JP2014060959A - Nucleic acid analysis method, and dna chip and assay kit for the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multiple nucleic acid reaction tool, a nucleic acid analysis method and an assay kit, capable of carrying out amplification reaction and hybridization reaction in one reaction field.SOLUTION: The method includes the steps of: preparing a sample, a base substance 2, a primer set 7 for amplifying target nucleic acid, and a nucleic acid probe 6 for detecting an amplified product; coating salt 8 with hardened oil or wax 9; immobilizing a nucleic acid probe on the surface of the base substance; immobilizing the primer set at the same location as the nucleic acid probe; bringing reaction mixture containing the sample and the coated salt capsule into contact with the nucleic acid probe and the primer set on the base substance, thereby amplifying target nucleic acid; melting hardened oil or wax by elevating the temperature of the reaction mixture, and eluting the salt; hybridizing the amplified product obtained by the amplification reaction with the nucleic acid probe; and measuring a produced hybridization signal to detect the presence or absence and/or quantity of target nucleic acid in the sample.

Description

  Embodiments described herein relate generally to a nucleic acid analysis method, a DNA chip therefor, and an assay kit.

  A DNA chip is a device in which nucleic acid probes are immobilized at high density on a glass or silicon substrate. When performing gene analysis with high accuracy using a DNA chip, gene amplification is required as pretreatment. The gene amplification is performed separately from the DNA chip, for example, in a separate reaction vessel such as a tube, and a salt or the like is appropriately added to the amplified product and reacted with the nucleic acid probe on the DNA chip. When reacting in a tube, it is difficult to multi-amplify a plurality of types of target nucleic acids, and in fact, multi-amplification of 3 to 4 or more types of target nucleic acids is difficult. Therefore, when the number of target nucleic acids to be detected increases, the number of tubes increases, the operation is complicated, and the reagent is expensive.

  In addition, optimal reaction conditions differ between the amplification reaction and the detection reaction in the DNA chip, that is, the hybridization reaction.

  The problem to be solved by the present invention is to provide a nucleic acid analysis method capable of performing an amplification reaction and a hybridization reaction in one reaction field, a DNA chip and an assay kit therefor.

  The nucleic acid analysis method according to the embodiment includes a step of preparing a specimen, a substrate, a primer set for amplifying a target nucleic acid, and a nucleic acid probe for detecting a product amplified using the primer set; Coating with oil or wax; immobilizing a nucleic acid probe on at least one surface of a substrate; immobilizing a primer set at or near the same position as the nucleic acid probe; reaction comprising an analyte and a coated salt capsule Contacting the solution with the nucleic acid probe and primer set of the substrate and amplifying the target nucleic acid by amplification reaction; after the amplification reaction, the temperature of the reaction solution is raised to melt the hardened oil or wax and elute the salt A step of hybridizing an amplification product obtained by the amplification reaction with a nucleic acid probe; and a resulting hybrid By measuring the size signal, comprising the step of detecting the presence and / or amount of a target nucleic acid in the sample.

The figure which shows the example of the DNA chip which concerns on embodiment. The figure which shows the example of the DNA chip which concerns on embodiment. The figure which shows the example of the DNA chip which concerns on embodiment. The figure which shows the chip | tip raw material of the array type primer probe chip which concerns on embodiment. The figure which shows the example of the array type primer probe chip which concerns on embodiment. The figure which shows the state at the time of use of the example of the array type primer probe chip which concerns on embodiment. The figure which shows the state at the time of use of the example of the array type primer probe chip which concerns on embodiment. FIG. 4 is a diagram showing the results of Example 1. 3 is a graph showing the results of Example 1. The graph which shows the result of Example 2.

1. Definitions “Nucleic acid” is a general term for substances capable of representing a part of the structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo. For example, the nucleic acid may be a partially or completely artificially synthesized and / or designed artificial nucleic acid, such as naturally occurring DNA and RNA, and mixtures thereof.

  The “specimen” is a target to be subjected to the analysis method according to the present embodiment, and may be any sample that may contain a nucleic acid. The specimen is preferably in a state that does not interfere with the amplification reaction and / or the hybridization reaction. For example, in order to use a material obtained from a living body or the like as a specimen according to the present embodiment, pretreatment may be performed by any means known per se. For example, the specimen may be a liquid.

