JP2013198417A - Device for detecting nucleic acid and nucleic acid detection apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device for detecting nucleic acid improving the efficiency of an amplification reaction of a nucleic acid, and a nucleic acid detection apparatus.SOLUTION: A device for detecting nucleic acid includes a first member provided with a groove on a first surface. The groove is provided with a channel type chamber for a nucleic acid sample to react. A cross section of the channel type chamber is larger than cross sections other than the channel type chamber in the groove.

Description

  Embodiments described herein relate generally to a nucleic acid detection device and a nucleic acid detection apparatus that perform a nucleic acid amplification reaction in a fine channel.

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

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

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

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

Japanese Patent No. 4127679 JP 2008-263959 A

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

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

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

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

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

  An object of the present invention is to provide a nucleic acid detection device and a nucleic acid detection apparatus that improve the efficiency of a nucleic acid amplification reaction.

  According to the embodiment, the nucleic acid detection device includes a first member including a groove on the first surface. The groove includes a flow channel chamber for allowing a nucleic acid sample to react. The cross-sectional area of the flow channel chamber is larger than the cross-sectional area of the groove portion other than the flow channel chamber.

1 is a schematic view of a nucleic acid detection device according to a first embodiment. 1 is a schematic view of a nucleic acid detection device according to a first embodiment. 1 is a schematic view of a nucleic acid detection device according to a first embodiment. 1 is a schematic view of a nucleic acid detection device according to a first embodiment. 1 is a schematic view of a support according to Example 1. FIG. 1 is a schematic view of a nucleic acid detection device according to Example 1. FIG. 1 is a schematic view of a nucleic acid detection device according to Example 1. FIG. 3 is a graph showing the results of a nucleic acid amplification reaction according to Example 1. The schematic of the device for nucleic acid detection concerning a 2nd embodiment. The schematic of the device for nucleic acid detection concerning a 2nd embodiment. The schematic of the device for nucleic acid detection concerning a 2nd embodiment. The schematic of the device for nucleic acid detection concerning a 2nd embodiment. The schematic of the device for nucleic acid detection concerning a 2nd embodiment. 4 is a schematic diagram of a nucleic acid detection device according to Example 2 and a graph showing the results of a nucleic acid amplification reaction.

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

(First embodiment)
An example of the nucleic acid detection device 1 according to the first embodiment will be described with reference to FIG. FIG. 1A is a plan view of an example of the nucleic acid detection device 1. FIG. 1B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. The nucleic acid detection device 1 is used to simultaneously and independently amplify a plurality of types of target nucleic acids using a plurality of types of primer sets 31 in one reaction field.

  The nucleic acid detection device 1 includes a support body (second member) 11 and a covering body (first member) 12. The support 11 has a substantially flat surface on the surface in contact with the covering 12. In the first embodiment, the direction in which the support 11 and the covering 12 are arranged in this order is referred to as a stacking direction. The covering 12 includes a groove 121 on a surface (first surface) that contacts the support 11. The groove portion 121 is provided on a surface in contact with the support 11. The groove 121 is hermetically sealed by the covering 12 and the support 11 in contact therewith. The groove 121 sealed with the covering 12 and the support 11 functions as a flow path for various solutions. As an example, the groove 121 has a shape meandering in a curved shape from the inlet 121a to the outlet 121b of various solutions, but is not particularly limited. The groove 121 is formed with substantially the same width from the inlet 121a to the outlet 121b. In the groove 121, for example, a plurality of chambers (channel type chambers) 1211 are arranged at equal intervals. The chamber 1211 is used for a nucleic acid amplification reaction, and an electrode (sensor) described later reacts with a nucleic acid sample. The chamber 1211 is formed in a shape that is recessed in the stacking direction from a region (part) other than the chamber 1211 in the groove 121. That is, the depth of the chamber 1211 is formed deeper than the depth of the region other than the chamber 1211 in the groove 121. Conversely, the depth of the region other than the chamber 1211 in the groove 121 is shallower than the depth of the chamber 1211. Therefore, the cross-sectional area of the chamber 1211 is larger than the cross-sectional area of the region other than the chamber 1211 in the groove 121. Note that the cross-sectional area of the chamber 1211 and the cross-sectional area of the region other than the chamber 1211 in the groove 121 are cross-sectional areas based on a surface orthogonal to the surface of the covering 12 where the groove 121 is provided. The cross-sectional area of the region other than the chamber 1211 in the groove 121 is preferably 90% or less of the cross-sectional area of the chamber 1211, but is not particularly limited. The chamber 1211 corresponds to the primer immobilization region 21. The primer immobilization region 21 is formed on, for example, the upper surface portion of the chamber 1211 (a portion recessed in the stacking direction from the region other than the chamber 1211). The plurality of primer immobilization regions 21 are arranged in the groove 121 independently of each other.

