JP2013198443A - Multiple nucleic acid reaction tool, multiple nucleic acid reaction carrier, and multiple nucleic acid reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multiple nucleic acid reaction tool capable of performing amplification and/or detection reaction of a plurality kinds of target sequences independently at the same time, and a reaction carrier, and a multiple nucleic acid reaction method.SOLUTION: This multiple nucleic acid reaction tool is equipped with: a support configured to support a liquid-phase reaction field; a plurality of primer immobilization regions independently arranged on at least one surface of the support which is in contact with the reaction field when the reaction field has been formed using the liquid phase; a plurality kinds of different primer sets fixed in a releasable manner to mutually independent primer immobilization regions for each kind of primer, and each configured to amplify the respective corresponding target sequence; and a thickener fixed to the primer immobilization regions in a releasable manner.

Description

  Embodiments described herein relate generally to a multinucleic acid reaction tool, a multinucleic acid reaction carrier, and a multinucleic acid reaction method.

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

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

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

  An object of the present invention is to provide a multinucleic acid reaction tool, a reaction carrier, and a multinucleic acid reaction method capable of independently and simultaneously performing amplification and / or detection reactions of a plurality of types of target sequences.

  According to the embodiment, the multi-nucleic acid reaction tool includes a support configured to support a liquid phase reaction field, and the support in contact with the reaction field when the reaction field is formed by the liquid phase. A plurality of primer-immobilized regions that are independently arranged on at least one surface of the body and a plurality of primer-immobilized regions that are independently releasably immobilized for each type and amplify a plurality of types of target sequences, respectively. A plurality of types of primer sets configured to be used, and a thickener releasably immobilized on the primer immobilization region.

The figure which shows an example of a multi-nucleic acid amplification reaction tool. The figure which shows an example of a multi-nucleic acid amplification reaction tool. The figure which shows an example of a multi-nucleic acid amplification reaction tool. The figure which shows an example of a multi-nucleic acid amplification reaction tool. The figure which shows an example of a multi-nucleic acid amplification reaction tool. The figure which shows one example of a multinucleic acid amplification detection reaction tool. The figure which shows one example of a multinucleic acid amplification detection reaction tool. The figure which shows an example of a chip | tip raw material. The figure which shows one example of a multinucleic acid amplification detection reaction tool. The figure which shows one example of a multinucleic acid amplification detection reaction tool. The figure which shows one example of a multinucleic acid amplification detection reaction tool. The figure which shows an example of a multi-nucleic acid reaction tool. The figure which shows the experimental result obtained by the multi-nucleic acid reaction tool.

    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 usable amplification method 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 and ICAN amplification Etc.

  “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.

2. Multi-Nucleic Acid Reactor <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.

  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. On the bottom surface of the support 2, a plurality of immobilization regions 3 independent from each other are arranged. FIG. 1B is an enlarged schematic view of the immobilization region 3. As shown there, one kind of primer set 4 is fixed to one immobilization region 3. A plurality of primer sets 4 are fixed to each of the plurality of immobilization regions 3 for each type.

  Plural types of primer sets 4 are prepared for amplifying a plurality of target nucleic acids. One kind of primer set 4 for amplifying one specific target nucleic acid is fixed to one immobilization region 3. For example, in the case of a PCR amplification reaction tool, a plurality of forward primers and reverse primers necessary for amplifying one type of specific target nucleic acid are included in one immobilization region. In the case of a reaction tool for LAMP amplification, in one immobilization region, FIP primer, BIP primer necessary for amplifying one kind of specific target nucleic acid, F3 primer, B3 primer as necessary, And a plurality of LP primers.

  The primer set 4 is fixed to the immobilization region 3 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 4 to the immobilization region 3 can be achieved, for example, by dropping a solution containing one set of primer sets into one immobilization region 3 and then drying. Furthermore, similarly, a solution containing a desired primer set 4 may be dropped and dried for the other immobilization regions 3 to fix a desired number of primer sets to the support 2. Thereby, the primer set 4 is fixed to all the fixing regions 3 that are independently arranged on one surface of the support 2. However, immobilization of the primer set 4 to the immobilization region 3 may be performed in a state where it can be released in contact with a liquid phase for providing a reaction field. Therefore, any immobilization method known per se capable of such immobilization may be used. In the method of dropping a solution containing a primer set, the solution containing a primer set may be, for example, water, a buffer solution, an organic solvent, or the like.

  The plurality of immobilization regions 3 arranged on the support 2 may be arranged independently of each other. Independently arranged means arranged at intervals that do not prevent amplification initiated and / or advanced for each primer set in the reaction field. For example, the adjacent immobilization regions 3 may be disposed in contact with each other, may be disposed in the vicinity of each other with a slight distance, or may be immobilized in a commonly used detection device such as a so-called DNA chip. The probes may be arranged at a distance similar to that of the probe. For example, the distance between adjacent immobilization regions 3 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. Good.

  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 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.

  In FIG. 1, the example in which the immobilization region 3 is disposed on the bottom surface of the support body 2 is illustrated, but the present invention is not limited thereto, and may be disposed on at least a part of the inner side surface of the support body 2. You may arrange | position to either an internal side surface, a ceiling surface, or all.

<Nucleic acid amplification reaction using multi-nucleic acid amplification reaction tool>
FIG. 2 is a diagram showing a state of a nucleic acid amplification reaction using the same multi-nucleic acid amplification reaction tool 21 as in the first embodiment. FIG. 2A shows the multi-nucleic acid amplification reaction tool 21 before the reaction. A plurality of primer sets 24 are respectively fixed to a plurality of immobilization regions 23 arranged on the bottom surface of the support 22. FIG. 2B shows a state in which the reaction solution 26 is added to the multi-nucleic acid amplification reaction tool 21 and accommodated therein.