  A “nucleic acid probe” is a nucleic acid comprising a sequence that is complementary to a target sequence. The nucleic acid probe may be immobilized on a known solid phase. A preferred nucleic acid probe is used by being immobilized on the surface of a substrate. An apparatus including such a substrate and a nucleic acid probe immobilized on the substrate may be generally performed by a DNA chip.

  A “DNA chip” is an apparatus that analyzes a nucleic acid using a hybridization reaction between a nucleic acid probe having a sequence complementary to the nucleic acid to be detected and the nucleic acid to be detected. The term DNA chip is synonymous with commonly used terms such as “nucleic acid chip”, “microarray” and “DNA array”, and is used interchangeably.

  An “amplification reagent integrated DNA chip” is a device in which a primer is immobilized in the vicinity of a nucleic acid probe of a DNA chip. If necessary, enzymes, substrates, and buffers necessary for amplification are immobilized. The primer is a primer set for amplifying a target nucleic acid detected by the nucleic acid probe, and is directly detected by the nucleic acid probe after amplification.

  "Amplification reagent and DNA chip integrated with salt concentration control mechanism" is a primer set near the nucleic acid probe of the DNA chip, and if necessary, enzyme, substrate and buffer are immobilized, and the salt concentration after amplification is adjusted. A DNA chip having a mechanism for As a method for adjusting the salt concentration, there is a method in which a salt having a high salt concentration is fixed and covered with an oily component such as hardened oil or wax. During the amplification reaction, the salt remains in the solid oily component. After amplification, for example, by raising the temperature, the oily component is melted and the retained salt is released into the solution. The salt and oily component are immobilized in the vicinity of the DNA probe and primer.

2. Nucleic Acid Analysis Method When performing an amplification reaction and a hybridization reaction continuously in one reaction field, a difference in reaction conditions between the amplification reaction and the hybridization reaction becomes a problem. In particular, when the amplification reaction and the hybridization reaction are continuously performed in one reaction field without transferring, replacing, exchanging, and / or adding the reaction solution, such a difference provides accurate data. This may hinder the detection sensitivity.

  The nucleic acid analysis method according to the embodiment is a method capable of performing an amplification reaction and a hybridization reaction in one reaction field, and obtaining an accurate analysis result with higher sensitivity.

  Of the reaction conditions for the amplification reaction and the hybridization reaction, the salt concentration was particularly noted. In general, the optimum salt concentration for gene amplification varies depending on the enzyme used. In a typical example, the optimum marginal concentration for a reaction for amplifying a nucleic acid is considered to be about 10 mM to 60 mM. On the other hand, the optimum salt concentration for hybridization is about 300 mM to 750 mM. Therefore, salt concentrations different from each other by 10 times or more in the amplification reaction and the hybridization reaction are the indicated concentrations. Therefore, if the amplification reaction and the hybridization reaction are continuously performed in one reaction field, it is considered that the hybridization efficiency becomes low, and as a result of intensive studies, this embodiment has been achieved.

  Embodiments relate to nucleic acid analysis methods for specimens. This nucleic acid analysis method is a reaction including performing a nucleic acid amplification reaction and a hybridization reaction in one reaction field. Therefore, the nucleic acid analysis method includes means for satisfying both the salt concentration necessary for the amplification reaction and the salt concentration necessary for the hybridization reaction.

  As described above, the salt concentration required for the hybridization reaction is about 10 times, or about 10 times higher than the salt concentration required for the amplification reaction. When the reaction solution necessary for the amplification reaction is present in the reaction field, the salt is insufficient when the hybridization reaction is subsequently performed. Therefore, a sufficient hybridization reaction cannot be performed. In order to eliminate this, a salt previously coated with hardened oil or wax is present in the reaction solution. Although details will be described later, the hardened oil and the wax do not melt under the temperature condition in which the amplification reaction is performed, but melt when the temperature is increased after the amplification.