  The material of the support 11 and the covering 12 may be the same or different. The support 11 and the cover 12 may be made of any material that does not participate in the amplification reaction or the like. The support 11 and the cover 12 may be made of any material that can perform an amplification reaction in the groove 121. The support 11 and the covering 12 may be arbitrarily selected from, for example, silicon, glass, resin, metal, and the like.

  Next, the fixing (holding) of the primer set 31 for amplifying the nucleic acid in the primer fixing region 21 will be described. The chamber 1211 holds the primer set 31 on the channel wall surface. The primer immobilization region 21 corresponds to the position of the chamber 1211 and may be appropriately read as the chamber 1211. As shown in FIG. 1, the primer set 31 is fixed in the vicinity of the upper surface portion (primer immobilization region 21) in the stacking direction in the chamber 1211 so as to be released when it comes into contact with the liquid phase for providing a reaction field. The In the plurality of chambers 1211 (primer immobilization regions 21), a plurality of primer sets 31 are fixed for each type of target nucleic acid. That is, the plurality of chambers 1211 (primer immobilization regions 21) hold a plurality of types of primer sets configured to amplify a plurality of types of target sequences, respectively. For example, the primer set 31 is immobilized by, for example, preparing only the covering 12 so that the groove 121 faces upward in the vertical direction, dropping a solution containing the primer set 31 into one primer immobilization region 21, and then drying it. Is possible. The method for holding the primer set 31 is not limited to drying, and other methods such as freeze drying may be used.

  As described above, since the chamber 1211 is formed deeper than the region other than the chamber 1211 in the groove 121, the solution containing the primer set 31 locally dripped into the primer immobilization region 21 is a region other than the chamber 1211. It doesn't move easily. Therefore, adjacent primer immobilization regions 21 can hold different primer sets 31 independently.

  Next, the addition of the reaction solution to the nucleic acid detection device 1 in which the plurality of primer sets 31 are fixed to different primer fixing regions 21 will be described. FIG. 2A is a plan view of an example of the nucleic acid detection device 1. FIG. 2B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. FIG. 2 shows a state where a reaction solution is added to the nucleic acid detection device 1. The nucleic acid detection device 1 shown in FIG. 2 is the same as that in FIG. 1 except that the reaction solution is added to the groove 121.

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

  As shown in FIG. 2, after adding the reaction solution from the inlet 121a, the primer set 31 fixed to the primer fixing region 21 starts to be released and diffused. The region where the primer is released and diffused is schematically shown as a primer release / diffusion region 22 in FIG.

  When the reaction solution is introduced into the nucleic acid detection device 1, it is desired that the primer set 31 that is liberated and diffused does not easily flow out to the other adjacent primer immobilization region 21. That is, the primer release / diffusion region 22 preferably corresponds to the primer immobilization region 21 (in other words, the chamber 1211).

  The nucleic acid detection device 1 can significantly reduce the flow velocity in the vicinity of the portion where the primer set 31 is fixed in the chamber 1211 (portion recessed in the stacking direction from the region other than the chamber 1211) when the reaction solution is introduced. . Therefore, the nucleic acid detection device 1 can prevent the primer set 31 from flowing out to the other primer immobilization region 21 adjacent thereto. Therefore, the nucleic acid detection device 1 according to the first embodiment can make the primer sets 31 of the adjacent primer immobilization regions 21 independent of each other.

  Further, even during the amplification reaction, the primer set 31 and the generated amplification product in a certain amplification region (region corresponding to the primer-immobilized region 21 (chamber 1211)) do not diffuse to other amplification regions and are independently It is desirable to be within the amplification region. In the nucleic acid detection device 1, the depth (channel cross-sectional area) of the groove 121 in a region other than the chamber 1211 is shallower (smaller) than the depth (channel cross-sectional area) of the chamber 1211. Therefore, the nucleic acid detection device 1 can suppress the diffusion of the primer in the amplification region and the generated amplification product to the other amplification region during the amplification reaction. Therefore, the nucleic acid detection device 1 according to the first embodiment can achieve amplification for a plurality of template sequences using a plurality of types of primer sets 31 independently (locally) and simultaneously with high efficiency. is there.

