B) In the support, when the reaction field is formed by the liquid phase, a plurality of primer-immobilized regions independently of each other are provided on at least one surface of the support in contact with the one reaction field. Placing,
(C) disposing a plurality of amplification reagent immobilization regions at the same position as and / or in the vicinity of each of the plurality of primer immobilization regions;
(D) to each of the plurality of primer immobilization region, be releasable immobilize the primer set for each type,
(E) to each of the plurality of amplification reagent immobilization region, to respective fixed releasable to said amplification reagent group for each composition,
(F) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(G) A method for amplifying a nucleic acid, including bringing a sample into the reaction field, and (h) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field. (A) a plurality of types of target nucleic acids, a primer set for amplifying each of the plurality of types of target nucleic acids, a plurality of types of amplification reagent groups corresponding to each of the primer sets , and each of the target nucleic acids Preparing a plurality of types of probe nucleic acids containing a sequence complementary to at least a part of an amplification product generated by the primer set, and a support configured to support a liquid phase reaction field,
(B) In the support, when the reaction field is formed by the liquid phase, a plurality of primer-immobilized regions independently of each other are provided on at least one surface of the support in contact with the one reaction field. Placing,
(C) placing the same position or more amplification reagents immobilization region near the with each of the plurality of primer immobilization region,
; (D) the same position or near the respective primer-immobilized regions, and before Symbol arranging a plurality of probe immobilization region in the same position or near the respective amplification reagent immobilization region,
(E) to each of the plurality of primer immobilization region, to respective fixed releasable to the primer set for each type,
(F) to each of the plurality of amplification reagent immobilization region, to respective fixed releasable to said amplification reagent group for each composition,
(G) to each of the plurality of probe immobilization region, to each immobilized detectably said probe nucleic acid independently hybridizing signal,
(H) adding a reaction solution for nucleic acid amplification to the support to form one reaction field;
(I) the bringing of the sample into the reaction field,
(J) performing an amplification reaction for each of the plurality of types of target nucleic acids in the one reaction field;
(K) the presence and / or amount of hybridized amplification reaction by the amplification products obtained and the probe nucleic acid, detecting independently the said amplification product obtained by each of the primer set, and (l ) A method for detecting a nucleic acid comprising determining the presence and / or amount of a target nucleic acid in the sample based on the result of (k). Each of the amplification reagent groups is selected from the group consisting of DNA synthase, reverse transcriptase, buffer, dNTP, ammonium sulfate, Tween 20, potassium chloride, magnesium sulfate, betaine, protein stabilizer and amplification inhibitor, and combinations thereof. The method according to claim 5, comprising at least one of the following. It said support is a support having a container form or the channel A method according to any one of claims 5-9 wherein the container or the flow path is a reaction field.

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