  Thus, an amplification reaction is achieved under an appropriate salt concentration condition in a certain reaction field, and a hybridization reaction is achieved under an appropriate salt concentration condition in the same reaction field. That is, the salt coated with the hardened oil or wax is maintained in the coated state during the amplification reaction. On the other hand, the hardened oil or wax is melted before or during the hybridization reaction. Thereby, the salt is eluted into the reaction solution, and thereby the salt concentration in the reaction field is adjusted to a concentration suitable for the hybridization reaction.

  Such a nucleic acid detection method including a continuous amplification reaction and a hybridization reaction may be performed in a reaction vessel. The reaction solution added to the inside of the reaction vessel becomes a reaction field. The reaction container may be, for example, a tube, a well, a chamber, a channel, a cup and a dish, and a plate including a plurality of them, such as a multiwell plate.

  The reagents necessary for the amplification reaction and the sample to be analyzed may be brought into the reaction field prior to the analysis by including them in the reaction solution. The nucleic acid probe necessary for the hybridization reaction may be immobilized in advance at a position in contact with the reaction solution on the inner surface of the container constituting the reaction field.

  In the analysis method, the nucleic acid contained in the specimen is amplified by an amplification reaction in the reaction field. Next, detection or quantification of the amplification product generated by amplification may be performed by detecting a hybridization signal generated by a hybridization reaction with a nucleic acid probe immobilized in advance in the reaction field.

  In addition, reagents necessary for the amplification reaction, such as primer set, DNA synthase, reverse transcriptase, buffer, dNTP, ammonium sulfate, Tween 20, potassium chloride, magnesium sulfate, and / or betaine, are used in the reaction field prior to the analysis method. It may be fixed to the device to be configured prior to analysis. Alternatively, it may be contained in the reaction solution and added to the container.

  In addition, in order to adjust the hybridization reaction to the optimum salt concentration, even if the salt coated with hardened oil or wax is contained in the reaction solution and brought into the reaction field, it reacts prior to analysis. Prior to analysis, it may be immobilized at a position in contact with the reaction solution on the inner surface of the container constituting the field.

  Prior to the analysis, when the salt is immobilized on the inner side surface of the container, the immobilization may be performed at a position different from the position where the nucleic acid probe is immobilized or at the same position. When a plurality of types of nucleic acid probes are immobilized on the inner surface of the container for each type, the salt is preferably immobilized in the vicinity of each nucleic acid probe. Further, when the salt is also immobilized on the inner side surface of the primer set, the salt immobilization position may be immobilized at a position different from the position where the primer set is immobilized. As will be described in detail later, when salt is immobilized at the same position as the position where the primer set is immobilized, the salt can be fixed and then covered with hardened oil or wax, and then the primer set can be released. It is preferable to fix to.

The nucleic acid analysis method may include, for example, the following steps;
(A) preparing a specimen, a substrate, a primer set for amplifying a target nucleic acid, and a nucleic acid probe for detecting a product amplified using the primer set;
(B) coating the salt with hardened oil or wax to prepare a salt capsule;
(C) immobilizing the nucleic acid probe on at least one surface of the substrate;
(D) immobilizing a primer set at or near the same position as the nucleic acid probe;
(E) contacting the reaction solution containing the specimen and the salt capsule with the nucleic acid probe and the primer set on the substrate and performing an amplification reaction to amplify the target nucleic acid;
(F) after the amplification reaction, raising the temperature of the reaction solution to melt the hardened oil or wax and eluting the salt;
(G) the step of hybridizing the amplification product obtained by the amplification reaction with the nucleic acid probe, and (h) the presence or absence of the target nucleic acid in the sample by measuring the hybridization signal generated in step (g) And / or detecting the amount.

Further nucleic acid analysis methods may include, for example, the following steps;
(A) preparing a specimen, a substrate, a primer set for amplifying a target nucleic acid, and a nucleic acid probe for detecting a product amplified using the primer set;
(B) immobilizing the nucleic acid probe on at least one surface of the substrate;
(C) immobilizing a primer at or near the same position as the nucleic acid probe;
(D) a step of immobilizing a salt in the vicinity of the nucleic acid probe;
(E) laminating a hardened oil or wax on the immobilized salt to coat the salt;
(F) contacting the reaction solution containing the specimen with the nucleic acid probe, primer set and coated salt of the substrate and amplifying the target nucleic acid by performing an amplification reaction;
(G) after the amplification reaction, raising the temperature of the reaction solution to melt the hardened oil or wax and eluting salts;
(H) the step of hybridizing the amplified product with a nucleic acid probe, and (i) the presence or absence and / or amount of the target nucleic acid in the sample is detected by measuring the amount of hybridization generated in step (h) Process.