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

  Next, detection of a hybridization signal by the nucleic acid detection device 1 will be described. FIG. 3A is a plan view of an example of the nucleic acid detection device 1. FIG. 3B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. The nucleic acid detection device 1 shown in FIG. 3 is the same as that shown in FIG. 1 except that the support 11 includes the probe immobilization region 111. For example, the probe immobilization region 111 is disposed on the support 11 at a position facing the primer immobilization region 21 (chamber 1211). However, the probe immobilization region 111 is not particularly limited. There may be. The probe immobilization region 111 is a region where, for example, an electrode for detecting a hybridization signal (electrode for nucleic acid detection) is provided. That is, the nucleic acid detection electrode in the probe immobilization region 111 is disposed at a position facing the surface in contact with the support 11 in the covering 12 and the groove 121 (particularly the primer immobilization region 21 (chamber 1211)). The

  In the probe immobilization region 111, a plurality of probe nucleic acids including a complementary sequence of a desired sequence to be detected are immobilized. The nucleic acid detection device 1 can obtain a hybridization signal in the probe immobilization region 111 after performing an amplification reaction in the primer immobilization region 21.

  Next, the case where a reaction solution is introduced into the nucleic acid detection device 1 shown in FIG. 3 and an amplification reaction is performed will be described. FIG. 4A is a plan view of an example of the nucleic acid detection device 1. FIG. 4B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. FIG. 4 shows a state in which a reaction solution is added to the nucleic acid detection device 1. The nucleic acid detection device 1 shown in FIG. 4 is the same as FIG. 3 except that the reaction solution is added to the groove 121. In the nucleic acid detection device 1, when a reaction solution is introduced, if a template nucleic acid is present, the template nucleic acid is amplified by the corresponding free and diffused primer set 31, and an amplification product 32 is generated. The amplification product 32 generated in the amplification reaction is locally generated in the chamber 1211 as shown in FIG. The nucleic acid detection device 1 can obtain a hybridization signal by reacting the amplification product 32 with the probe immobilized on the probe immobilization region 111.

Example 1
An example of nucleic acid detection using the nucleic acid detection device 1 according to the first embodiment will be specifically described below. In Example 1, a packing 12a made of silicon rubber was used as the covering 12, and the primer set 31 was fixed to the primer fixing region 21 on the entire surface of the packing 12a. FIG. 5 is a schematic diagram illustrating an example of the support 11 according to the first embodiment. In Example 1, the support 11
As an example, an array type chip (substrate) 11a for electrochemical detection using the probe-immobilized region 111 as an electrode 111a was used as a sensor for detecting a current response generated depending on the presence of hybridization. The pad portion 112a is for transmitting a hybridization signal of the electrode 111a to the nucleic acid detection device (not shown) via the wiring 113b. That is, the nucleic acid detection device detects nucleic acid based on the current value from each electrode 111a.

1. Preparation of nucleic acid detection device 1-1 Production of array chip for electrochemical detection The array chip 11a for electrochemical detection was formed by sputtering a thin film of titanium and gold on the surface of Pyrex (registered trademark) glass. Thereafter, an electrode pattern of titanium and gold was formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and the electrode 111a was exposed by an etching process.

Next, six types of nucleic acid probes (sequences A to E, NC) shown in Table 1 were immobilized on each electrode 111a (AE, NC (negative control) in FIG. 5) on the chip material. A solution containing each nucleic acid probe was dropped onto each electrode 111a, and then the excess nucleic acid probe was washed and removed to be immobilized.

1-2 Preparation of Primer-Fixed Packing 12a, Assembly of Device for Nucleic Acid Detection First, primer DNA used as the primer set 31 was prepared. The primer DNA used is a primer set 31 for amplification by a loop-mediated isothermal amplification (LAMP) method. Table 2 shows the base sequences of the primer DNAs used. The solution containing the primer DNA was spotted on the bottom surface of the packing 12a facing the region where the corresponding probe DNA was fixed, and dried at 40 ° C. for 2 minutes. Thereby, primer-fixed packing 12a was obtained. The packing 12a was attached to the array type chip 111a to obtain the nucleic acid detection device 1 shown in FIG. FIG. 6A is a plan view of an example of the nucleic acid detection device 1 according to the first embodiment. FIG. 6B is a cross-sectional view of the nucleic acid detection device 1 taken along line XX in FIG.