  The reaction solution 26 may contain components necessary for a desired amplification reaction. Although not limited thereto, for example, in the case of simultaneously performing reverse transcription and a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from an enzyme such as a polymerase or a primer, A buffer such as reverse transcriptase and its necessary substrate, and salts for maintaining an appropriate amplification environment may be included.

  The reaction solution may be a liquid phase in which an amplification reaction between the primer set and the target nucleic acid is possible after the fixed primer set is released. This reaction solution may be injected into the reaction field (initially filled with air) where the primer is immobilized by some method mechanically or artificially before starting the amplification reaction. It is preferable that the reaction solution contains a thickener that does not inhibit the amplification reaction so that the immobilized primer stays in the local diffusion after being released. Details of the thickener will be described later.

  As shown in FIG. 2 (b), in the multi-nucleic acid amplification reaction tool after the addition of the reaction solution 26, the primer fixed on the bottom surface is liberated as shown schematically in FIG. 2 (c). Spread. The free and diffused area is schematically represented by area 27. The released and diffusing primer encounters other components necessary for amplification such as template nucleic acid, polymerase and substrate existing in the vicinity thereof, and the amplification reaction is started. A plurality of primer sets immobilized independently for each type can start and proceed with an amplification reaction for the template nucleic acid independently for each type. Thereby, amplification of a plurality of template sequences using a plurality of types of primer sets is achieved independently and simultaneously. Here, the “reaction field” is theoretically referred to as a region defined by the reaction solution 26 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 27, the region 27 is interpreted as a reaction region.

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

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

  When performing the multi-amplification reaction using the first embodiment described above, it is possible to achieve the multi-amplification reaction more efficiently by including a thickener in the reaction solution to be used.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

<Third Embodiment>
A further example of the multi-nucleic acid amplification reaction tool will be described with reference to FIG.

  FIG. 3 is a plan view showing a further example of a multi-nucleic acid amplification reaction tool. The multi-nucleic acid amplification reaction tool 31 described in FIG. 3 is an example in which a substrate is used as the support 32. A plurality of immobilization regions 33 independent of each other are disposed on one surface of the support 32. As in FIG. 1, one kind of primer set 34 is fixed to one immobilization region 33 in the immobilization region 33. A plurality of primer sets 34 are fixed to each of the plurality of immobilization regions 33 for each type. The configuration of the primer set 34 included in one immobilization region 33 includes different types of primers necessary for amplifying one type of specific target nucleic acid, as in the first embodiment. Further, a thickener 35 is fixed to the fixing region 33 by coating so as to cover the primer set 34.

  In the amplification using the second embodiment, a reaction field may be formed by placing the reaction solution on at least the region where the primer set 34 of the support 32 is immobilized.

  The immobilization region 33 may be disposed on a concave surface formed in advance on the surface of the support 32 or an inner wall of a flow path formed by the concave portion.

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

  Furthermore, the multi-nucleic acid amplification reaction tool of the second embodiment may be arranged in a container capable of maintaining it, and a reaction solution may be added to the container to form a reaction field. In this case, the primer set 34 may be fixed on both surfaces of the support 32. Furthermore, the thickener 35 may be fixed by coating so as to cover the primer set 34. Thereby, it becomes possible to fix more kinds of primer sets to the present multi-nucleic acid amplification reaction tool, and it becomes possible to amplify more target sequences. In this aspect, the multi-nucleic acid amplification reaction tool may be a support having any shape as long as it is a support in which a plurality of primer sets are independently fixed on at least one surface. In this case, the material of the support and the method for immobilizing the primer may be the same as those in the first embodiment and the second embodiment. Such a multi-nucleic acid amplification reaction tool is used as a multi-nucleic acid amplification reaction carrier comprising a plurality of types of primer sets that are releasably immobilized for each type in a substrate and at least one surface of the immobilization region independent of each other. May be. In this case, the practitioner may arbitrarily select the size and shape of the substrate. For example, the shape may be a plate shape, a spherical shape, a rod shape, or a part thereof.

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

<Fourth Embodiment>
(1) Multinucleic acid amplification reaction tool A further example of a multinucleic acid amplification reaction tool will be described with reference to FIG. The multi-nucleic acid amplification reaction tool 41 shown in FIG. 4 is an example using a support having a flow path. 4A is a plan view, and FIG. 4B is a cross-sectional view taken along line mm ′.

  The multi-nucleic acid amplification reaction tool 41 is provided with a plurality of primers which are releasably fixed for each type in a plurality of independent immobilization regions 43 arranged on the bottom surface of the flow path 44 formed in the support 42. It has.

  This multi-nucleic acid amplification reaction tool is manufactured using, for example, a first substrate and a second substrate. First, a plurality of primer sets 44 and a thickener mixture 45 are releasably fixed to a predetermined immobilization region 43 of the first substrate. The primer set 44 is fixed for each type. This immobilization can be performed as described above. On the other hand, a recess 46 is formed in the second substrate so as to correspond to the shape of a desired flow path. The recess 46 can be formed by a method known per se according to the material of the substrate to be used. In addition, the arrangement of the immobilization region 43 is determined so as to be included in the flow path formed by the recess 46 formed in the second substrate. Next, the first substrate and the second substrate are integrated. As a result of the integration, a reaction portion 47 having a channel shape is formed by the inner wall of the recess 46 and the surface of the first substrate on the second substrate side. At this time, the concave portion 46 of the second substrate is integrated so as to face the first substrate side. Further, a through hole (not shown) may be provided in a part of the recess 46 of the second substrate. This through hole may be used as an inlet / outlet for a reaction liquid to / from the flow path.