  Furthermore, the nucleic acid analysis method will be described in detail.

(1) Template nucleic acid A sample is prepared before performing the nucleic acid analysis method of the embodiment. The specimen may contain a nucleic acid, and may be an object for which it is desired to analyze the nucleic acid contained therein. Since the sample needs to be appropriately subjected to an amplification reaction, the sample may be subjected to pretreatment on a crude sample obtained from a subject to be analyzed as necessary. In addition, when appropriate for the amplification reaction, the crude sample may be used as a sample as it is without pretreatment. If nucleic acids having the sequence to be analyzed exist in the specimen, they are used as the initial template nucleic acid. The test nucleic acid used as the template nucleic acid may be single-stranded or double-stranded.

(2) DNA chip An example of a DNA chip will be described with reference to FIG. FIG. 1A is a plan view of the DNA chip 1. FIG. 1B is a sectional view taken along line BB of the DNA chip 1 shown in FIG. The DNA chip described below may also be referred to as an amplification reagent and a salt concentration control mechanism integrated DNA chip.

  With reference to FIG. 1C, a further embodiment of an amplification reagent and a salt concentration adjusting mechanism integrated DNA chip will be described.

  The DNA chip 1 includes a substrate 2, a probe immobilization region 3 disposed on one surface of the substrate, a primer immobilization 4 disposed in the vicinity of the probe immobilization region 3, a probe immobilization region 3, and A salt-immobilized region 5 disposed at a position other than the primer-immobilized region 4, a nucleic acid probe 6 immobilized on the probe-immobilized region 3, a primer set 7 releasably immobilized on the primer-immobilized region 4, and a salt A salt 8 fixed in the fixing region and a hardened oil or wax 9 covering the salt are provided.

  A plurality of nucleic acid probes 6 immobilized on the probe immobilization region 3 are immobilized on each probe immobilization region 3 for each type. A plurality of primer sets 7 immobilized on the primer immobilization region 4 are immobilized on each primer immobilization region 4 for each type.

  The nucleic acid probe 6 for detecting one type of amplification product may be immobilized on one probe immobilization region 3. For this purpose, a sequence complementary to at least a part of the amplification product to be detected may be included. An amplification product is formed by the primer set 7 using the target nucleic acid as a template. Therefore, the amplification product includes the sequence of at least a part of the target nucleic acid and the sequence of primer set 7.

  The probe immobilization region 3 is arranged so that a hybridization signal between the nucleic acid probe 6 and the amplification product can be detected independently between the plurality of probe immobilization regions 3.

  For fixing the nucleic acid probe 6 to the probe immobilization region 3, any general technique for fixing the nucleic acid probe to the substrate surface in a so-called DNA chip known per se may be used.

  For example, the distance between adjacent probe 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 It may be.

  Primers necessary for amplifying the target nucleic acid to be amplified are immobilized on one primer-immobilized region. For example, in the case of the DNA chip 1 for PCR amplification, one primer-immobilized region 4 includes a plurality of forward primers and reverse primers necessary for amplifying one type of specific target nucleic acid. In the case of the DNA chip 1 for LAMP amplification, the FIP primer, BIP primer necessary for amplifying one kind of specific target nucleic acid in one primer immobilization region 4, F3 primer as necessary, A plurality of B3 primers and LP primers are included.