2. Detection of Nucleic Acid 2-1 Preparation of Template Solution As shown in Table 3, a template solution was prepared by mixing three types of templates of genes A, B, and D and a reagent for LAMP amplification.

The template solution used contained Bst DNA polymerase and reaction mix and was added with distilled water (ie, DW) so that the total amount was 50 μL. As shown in Table 4, the template solution was subjected to amplification reaction by the LAMP method with the primer DNA (set A) and hybridized with the probe DNA (A) and detected with the primer DNA (set B). ) Causes an amplification reaction by the LAMP method, and a template B detected by hybridization with the probe DNA (B) and the primer DNA (set D) cause an amplification reaction by the LAMP method, and the probe DNA (D And a template D detected by hybridization.

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

2-3 Reaction of Nucleic Acid Under the conditions shown below, the flow channel region of the nucleic acid detection device 1 was heated or cooled to perform various reactions. FIG. 7A is a plan view of an example of the nucleic acid detection device 1 according to the first embodiment. FIG. 7B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. As shown in FIG. 7, when a template containing a target sequence to be amplified is contained in a LAMP reaction solution for a specific primer, the LAMP reaction proceeds locally at the location where the primer is fixed, and the resulting amplification The product 32 hybridizes with the probe DNA in the vicinity thereof.

Nucleic acid amplification reaction: 64 ° C., 60 minutes Hybridization reaction: 50 ° C., 10 minutes Washing reaction: 30 ° C., 5 minutes Current detection reagent (Hoechst 33258) reaction: 25 ° C., 3 minutes 2-4 Nucleic acid detection Each probe nucleic acid The potential was swept to the immobilized working electrode, and the oxidation current of Hoechst 33258 molecule specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The 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.

Results The results obtained are shown in FIG. FIG. 8 is a graph showing the results of the nucleic acid amplification reaction according to Example 1. In the electrodes A, B, and D on which the probes A, B, and D corresponding to the genes A, B, and D to which the template was added were immobilized, a current value larger than that of the NC was obtained. On the other hand, the current values of the electrodes C and E corresponding to the genes C and E to which no template was added were about the same as those of the NC. This revealed that the template in the template solution could be reliably detected.

(Second Embodiment)
The second embodiment will be described in detail with reference to the drawings. In addition, about the same structure as the structure demonstrated in 1st Embodiment, a same sign is attached | subjected and description is abbreviate | omitted. An example of the nucleic acid detection device 1 according to the second embodiment will be described with reference to FIG. FIG. 9A is a plan view of an example of the nucleic acid detection device 1. FIG. 9B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. The nucleic acid detection device 1 is used to simultaneously and independently amplify a plurality of types of target nucleic acids using a plurality of types of primer sets 31 in one reaction field. The second embodiment is different from the first embodiment in the shape of the groove 121.

  The groove 121 is formed with substantially the same depth in the stacking direction from the inlet 121a to the outlet 121b. In the groove 121, a plurality of chambers 1211 are arranged at predetermined intervals. The width of the chamber 1211 is formed to be wider than the width of the region other than the chamber 1211 in the groove 121. Conversely, the width of the region other than the chamber 1211 in the groove 121 is narrower than the width of the chamber 1211. The chamber 1211 only needs to be wider than the region other than the chamber 1211 in the groove 121, and may protrude in an arc shape in a direction orthogonal to the stacking direction or may protrude in a square shape. Therefore, the cross-sectional area of the chamber 1211 is larger than the cross-sectional area of the region other than the chamber 1211 in the groove 121. Note that the cross-sectional area of the chamber 1211 and the cross-sectional area of the region other than the chamber 1211 in the groove 121 are cross-sectional areas based on a surface orthogonal to the surface of the covering 12 where the groove 121 is provided. The cross-sectional area of the region other than the chamber 1211 in the groove 121 is preferably 90% or less of the maximum value of the cross-sectional area of the chamber 1211, but is not particularly limited. The chamber 1211 corresponds to the primer immobilization region 21. The primer immobilization region 21 is formed in the vicinity of the upper surface portion of the groove portion 121 in the stacking direction. The plurality of primer immobilization regions 21 are arranged in the groove 121 independently of each other.

  As described above, since the chamber 1211 is formed wider than the region other than the chamber 1211 in the groove 121, the solution containing the primer set 31 locally dripped into the primer immobilization region 21 is not included in the chamber 1211. It does not move easily into the area. Therefore, adjacent primer immobilization regions 21 can hold different primer sets 31 independently.