  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, an example is shown in which the primer set is fixed to the bottom surface of the support body having the flow channel, but the arrangement and shape of the flow channel are not limited to this. In addition, the surface on which the probe set is fixed may be any surface that defines the flow path, the probe set may be fixed to all surfaces that define the flow path, or may be fixed to a plurality of surfaces. Also good.

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

(2) Primer 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 or more, about 20 bases or more, about 25 bases or more, about 30 bases or more, about 35 bases or more, about 40 bases or more, about 45 bases or 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 may be sufficient, and the range which combined either of these minimums and upper limits may be sufficient. 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.

(3) Multinucleic Acid Amplification and Detection As shown in FIG. 5, the multinucleic acid amplification reaction tool 51 includes a tube 52 as a support and a plurality of primer sets that are independently fixed to the inner surface of the tube 52. Even in such a tube-type multi-nucleic acid amplification reaction tool 51, it is possible to amplify a plurality of types of target nucleic acids. Thereafter, the obtained amplification product can be detected by adding it to a device such as a DNA chip 55 to which a plurality of different nucleic acid probes 56 are fixed. As described above, the multi-nucleic acid amplification reaction tool shown as an example in the embodiment is capable of preparing a detection sample more easily and performing a plurality of amplifications independently as compared with the conventional case.

(4) Multi-nucleic acid amplification method A method for amplifying a plurality of target nucleic acids using a multi-nucleic acid amplification reaction tool as described above as an example is also provided as one embodiment.

  In addition, multiple types of primer sets designed to amplify multiple types of target nucleic acids are released to at least one surface of a support such as a substrate on which a specific container, tube, dish or flow path is formed. A multi-nucleic acid amplification reaction comprising a possible immobilization step is also provided as a further embodiment.

  Such a multi-nucleic acid amplification reaction, for example, releasably immobilizes a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on at least one surface of a desired support. And a reagent necessary for amplification, for example, an enzyme such as a polymerase, and a deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer so that a plurality of primer sets are included in one reaction field. When a substrate such as an acid and reverse transcription are simultaneously performed, a reaction solution containing a buffer such as reverse transcriptase and a substrate necessary for the reverse transcriptase and salts for maintaining an appropriate amplification environment is added. Adjusting the reaction environment suitable for the amplification reaction, such as adjusting the temperature by heating or cooling the support on which the primer is fixed; It may comprise and performing a multi-nucleic acid amplification reaction by Les.

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

  Furthermore, a multi-nucleic acid amplification reaction including a step of releasably fixing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on the surface of a substrate such as a microbead, a plate piece, or a rod. Provided as a further embodiment.

  Such multi-nucleic acid amplification reaction includes, for example, releasably immobilizing a plurality of types of primer sets designed to amplify a plurality of types of target nucleic acids, respectively, on at least one surface of a desired substrate. When a substrate is used as a reagent necessary for amplification, for example, an enzyme such as polymerase, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, and reverse transcription at the same time Such as reverse transcriptase and its necessary substrates, and further placing it in a reaction solution containing a buffer such as salts to maintain an appropriate amplification environment, and adjusting the temperature by heating or cooling the reaction solution, etc. Adjusting the reaction environment suitable for the amplification reaction and thereby performing the multi-nucleic acid amplification reaction.

<Fifth Embodiment>
(1) Multi-nucleic acid amplification detection reaction tool The multi-nucleic acid reaction detection tool may be provided as a multi-nucleic acid amplification detection reaction tool. In addition to the first to fourth embodiments as described above, the multi-nucleic acid amplification detection reaction tool further includes a probe-immobilized region and a probe nucleic acid immobilized thereon. Next, an example of the multinucleic acid amplification detection reaction tool will be described.

  One example of the multinucleic acid amplification detection reaction tool will be described with reference to FIGS. 6 (a), (b) and (c).

  FIG. 6A is a perspective view of an example of a multi-nucleic acid amplification detection reaction tool. The multi-nucleic acid amplification detection reaction tool 61 shown in FIG. 6A has a container-shaped support 62. A plurality of primer immobilization regions 64 that are independent from each other are disposed on the bottom surface 63 of the support 62. A plurality of probe immobilization regions 65 are arranged adjacent to the plurality of primer immobilization regions 64 and corresponding to each primer region.

  FIG. 1B is an enlarged schematic view of the cross section of the primer immobilization region 64. As shown there, one kind of primer set 66 is fixed to one primer fixing region 64. In each of the plurality of primer immobilization regions 64, a plurality of primer sets 64 are fixed for each type.

  Plural types of primer sets 66 are prepared for amplifying a plurality of target nucleic acids. One primer set 66 for amplifying one specific target nucleic acid is fixed to one primer fixing region 64. For example, in the case of a PCR amplification reaction tool, a plurality of forward primers and reverse primers necessary for amplifying one type of specific target nucleic acid are included in one primer-immobilized region. In the case of a LAMP amplification reaction tool, one primer-immobilized region has an FIP primer, a BIP primer, and an F3 primer and a B3 primer as necessary to amplify one specific target nucleic acid. , And a plurality of LP primers.