  The primer set 7 is fixed to the primer fixing region 4 in a releasable state so as to be released in contact with a liquid phase for providing a reaction field. Immobilization of the primer set 7 to the primer immobilization region 4 may be achieved, for example, by dropping a solution containing one set of primer sets into one primer immobilization region 4 and then drying it. By repeating this, the primer set 7 is fixed to all the primer fixing regions 4 arranged so that the amplification reaction can be achieved independently on one surface of the substrate 2. However, the primer set 7 may be immobilized in the immobilization region 4 in a state in which 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 4 arranged on the substrate 2 may be arranged independently of each other. The term “independently arranged” means that they are arranged at intervals that do not interfere with amplification initiated and / or advanced for each primer set 7 in the reaction field. For example, the adjacent primer immobilization regions 4 may be arranged in contact with each other, may be arranged in the vicinity of each other at a slight distance, or fixed in a commonly used detection device such as a so-called DNA chip. The nucleic acid probes may be arranged at the same distance from each other at a similar distance.

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

  The primer set 7 may be immobilized after the nucleic acid probe 6 is immobilized, the nucleic acid probe 6 may be immobilized after the primer set 7 is immobilized, and the primer set 7 and the nucleic acid probe 6 are immobilized. You may do it at the same time.

  For example, the distance between the probe immobilization region 3 and the primer immobilization region 4 is 0 μm to 0.1 μm, 0.1 μm to 1 μm, 1 μm to 10 μm, 10 μm to 100 μm, 100 μm to 1 mm, 1 mm to 10 mm, or The above may be sufficient, Preferably, it may be 100 micrometers-10 mm.

  For example, when the distance between the probe immobilization region 3 and the primer immobilization region 4 is 0 μm, it may be understood that the probe immobilization region 3 and the primer immobilization region 4 are at the same position on the substrate surface. . The probe immobilization region 3 may be included in the primer immobilization region 4, and the primer immobilization region 4 may be included in the probe immobilization region 3. A plurality of probe immobilization regions 3 may be arranged for one primer immobilization region 4.

  In the salt immobilization region 5, a salt 8 necessary for achieving an optimum salt concentration for the hybridization reaction from a salt concentration at a level necessary for the amplification reaction is immobilized.

  The salt 8 may be, for example, sodium chloride, sodium citrate, potassium chloride, sodium acetate, SSC prepared using sodium chloride and sodium citrate, but is not limited thereto. Hybridization promoters such as dextran sulfate, salmon sperm DNA, and bovine thymus DNA, buffer, EDTA, or surfactant are immobilized together with the salt 8 on the salt immobilization region 5 together with the salt. May be.

  The hardened oil or wax 9 completely covers the salt so that the salt does not elute during the amplification reaction. Immobilization of the salt 8 may be performed by dissolving the desired salt 8 in the solution, spotting on the salt immobilization region 5, and then drying. The immobilization of the salt 8 may be performed using, for example, a salt dissolved in a gelling agent such as agar, agarose, or gelatin. The spotted salt solution may be dried by a heat drying method, a vacuum drying method, freeze drying, or the like.

  The hardened oil may be, for example, rapeseed hardened oil, soybean hardened oil, palm hardened oil, castor hardened oil, etc., but is not limited thereto. The wax may be an animal-derived, plant-derived, petroleum-derived or mineral-derived natural wax, or a synthetic wax. For example, beeswax, spermaceti, shellac wax, carnauba wax, wood wax, rice wax, candelilla wax, paraffin wax, stearic acid, microcrystalline wax, montan wax and / or ozokerite may be used. Is not to be done.

  The method of coating the salt 7 may be performed as follows. These hardened oils or waxes 9 are spotted at the positions where the salt 7 is fixed. In order to prevent leakage of the salt 7, it is preferable to increase the spot amount of the oily substance, that is, the hardened oil or the wax 9, as compared with the salt 7 solution. However, the quantity ratio may be adjusted as appropriate. The hardened oil or wax 9 preferably has a melting point higher than the amplification temperature. It is in a solid state at the time of amplification. Furthermore, it liquefies when the temperature is raised after amplification. Since the optimum amplification temperature differs depending on the amplification method, the hardened oil or wax 9 may be selected as appropriate depending on the amplification method.

  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 material of the substrate 2 may be any material that does not itself participate in the reaction, and may be any material that can perform the amplification reaction there. For example, it may be arbitrarily selected from silicon, glass, resin and metal.