  Next, the addition of the reaction solution to the nucleic acid detection device 1 in which the plurality of primer sets 31 are fixed to different primer fixing regions 21 will be described. FIG. 10A is a plan view of an example of the nucleic acid detection device 1. FIG. 10B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. FIG. 10 shows a state in which a reaction solution is added to the nucleic acid detection device 1. The nucleic acid detection device 1 shown in FIG. 10 is the same as FIG. 9 except that the reaction solution is added to the groove 121. The reaction solution may be the same component as in the first embodiment.

  As shown in FIG. 10, after adding the reaction solution from the inlet 121a, the primer set 31 fixed to the primer fixing region 21 starts to be released and diffused. The region where the primer is released and diffused is schematically shown as a primer release / diffusion region 22 in FIG. In the nucleic acid detection device 1, the width (channel cross-sectional area) of the groove 121 in a region other than the chamber 1211 is narrower (smaller) than the width (channel cross-sectional area) of the chamber 1211. Therefore, the nucleic acid detection device 1 can suppress the diffusion of the primer in the amplification region and the generated amplification product to the other amplification region during the amplification reaction. Therefore, the nucleic acid detection device 1 according to the second embodiment can achieve amplification for a plurality of template sequences using a plurality of types of primer sets 31 independently (locally) and simultaneously with high efficiency. is there.

  9 and 10, the example in which the primer set 31 is fixed to the channel wall surface in the vicinity of the upper surface of the chamber 1211 has been described. However, the present invention is not limited to this. FIG. 11 is a plan view of an example of the nucleic acid detection device 1 showing other fixing locations of the primer set 31 in the chamber 1211 (primer immobilization region 21). The primer set 31 may be fixed to the flow path wall surface of the chamber 1211 orthogonal to the stacking direction. That is, the primer set 31 is provided in a portion of the chamber 1211 that protrudes in a direction orthogonal to the stacking direction from the region of the groove 121 other than the chamber 1211.

  As shown in FIG. 11, when the primer set 31 is fixed in the chamber 1211, the nucleic acid detection device 1 is configured so that when the reaction solution is introduced, the portion where the primer set 31 in the chamber 1211 is fixed (the groove 121 other than the chamber 1211 The flow velocity in the vicinity of the portion protruding in the direction perpendicular to the stacking direction than the region can be greatly reduced. Therefore, the nucleic acid detection device 1 can prevent the primer set 31 from flowing out to the other primer immobilization region 21 adjacent thereto. Therefore, the nucleic acid detection device 1 according to the second embodiment can make the primer sets 31 of the adjacent primer immobilization regions 21 independent of each other.

  In the example shown in FIGS. 9, 10, and 11, the portion of the groove 121 that connects the chambers 1211 is formed in a straight line shape that connects at the shortest distance, but is not limited thereto. FIG. 12 is a plan view of an example of the nucleic acid detection device 1 showing another shape of the groove 121 that connects the chambers 1211 to each other. In the example shown in FIG. 12, the portion of the groove 121 that connects the chambers 1211 is configured to have a shape (for example, a curved shape) in which the distance of the flow path is longer than the linear shape. Therefore, the groove part 121 shown in FIG. 12 can improve the independence of each chamber 1211 compared with the structure of the groove part 121 which connects each chamber 1211 with the linear shape shown to FIG. Efficient nucleic acid amplification can be performed. In addition, although the structure of the groove part 121 which connects each chamber 1211 was demonstrated regarding 2nd Embodiment here, the structure as shown in FIG. 12 is applied also to 1st Embodiment, and the above-mentioned. The effect of can be obtained.

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

  Next, detection of a hybridization signal by the nucleic acid detection device 1 will be described. FIG. 13A is a plan view of an example of the nucleic acid detection device 1. FIG. 13B is a cross-sectional view of the nucleic acid detection device 1 taken along line XX in FIG. The nucleic acid detection device 1 shown in FIG. 13 is the same as that shown in FIG. 9 except that the support 11 includes the probe immobilization region 111. The probe immobilization region 111 is not particularly limited. For example, the probe immobilization region 111 is disposed on the support 11 at a position facing the chamber 1211 (primer immobilization region 21). The probe immobilization region 111 is a region where an electrode for detecting a hybridization signal is provided.