  The primer set 66 is fixed to the primer immobilization region 64 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 66 to the primer immobilization region 64 can be achieved, for example, by dropping a solution containing one set of primer sets onto one primer immobilization region 64 and then drying. Further, similarly, a solution containing a desired primer set 66 may be dropped and dried for each of the other primer immobilization regions 64, and a desired number of primer sets may be fixed to the support 62. As a result, the primer set 66 is fixed to all the fixing regions 64 that are independently arranged on one surface of the support 62. However, the primer set 66 may be immobilized on the immobilization region 64 in such a manner that it can be released in contact with a liquid phase for providing a reaction field. Therefore, any immobilization method known per se capable of such immobilization may be used. In the method of dropping a solution containing a primer set, the solution containing a primer set may be, for example, water, a buffer solution, an organic solvent, or the like.

  The plurality of primer immobilization regions 64 arranged on the support 62 may be arranged independently of each other. Independently arranged means arranged at intervals that do not prevent amplification initiated and / or advanced for each primer set in the reaction field. For example, the adjacent primer immobilization regions 64 may be arranged in contact with each other, may be arranged in the vicinity of each other with a slight distance, or fixed in a commonly used detection device such as a so-called DNA chip. The probe nucleic acids may be arranged at the same distance from each other at the same distance.

  For example, the distance between adjacent primer immobilization regions 64 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 body 62 may be, for example, a tube, a well, a chamber, a flow path, a cup, 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.

  In FIG. 1, the example in which the primer immobilization region 64 is disposed on the bottom surface 63 of the support 62 is shown, but the present invention is not limited thereto, and may be disposed on at least a part of the inner side surface of the support 62. It may be arranged on any or all of the bottom surface, the inner side surface, and the ceiling surface defined by the covering.

  FIG. 6C is an enlarged view of the probe immobilization region 65 arranged in the vicinity of the primer immobilization region 64. A plurality of probe nucleic acids 67 including a complementary sequence of a desired sequence to be detected are immobilized on the probe immobilization region 65.

  The desired sequence to be detected may be the target sequence. The probe immobilization region 65 is arranged such that a hybridization signal between the probe nucleic acid 67 and the target sequence chain is detected independently between the plurality of probe immobilization regions 65.

  For fixing the probe nucleic acid 67 to the probe immobilization region 65, any general technique for immobilizing the probe nucleic acid to the substrate surface in a so-called DNA chip known per se may be used. The primer 66 may be fixed after the probe nucleic acid 67 is fixed, the probe nucleic acid 67 may be fixed after the primer 66 is fixed, or the primer 66 and the probe nucleic acid 67 may be fixed simultaneously.

  For example, the distance between adjacent probe immobilization regions 65 may be 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or more, preferably 100 μm to 10 mm It may be.

  Further, for example, the distance between the probe immobilization region 65 and the primer immobilization region 64 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 65 and the primer immobilization region 64 is 0 μm, it is understood that the probe immobilization region 65 and the primer immobilization region 64 are at the same position on the support surface. Good. The probe immobilization region 65 may be included in the primer immobilization region 64, and the primer immobilization region 64 may be included in the probe immobilization region 65.

(2) Nucleic Acid Amplification Detection Method Using Multinucleic Acid Amplification Detection Reactor FIG. 7 shows a reaction field after a nucleic acid amplification reaction performed using a multinucleic acid amplification detection reaction tool 70 similar to that of the fifth embodiment. It is a schematic diagram which shows the mode of. FIGS. 7A-1 and 7B-1 show the multi-nucleic acid amplification detection reaction tool 70 before the reaction. A plurality of primer immobilization regions 73 are disposed on the bottom surface 72 of the support 71. Probe immobilization regions 74 are arranged in the vicinity of the plurality of primer immobilization regions 73. A plurality of primer sets 75 are fixed to the plurality of primer fixing regions 73, respectively. Corresponding to each primer immobilization region 73, a plurality of probe nucleic acids 76 are immobilized for each desired type in the probe immobilization region 74 arranged in the vicinity thereof.

  A state in which the reaction solution 78 is added to the multi-nucleic acid amplification detection reaction tool 70 and accommodated therein is shown in FIGS.

  The reaction solution 78 may contain components and a thickener necessary for a desired amplification reaction. Although not limited thereto, for example, in the case of simultaneously performing reverse transcription and a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from an enzyme such as a polymerase or a primer, A buffer such as reverse transcriptase and its necessary substrate, and salts for maintaining an appropriate amplification environment may be included.

  The thickener may contain the same kind of substance as in Example 1 at the same concentration.

  The sample may be added to the reaction field by adding the reaction solution 78 to the reaction solution 78 in advance before adding the reaction solution 78 to the multi-nucleic acid amplification detection reaction device 70. Alternatively, the sample may be added to the multi-nucleic acid amplification detection reaction tool 70 before the reaction solution 78 is added to the multi-nucleic acid amplification detection reaction tool 70.

  As shown in FIGS. 7A-2 and 7B-2, the multi-nucleic acid amplification detection reaction tool 70 after the reaction solution 78 is added is schematically shown in FIGS. 7A-3 and 7B-3. As shown, the primer 76 fixed to the bottom surface 72 inside the support 71 is released and gradually diffuses. The free and diffused area is schematically indicated by area 79. The primer set 76 that is released and diffused 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. Plural primer sets 76 that are independently fixed for each type can start and proceed with the 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 76 is achieved independently and simultaneously. Here, the “reaction field” is theoretically referred to as a region defined by the reaction solution 78 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 79, the region 79 is interpreted as a reaction region. The diagram of FIG. 7A-3 is a schematic diagram when an amplification reaction is caused by the primer set 76 fixed to all the primer fixing regions 73. FIG. FIG. 7 (b-3) shows a part of all the primer-immobilized regions 73 fixed to the bottom surface 72 by the fixed primer set 76, and FIG. 7 (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 79, the probe-immobilized region 74 hybridizes with the nucleic acid. The probe nucleic acid 77 immobilized on the probe immobilization region 74 is immobilized so as to hybridize only with the amplification product in the corresponding primer immobilization region 73. That is, the probe nucleic acid 77 fixed to one probe immobilization region 74 is maintained at a distance so as to hybridize only with the amplification product in the corresponding primer immobilization region 73, and each probe immobilization region 74 and primer An immobilization region 73 is disposed.