  When the DNA chip 1 is used in the nucleic acid analysis method, at least one surface on which the nucleic acid probe 6, the primer set 7 and the coated salt 7 are immobilized comes into contact with a liquid phase for reaction, for example, a reaction solution. For example, the reaction solution may be placed on the surface on which at least the nucleic acid probe 6, the primer set 7 and the coated salt 7 are immobilized, or the DNA chip 1 may be immersed in a container containing the reaction solution.

  A method of using such a DNA chip 1 will be described with reference to FIG. FIG. 1C is a cross-sectional view showing a state in which the covering 10 is attached to the DNA chip 1 shown in FIG.

  The covering 10 may be attached to the substrate 2 by any method known per se in which the liquid contained therein is maintained without leaking to the outside.

  The covering 10 is made of, for example, a silicon resin such as silicon rubber and / or a resin such as a fluororesin, and the resin is made of, for example, extrusion, injection molding or stamping and / or, for example, It may be formed by any known resin molding method such as bonding of each member with an adhesive.

  By attaching the covering body 10 to the base body 2, it is possible to form a reaction part by the surfaces of the covering body 10 and the base body 2. A reaction liquid may be supplied to the reaction part by injecting a liquid from an injection part (not shown) arbitrarily opened in the covering 10. Or you may inject | pour the reaction liquid with the injector which can permeate | transmit the liquid to a needle | hook after piercing | piercing the covering body 1 with the needle | hook with a sharp tip and penetrating.

(3) Nucleic acid analysis method A sample is injected into the integrated DNA chip 1. If there is an amplification reagent that is not held in the integrated DNA chip 1, it may be injected together with the specimen. Amplification is performed by a known amplification method, and the reaction temperature can also be appropriately selected. During the amplification reaction, the hardened oil or wax is solidified and the salt is retained inside. After amplification, the temperature of the amplification liquid is made higher than the melting point of the hardened oil or wax to liquefy the hardened oil or wax. The liquefied hydrogenated oil or wax moves to the upper layer, and the internal salt is released. Thereafter, the amplification product and the nucleic acid probe are hybridized under optimum conditions under a high salt concentration. Thereafter, a hybridizing signal is detected. Based on the obtained results, the sequence information and / or amount of the test nucleic acid contained in the specimen is analyzed.

  Items to be analyzed may be, for example, measurement of gene expression level, quantification of nucleic acids derived from viruses and / or bacteria, identification of genotypes for polymorphisms and / or detection of mutations. However, it is not limited to these.

  Further, a nucleic acid analysis method using the DNA chip 1 will be described below.

<Sample injection process>
First, the specimen is injected into the reaction part. The sample solution may be frozen and refrigerated if necessary, and heat-denatured before injection. In addition, a special reagent for trapping a substance that inhibits amplification may be added. A part of the amplification reagent that is not immobilized on the DNA chip may be contained.

<Amplification process>
Any known amplification reaction may be used. Examples include, but are not limited to, the LAMP method, the SMAP method, the NASBA method, the ICAN method, the TRC method, the SDA method, the TMA method, the RCA method, and the PCR method. In order to increase the hybridization efficiency with the nucleic acid probe, a primer configured to form a single strand may be used. If desired, a reverse transcription reaction using reverse transcriptase may be performed in combination with the amplification reaction. As a technique for performing the amplification reaction and the reverse transcription reaction at the same time, any technique known per se may be used.

<Hardened oil, wax dissolution process>
After the amplification reaction, the hardened oil and wax are liquefied by raising the temperature. If it is a hardened oil or wax having a melting point close to the amplification temperature, it immediately becomes a liquefied state. The liquefied oily substance naturally moves to the upper part of the reaction vessel, but if the oily substance does not move, the chip may be vibrated slightly. Furthermore, in order to confirm the elution of the salt, a dye may be contained together with the salt, and the elution of the dye may be confirmed.

<Hybridization process>
Hybridization is performed in a state where the salt incorporated in the hardened oil or wax is sufficiently diffused. The reaction temperature is appropriately set. The reaction efficiency may be increased by stirring or shaking.