  In the probe immobilization region 111, a plurality of probe nucleic acids including a complementary sequence of a desired sequence to be detected are immobilized. The nucleic acid detection device 1 can obtain a hybridization signal in the probe immobilization region 111 after performing an amplification reaction in the primer immobilization region 21.

  In addition, when the support 11 is a current detection type sensor that uses an intercalator in particular, amplification inhibition occurs due to an eluate from the sensor protective film accompanying heating during the amplification reaction. However, since the nucleic acid detection device 1 according to the second embodiment has a configuration in which the groove 121 other than the amplification / detection region (chamber 1211) is narrowed (narrow in width), it is in contact with the sensor provided in the probe immobilization region 111. Since the liquid area can be reduced, elution of amplification inhibitors can be effectively suppressed.

(Example 2)
The nucleic acid detection by the nucleic acid detection device 1 according to the second embodiment described above is performed as follows, for example. A reaction solution containing a nucleic acid to be inspected is introduced into a groove 1211 formed in the nucleic acid detection device 1 using an instrument such as a pipette. When the reaction solution is introduced and the template nucleic acid is present, the template nucleic acid is amplified by the corresponding free-diffused primer set 31 to generate an amplification product.

  FIG. 14A is a plan view of an example of the nucleic acid detection device 1 according to the second embodiment. FIG. 14B is a cross-sectional view of the nucleic acid detection device 1 along the line XX in FIG. FIG. 14C is a graph showing the results of the nucleic acid amplification reaction according to Example 2. FIG. 14C shows an amplification reaction occurring in the regions A, B, and D (each chamber 1211) of the nucleic acid detection device 1 shown in FIGS. 14A and 14B, and the amplification product corresponding to the probe nucleic acid. The results obtained when the target sequence complementary to the sequence is included are shown. As shown in FIG. 14C, the current values obtained for the regions A, B, and D are larger than those of the NC. On the other hand, the detection signals obtained for the regions C and E are approximately the same as the NC. This revealed that the template in the template solution could be reliably detected. Note that the detection signals are also obtained particularly in the adjacent regions A and B. Therefore, the nucleic acid detection device 1 according to the second embodiment has no interference between adjacent regions, and can independently perform from nucleic acid amplification reaction to detection. As described above, according to Example 2, using the nucleic acid detection device 1 according to the second embodiment, multiple types of target nucleic acids can be amplified simultaneously and independently until detection. It was shown that it can be done.

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

  It is possible to combine the shape of the chamber 1211 according to the first embodiment and the shape of the chamber 1211 according to the second embodiment.

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

  DESCRIPTION OF SYMBOLS 1 ... Device for nucleic acid detection, 11 ... Support, 12 ... Covering body, 12a ... Packing, 21 ... Primer immobilization region, 22 ... Primer release / diffusion region, 31 ... Primer set, 32 ... Amplification product, 111a ... Electrode, 112a ... pad part, 113a ... wiring, 121 ... groove part, 121a ... inlet, 121b ... outlet, 1211 ... chamber.

Claims (8)

A first member having a groove on the first surface;
The groove includes a flow channel chamber for a nucleic acid sample to react,
The cross-sectional area of the flow channel chamber is larger than the cross-sectional area of the groove other than the flow channel chamber,
Nucleic acid detection device.   The nucleic acid detection device according to claim 1, wherein a depth of the flow channel chamber is deeper than a depth of a region other than the flow channel chamber in the groove.   The nucleic acid detection device according to claim 1, wherein a width of the flow channel chamber is wider than a width of a region other than the flow channel chamber in the groove.   The nucleic acid detection device according to claim 1, wherein a cross-sectional area of the flow channel chamber and a cross-sectional area of the groove portion other than the flow channel chamber are cross-sectional areas based on a surface orthogonal to the first surface.   The nucleic acid detection device according to claim 1, wherein the plurality of flow channel chambers hold a plurality of types of primer sets configured to amplify a plurality of types of target sequences, respectively.   The nucleic acid detection device according to claim 1, wherein the flow channel chamber holds the primer set on a wall surface.   The nucleic acid detection according to any one of claims 1 to 6, further comprising a second member that is opposed to the first surface of the first member and includes a nucleic acid detection electrode at a position facing the groove. Device. A nucleic acid detection apparatus using the nucleic acid detection device according to any one of claims 1 to 7,
A nucleic acid detection apparatus for detecting a nucleic acid based on a current value from the electrode.

Images