  Detection of hybridization between the probe nucleic acid 77 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 deoxynucleoside 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 amplification detection reaction tool 70 is cleaned, or may be performed without cleaning. When detecting by electrochemical means, the hybridizing signal may be detected using an intercalator. In this case, for example, an intercalator may be included in the reaction solution 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 amplification detection reaction tool 70 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 may be the same as that described in the fourth embodiment.

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

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

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

  The type of primer set 76 fixed to one primer immobilization region 73 may be one type for amplifying one type of target nucleic acid, or a plurality of types for amplifying two or more types of target nucleic acids. It may be.

  The type of the probe nucleic acid 77 group immobilized on one probe immobilization region 74 may be one type for hybridizing with one type of target sequence, in order to amplify two or more types 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 immobilization regions 73 arranged in one array type multi-nucleic acid amplification detection reaction tool 70 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, upper limit 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 is a range in which any of these upper and lower limits is combined. Also good.

  The number of primer-immobilized regions 73 and probe-immobilized regions 74 arranged in one multi-nucleic acid amplification detection reaction tool 70 may be the same or different. That is, the same number of probe immobilization regions 74 may be arranged so as to correspond to all the primer immobilization regions 73, and the number of primer immobilization regions 73 may be larger than the number of probe immobilization regions 74, The number of primer immobilization regions 73 may be smaller than the number of probe immobilization regions 74. 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, the example in which only the primer set 76 is fixed to the support 71 is shown. However, the present invention is not limited to this, and other components necessary for amplification, such as an enzyme such as a polymerase or reverse transcriptase, a substrate, A substrate and / or a buffering agent may be fixed to the support 71 together with the primer set 76. In that case, the substance to be fixed may be included in a desired liquid medium together with the primer set 76, and fixed by dropping, drying, or the like in the same manner as described above. In such a multi-nucleic acid amplification detection reaction tool 70, when performing an amplification reaction, the composition of the reaction solution added thereto may be selected according to the fixed component.

  Moreover, although the example which adds a thickener to a reaction liquid was shown in the above-mentioned example, you may fix to the support body 71, without including a thickener in a reaction liquid. Fixing may be performed as described above.

  The support 71 is not limited to the container shape, and as described above, the support 71 may have a plate shape, a spherical shape, a rod shape, or a part thereof, and the size and shape of the substrate can be arbitrarily determined by the practitioner. You may choose. Moreover, it is preferable to use a substrate having a flow path as the support 71 as in the fourth embodiment.

<Sixth Embodiment>
A multi-nucleic acid amplification detection reaction tool according to a sixth embodiment will be described with reference to FIGS.

(1) Chip Material First, an example of the structure and manufacturing method of a chip material of a multi-nucleic acid amplification detection reaction tool that detects a hybridization signal by electrochemical detection will be described with reference to FIGS. 8 (a) and 8 (b). . 8A is a plan view of the chip material, and FIG. 8B is a cross-sectional view taken along line BB of the chip material of FIG. 8A.

  The chip material 91 includes, for example, four electrodes 84a to 84d arranged on the rectangular substrate 81 along the longitudinal direction thereof. Each of the electrodes 84a to 84d has a structure in which a first metal thin film pattern 82 and a second metal thin film pattern 83 are stacked in this order. Each of the electrodes 84 a to 84 d has a shape in which a large rectangular portion 85 and a small rectangular portion 86 are connected by a thin wire 87. The insulating film 88 is covered on the substrate 81 including the electrodes 84a to 84d. The circular window 89 is opened in the insulating film 88 corresponding to the large rectangular portion 85. The rectangular window 90 is opened in the insulating film 88 corresponding to the small rectangular portion 86. The large rectangular portion 85 exposed from the circular window 89 of the electrode 84a functions as the first working electrode 92a. The large rectangular portion 85 exposed from the circular window 89 of the electrode 84b functions as the second working electrode 92b. The large rectangular portion exposed from the circular window 89 of the electrode 84 c functions as the counter electrode 93. The large rectangular portion exposed from the circular window 89 of the electrode 84d functions as the reference electrode 94. The small rectangular portion 86 exposed from the rectangular windows 90 of the electrodes 84a to 84d 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 81 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 82 and a second metal thin film pattern 83 are laminated in this order. 84 d is formed along the longitudinal direction of the substrate 81. These electrodes 84 a to 84 d have a shape in which a large rectangular portion 85 and a small rectangular portion 86 are connected by a thin wire 87.

  Next, an insulating film 88 is deposited on the substrate 81 including the electrodes 84a to 84d by, for example, a sputtering method or a CVD method. Subsequently, the insulating film 88 portion corresponding to the large rectangular portion 85 of each electrode 84a to 84d and the insulating film 88 portion corresponding to the small rectangular portion 86 are selectively etched using the resist pattern as a mask to form the large rectangular portion 85. A circular window 89 is opened in the corresponding insulating film 88 portion, and a rectangular window 90 is opened in the insulating film 88 portion corresponding to the small rectangular portion 86. Thereby, the above-described chip material 91 is produced.