<Washing process>
After hybridization, the DNA chip may be washed. A washing solution for washing the DNA chip after hybridization has an ionic strength in the range of 0.01 to 5, and a buffer solution in the range of pH 5 to 9 is preferably used. The cleaning liquid preferably contains a salt and a surfactant. For example, an SSC solution, a Tris-HCl solution, a Tween 20 solution, or an SDS solution is preferably used. The washing temperature is, for example, in the range of 10 ° C to 70 ° C. The washing solution passes or stays on the surface of the probe-immobilized substrate or the region where the nucleic acid probe is immobilized.

<Detection process>
Detection of hybrids generated by the hybridization step can utilize the following fluorescence detection method and electrochemical detection method. In order to use these detection methods, a detection device suitable for those detection methods may be selected and prepared. Those detection devices will be described later.

(A) Fluorescence detection method Detection is performed using a fluorescent labeling substance. Primers used in the nucleic acid amplification step may be labeled with a fluorescently active substance such as a fluorescent dye such as FITC, Cy3, Cy5, or rhodamine. Alternatively, a second probe labeled with these substances may be used. A plurality of labeling substances may be used simultaneously. The label in the labeled sequence or the secondary probe is detected by the detection device. Use an appropriate detection device depending on the label used. For example, when a fluorescent substance is used as a label, it is detected using a fluorescence detector.

(B) Electrochemical detection method A double-stranded recognition substance well known in the art is used. The double-stranded recognition substance may be selected from Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator. Further, these double strand recognition substances may be modified with an electrochemically active metal complex such as ferrocene or viologen.

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

  In the measurement, for example, a potential equal to or higher than the potential at which the double strand recognition substance reacts electrochemically is applied, and the reaction current value derived from the double strand recognition substance is measured. At this time, the potential may be applied at a constant speed, may be applied in pulses, or a constant potential may be applied. The current and voltage may be controlled using devices such as a potentiostat, a digital multimeter, and a function generator. Electrochemical detection can be performed by methods well known in the art. A DNA chip for performing electrochemical detection will be described below.

<Detection device>
The amplification product may be detected by detecting the hybridization signal as described above. A DNA chip according to the above-described embodiment is also provided as a detection device for performing such detection. For example, the DNA chip substrate may be any conventionally known microarray substrate such as an electrochemical detection type represented by a current detection type, a fluorescence detection type, a chemical color development type, and a radioactivity detection type. Good.

  Any kind of microarray can be produced by a method known per se. For example, in the case of a current detection type microarray, the negative control probe immobilization region and the detection probe immobilization region may be arranged on different electrodes.

  An example of a DNA chip as a detection device is shown in FIG. 2 as a schematic diagram. However, the present invention is not limited to this.

  As shown in FIG. 2, the DNA chip 100 includes a probe immobilization region 102 on a base 101. The nucleic acid probe 103 is immobilized on the probe immobilization region 102. Such a DNA chip 100 can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and arrangement of the probe immobilization regions 102 arranged on the base 101 as necessary. Such a DNA chip may be suitably used for a detection method using fluorescence.

  Another example of a further DNA chip is shown in FIG. A DNA chip 200 shown in FIG. 3 includes an electrode 202 on a base body 201. A nucleic acid probe 203 is immobilized on the electrode 202. The electrode 202 is connected to the pad 204. Electrical information from the electrode 202 is acquired via the pad 204.

  Such a DNA chip 200 can be manufactured by a method well known in the art. The number of electrodes 202 arranged on the base 201 and the arrangement thereof can be appropriately changed by those skilled in the art as needed. Furthermore, the DNA chip of this example may include a reference electrode and a counter electrode as necessary.

  The electrode is composed of a simple metal such as gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium or tungsten, or an alloy thereof, or carbon such as graphite or glassy carbon, or a material thereof. An oxide or a compound can be used, but is not limited thereto. Such a DNA chip may be suitably used for an electrochemical detection method.

  A primer set is further immobilized on the detection device as described above. The primer set is immobilized in the vicinity of the position where the nucleic acid probe of the substrate of the DNA chip, which is a detection device as described above, is immobilized. Furthermore, amplification reagents other than the primer set, such as enzymes, substrates, and buffers, may be similarly immobilized.