  The substrate 81 is made of glass such as Pyrex (registered trademark) glass or resin, 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 81, 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 88 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 88 include plasma etching using an etching gas or reactive ion etching.

(2) Multi-Nucleic Acid Amplification Detection Reactor Next, an example of the configuration and production method of the multi-nucleic acid amplification detection reaction tool in which the primer set and the probe nucleic acid are fixed to the chip material 91 manufactured in (1) above is shown in FIG. Description will be made with reference to a) and FIG. 9A is a plan view of the multi-nucleic acid amplification detection reaction tool, and FIG. 9B is a cross-sectional view taken along line BB of the multi-nucleic acid amplification detection reaction tool of FIG. 9A.

  The first working electrode 92a of the electrode 84a formed on the chip material 91 is used as a first probe fixing region, and the first probe including a complementary sequence of the first target sequence in the first probe fixing region. The nucleic acid 95a is fixed. A plurality of the first probe nucleic acids 95a to be fixed are fixed as one probe nucleic acid group. Similarly, the second working electrode 92b of the electrode 84b is used as a second probe immobilization region, and the second probe immobilization region includes a second target sequence that is different from the first target sequence. The second probe nucleic acid 95b is immobilized.

  Examples of the method for immobilizing the probe nucleic acids 95a and 95b to the probe immobilization region include a method of introducing a thiol group at the 3 'end into the first probe nucleic acid 96a for the chip material 91 having a gold electrode.

  Next, the first primer immobilization region 96a is disposed in the vicinity of the first working electrode 92a, and the second primer immobilization region 96b is disposed in the vicinity of the second working electrode 92b. A first primer set 98a is fixed on the first primer fixing region 96a, and a second primer set 98b is fixed on the second primer fixing region 96b. Thereby, a multi-nucleic acid amplification detection reaction tool is prepared.

  The first primer set 98a has a sequence designed to amplify the first target sequence, and the second primer immobilization region 96b is a second target sequence comprising a sequence different from the first target sequence. Has a sequence designed to amplify.

  For fixing the first and second primer sets 98a and 98b to the first and second primer fixing regions 96a and 98b, respectively, the primer set is contained in a liquid such as water, a buffer solution or an organic solvent. For example, in the case of room temperature, it is allowed to stand for 10 minutes until the film is dried under an appropriate temperature condition such as room temperature.

(3) Multi-nucleic acid amplification detection reaction tool at the time of use The usage method of the multi-nucleic acid amplification detection reaction tool prepared in the above (2) will be described with reference to FIGS.

  FIG. 10A is a plan view of the multi-nucleic acid amplification detection reaction tool in use, and FIG. 10B is a cross-sectional view taken along line BB of the multi-nucleic acid amplification detection reaction tool of FIG. It is.

  When the multi-nucleic acid amplification detection reaction tool 91 of this embodiment is used, the first working electrode 92a, the second working electrode 92b, the counter electrode 93 and the reference electrode 94 formed on the electrodes 84a to 84d, respectively, and the first The reaction solution is maintained so that the primer region 96a and the second primer region 96b are included in the same one reaction field. For this purpose, for example, a silicone resin such as silicone rubber and / or a resin such as a fluororesin is used, for example, any one known per se, such as extrusion, injection molding or stamping and / or adhesion with an adhesive. The covering 101 molded by the resin molding method is mounted on the multi-nucleic acid amplification detection reaction tool 91 before the multi-nucleic acid amplification detection reaction tool 91 is used. After the covering 101 is mounted, the reaction solution 102 containing the template nucleic acid 103 is added to the space formed by the multi-nucleic acid amplification detection reaction tool 91 and the covering 101.

  In the multi-nucleic acid amplification detection reaction tool 91 to which the covering 101 is attached, the small rectangular portion 86 exposed from each rectangular window 90 of the electrodes 84a to 84d is exposed.

  Examples of mounting the covering 101 on the multi-nucleic acid amplification detection reaction tool 91 include, for example, pressure bonding, bonding with an adhesive, and the like.

  Next, the reaction solution 102 is added after the covering 101 is mounted on the multi-nucleic acid amplification detection reaction tool 91.

  For example, the liquid may be added to the space formed by the multi-nucleic acid amplification detection reaction tool 91 and the covering 101 by, for example, providing an opening in a part of the covering 101 in advance and adding the liquid from the opening. Alternatively, it may be added by being inserted into a part of the covering 101 using an injector having a sharp tip such as a needle.

  The reaction solution 102 comprises a sample, a thickener, an amplification reagent, for example, an enzyme such as a polymerase, a substrate such as deoxynucleoside triphosphate necessary for forming a new polynucleotide chain starting from a primer, reverse transcription, and the like. Are simultaneously recognized, the reverse transcriptase and the necessary substrate, and other buffers such as salts for maintaining an appropriate amplification environment and double-stranded nucleic acid such as Hoechst 33258 are recognized and signaled. May include an intercalator that produces When the template nucleic acid containing the target sequence to be amplified by the primer set immobilized on the specific primer immobilization region is present in the sample to be examined, the primer immobilization region and the corresponding probe immobilization region are included. An amplification product is formed in the reaction field. This is schematically shown in FIG.

  FIG. 11A schematically shows a state in which an amplification product is formed in the reaction field 111. FIG. 11A is a plan view of the multi-nucleic acid amplification detection reaction tool in use, and FIG. 11B is a cross-sectional view taken along line BB of the multi-nucleic acid amplification detection reaction tool of FIG. It is. As described above, the sample added in FIG. 10 contains a nucleic acid containing a sequence that can be bound by the second primer set 98b, and as shown in FIGS. 11 (a) and 11 (b). In the reaction field 111, the second primer set is released and diffused, and after encountering the template nucleic acid, an amplification reaction is performed, whereby an amplification product is formed. The amplification product by the second primer set 98b diffuses around the second primer immobilization region 97b and reaches the second probe immobilization region 92b. When the reached amplification product contains the target sequence, the second probe nucleic acid 95b and the amplification product hybridize to form a double-stranded nucleic acid. The intercalator contained in the reaction solution 102 is combined with this double-stranded nucleic acid to generate a hybrid signal.

  The hybridization signal is generated by, for example, bringing a prober into contact with the small rectangular portion 86 exposed from each rectangular window 90 of the electrodes 84a to 84d and measuring the current response of an intercalator such as Hoechst 33258.

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

<Method of detecting target nucleic acid>
A method for amplifying a plurality of target nucleic acids using the multi-nucleic acid amplification detection reaction tool as described above as an example and detecting a target nucleic acid using a hybridization signal as an index is also provided as a further embodiment.

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

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

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

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

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

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

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

  Further, instead of adding the thickener to the reaction solution, the thickener may be fixed to the support as described above.

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

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

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

[Example]
Example 1
Using the fourth embodiment, the nucleic acid status of the primers was evaluated.

  A fluorescently labeled primer set was prepared. FIG. 12 shows the multi-nucleic acid reaction device 121 used.

  The prepared primer set was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) to a final concentration of 200 μM. A fixed solution was prepared by dissolving agarose in this solution to a final concentration of 0.3%. A glass substrate was used as the support 122. This fixing solution is dropped at three points on the center of the support 122 (indicated by 123 in the figure) and two corners of the support 122 (indicated by 124 and 125 in the figure) and left at room temperature. And dried. A silicon rubber plate having a groove 126 formed on one surface thereof was used as the covering 127. Through holes 128 and 129 are formed at two ends of the groove 126 of the covering body 127, respectively. The covering body 127 was adhered to the supporting body 122 so that the region where the primer set and the agarose of the supporting body 122 were fixed was included in the groove portion 126 of the covering body 127. Thereby, a multi-nucleic acid reaction tool was obtained. In this multi-nucleic acid reaction tool, a channel 130 is formed by the groove 126 of the covering 127 and the surface of the support 122.

  The two through holes of the covering 127 were used as the inlet 128 and the outlet 129, respectively. PBS was added from the inlet 128. Thereafter, the diffusion state of the primer was observed using the fluorescence intensity as an index.

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

  The results are shown in FIGS. 13 (a) and (b). FIG. 13 (a) shows the results obtained in the control multi-nucleic acid reaction device in which only the primers are fixed. FIG. 13 (b) shows the results obtained in the multi-nucleic acid reaction device in which the primer set and agarose are fixed.

  The distance of the control multinucleic acid reaction tool moved by adding TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was larger than that of the multinucleic acid reaction tool in which the primer set and agarose were fixed. From this result, it was clarified that the primer set and the multi-nucleic acid reaction device to which agarose is immobilized can diffuse the primer more locally.

Example 2
A multi-nucleic acid amplification detection reaction tool for electrochemical detection similar to that of the sixth embodiment was produced.

(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. Table 1 shows the base sequence of the probe DNA used.

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

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

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

First, primer DNA to be used was prepared. The primer DNA to be used is a primer set for amplification by a loop-mediated isal amplification (LAMP) method. The base sequences of the primer DNA are shown in Tables 2A, 2B, and 2C.

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

  Specifically, 0.200 μL of each prepared solution was spotted on the bottom of the groove of the covering and dried at 40 ° C. for 2 minutes. Spotting was performed so that each probe DNA would be at a position facing the corresponding primer set when the covering was attached to the DNA chip. The covering and the chip material prepared above were bonded so that the groove portion of the covering and the surface on which the probe DNA was immobilized faced each other. Thereby, 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 is shown in Tables 3 to 6.

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

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

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

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

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

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

  Composition (4) does not contain a template.

Table 7A, Table 7B and Table 7C show the nucleotide sequences of Template A, Template B, Template C, Template D, Template E, Template F, Template G, Template H, Template I, Template J, Template K, Template L and Template M. Shown in

(4) LAMP amplification reaction on 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 results The detection results are shown in Table 8.

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

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

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

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

  From these results, the template A, template C, template E, template G, template I, template K, and template M contained in the LAMP reaction solution were converted to primer sets corresponding to the multinucleic acid amplification detection reaction tool of the embodiment. It was detected that it was amplified and hybridized with the corresponding probe nucleic acid.

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

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

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

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

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

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

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

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

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

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

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

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

The results are shown in Table 9.

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

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

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

  From the results of Examples 2 and 3 above, the following was shown. As described in this example, multiple types of templates contained in the LAMP reaction solution can be detected with higher accuracy by adding a thickener together with the fixation of the primer set in the multi-nucleic acid amplification detection reaction device according to the embodiment. It has been shown that it is possible to identify the sequence.

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

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

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

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

Claims (19)

  A support configured to support a reaction field in a liquid phase, and when the reaction field is formed by the liquid phase, the support is independently disposed on at least one surface of the support in contact with the reaction field. A plurality of primer-immobilized regions, and a plurality of types of primer sets that are independently and releasably immobilized for each type in the plurality of primer-immobilized regions, and are configured to amplify a plurality of types of target sequences, respectively. A multi-nucleic acid reaction device comprising a thickener releasably immobilized on the primer immobilization region.   The multi-nucleic acid amplification reaction tool according to claim 1, further comprising a covering attached to a surface of the support that supports the reaction field, wherein the covering is fixed to at least all primers of the support. A groove portion formed in a portion corresponding to the region including the control region, and a through-hole opened at one end and the other end of the groove portion, and the groove portion of the covering body and the reaction portion of the support body A multi-nucleic acid reaction tool in which a reaction part is formed by a supporting surface.   3. The method according to claim 1, further comprising: a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions; and a plurality of probe nucleic acids immobilized on the plurality of probe immobilization regions. Multi-nucleic acid reaction tool.   The multi-nucleic acid reaction device according to any one of claims 1 to 3, wherein the thickener is agar or gelatin.   The multi-nucleic acid reaction tool according to any one of claims 1 to 4, wherein the thickener is fixed so as to cover the primer.   The multi-nucleic acid reaction tool according to any one of claims 1 to 4, wherein the thickener is fixed together with the primer in the primer-immobilized region.   A plurality of target sequences, each of which is releasably immobilized on a support and a plurality of primer-immobilized regions independent of each other on at least one surface of the support, each of which is configured to amplify each of the target sequences. A multi-nucleic acid reaction carrier comprising a kind of primer set and a thickener releasably immobilized in the primer immobilization region.   The multi of claim 7, further comprising a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions, and a plurality of probe nucleic acids immobilized on the plurality of probe immobilization regions. Nucleic acid reaction carrier.   The multi-nucleic acid reaction carrier according to claim 7 or 8, wherein the thickener is agar or gelatin.   The multi-nucleic acid reaction carrier according to any one of claims 7 to 9, wherein the thickener is immobilized so as to cover the primer.   The multi-nucleic acid reaction carrier according to any one of claims 7 to 9, wherein the thickener is immobilized in the primer immobilization region together with the primer.   A plate-like support, a cover fixed to one surface of the support and extending in the axial direction to a groove extending in the surface on the support, the groove of the cover, and the one of the supports A flow path constituted by a surface, a first through hole opened at one end of the flow path, a second through hole opened at the other end of the flow path, and primer fixing of the inner wall of the flow path A multi-nucleic acid amplification reaction tool comprising a plurality of primer sets that are releasably fixed to the activating region and a thickener that is releasably fixed to the primer-immobilizing region, wherein the plurality of primer sets are A multi-nucleic acid reaction tool that is fixed to the primer-immobilized region independently for each type, and that one primer set includes a plurality of primers for amplifying one target nucleic acid.   The multi of claim 12, further comprising a plurality of probe immobilization regions arranged in the vicinity of the plurality of primer immobilization regions, and a plurality of probe nucleic acids immobilized in the plurality of probe immobilization regions. Nucleic acid reaction tool. (A) a plate-like support, a covering that is fixed to one surface of the support and that extends in the axial direction to the surface on the support, the groove of the covering, and the support A flow path constituted by one surface, a first through-hole opened at one end of the flow path, a second through-hole opened at the other end of the flow path, and an inner wall of the flow path A plurality of primer-immobilized regions arranged independently of each other; a plurality of primer sets releasably fixed to the plurality of primer-immobilized regions; and a thickener releasably fixed to the primer-immobilized region. Providing a multi-nucleic acid reaction device comprising an agent;
Here, the plurality of primer sets are independently fixed to the primer fixing region independently for each type, and one primer set includes a plurality of primers for amplifying one target nucleic acid,
(B) adding a reaction solution containing a target nucleic acid to the channel from the first opening;
(C) amplifying the target nucleic acid;
A multi-nucleic acid reaction method comprising: (A) a plate-like support, a cover that is fixed to one surface of the support, and that has an axially extending groove that opens to the surface on the support, the groove of the cover, and the surface of the support A flow path constituted by one surface, a first through-hole opened at one end of the flow path, a second through-hole opened at the other end of the flow path, and an inner wall of the flow path Preparing a multi-nucleic acid reaction device comprising a plurality of primer-immobilized regions arranged independently of each other, and a plurality of primer sets fixed releasably to the plurality of primer-immobilized regions, respectively.
Here, the plurality of primer sets are independently fixed to the primer immobilization region for each type, and one primer set includes a plurality of primers for amplifying one target nucleic acid,
(B) adding a reaction liquid containing a target nucleic acid and a thickener to the channel from the first opening;
(C) amplifying the target nucleic acid;
A multi-nucleic acid reaction method comprising: The multi-nucleic acid reaction device further comprises a plurality of probe immobilization regions arranged in the vicinity of each of the plurality of primer immobilization regions, and a probe nucleic acid immobilized on the plurality of probe immobilization regions,
(E) detecting a hybridization signal between the amplification product obtained in (c) and the probe nucleic acid;
The multi-nucleic acid reaction method according to claim 14 or 15, further comprising:   The multi-nucleic acid reaction method according to any one of claims 14 to 16, wherein the thickener is immobilized on the primer-immobilized region after the primer set is immobilized on the primer-immobilized region.   The multi-nucleic acid reaction method according to claim 14, wherein a mixture of the primer set and the thickener is immobilized on the primer immobilization region.   The multi-nucleic acid reaction method according to any one of claims 14 to 18, wherein the reaction solution is added at a flow rate of 10 mm / second or more.

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