  Immobilization of the primer set may be performed, for example, by dissolving the primer set in water, a buffer and / or an organic solvent, spotting the obtained solution, and then drying. Drying may be performed by, for example, a heat drying method, a vacuum drying method, or a freeze drying method. The concentration of the primer set and each reaction reagent may be appropriately changed, and may be selected so as to satisfy the conditions for obtaining a sufficient amplification product. Further, a salt having a high salt concentration is immobilized in the vicinity of the nucleic acid probe, and then the salt is coated with an oily substance such as hardened oil or wax.

  In such a DNA chip, an amplification reaction, a hybridization reaction, and detection of a hybridization signal are continuously performed, whereby the nucleic acid analysis method can be performed simply and accurately in a short time.

  In addition, a probe immobilization region, a primer immobilization region and a salt immobilization region are channels formed by recesses and / or projections formed in advance on the substrate, or spaces formed by attaching a covering to the substrate. The operation such as addition and / or discharge of liquid becomes easier by carrying out in the reaction section constituted by

3. Multi-Nucleic Acid Reactor The nucleic acid analysis method as described above is designed to immobilize an amplification reagent such as a primer near the position where the nucleic acid probe is immobilized, and perform a minute reaction from the amplification reaction to the detection of hybridization and hybridization signals. It may be a method that is performed directly in a section. Such a DNA chip may be provided as a multi-nucleic acid reaction device in which a plurality of types of nucleic acids can be simultaneously subjected to the reaction.

  The multi-nucleic acid reaction tool comprises a substrate, a plurality of primer-immobilized regions arranged independently of each other on at least one surface of the substrate, and a plurality of primers arranged at or near the same position as each of the plurality of primer-immobilized regions. And a plurality of salt-immobilized regions in the vicinity of the plurality of primer-immobilized regions. In each primer immobilization region, a plurality of types of primer sets each configured to amplify a plurality of types of target sequences are immobilized in a releasable manner for each type. In each probe immobilization region, a plurality of types of nucleic acid probes containing sequences complementary to at least a part of the amplification product are immobilized for each type so that the hybridized signal can be detected independently. The salt and the hardened oil or wax covering the salt are fixed in the salt fixing region.

  That is, the multi-nucleic acid reaction tool comprises: a substrate; a plurality of primer-immobilized regions arranged independently of each other on at least one surface of the substrate; and the same position as or near each of the plurality of primer-immobilized regions. A plurality of arranged probe-immobilized regions; a plurality of salt-immobilized regions in the vicinity of the plurality of primers and the probe-immobilized region; and a plurality of primer-immobilized regions that are releasably immobilized for each type. A plurality of types of primer sets configured to amplify each type of target sequence; and the target immobilized on the plurality of probe immobilization regions for each type so that a hybridization signal can be independently detected A plurality of nucleic acid probes comprising a sequence complementary to at least a part of an amplification product generated by the amplification of the sequence; and immobilized on the plurality of salt-immobilized regions. , A coated salt by hardened oils or waxes; may comprise.

  Furthermore, the multi-nucleic acid reaction tool comprises: a substrate; a primer immobilization region disposed on at least one surface of the substrate; a probe immobilization region disposed at or near the same position as the primer immobilization region; A salt-immobilized region disposed in the vicinity of the primer-immobilized region; a primer set that is releasably immobilized on the primer-immobilized region and configured to amplify a target sequence; and immobilized on the probe-immobilized region A nucleic acid probe comprising a sequence complementary to at least a part of an amplification product generated by amplification of the target sequence; and a salt immobilized on the salt-immobilized region and coated with hardened oil or wax; You may prepare.

<Array primer probe chip>
An array type primer probe chip which is an example of a multi-nucleic acid reaction tool will be described as one embodiment with reference to FIGS.

(1) Chip Material First, an example of the structure and manufacturing method of an array type primer probe chip that detects a hybridized signal by electrochemical detection will be described with reference to FIGS. 4 (a) and 4 (b). FIG. 4A is a plan view of the chip material, and FIG. 4B is a cross-sectional view taken along line BB of the chip material of FIG.

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

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

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

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

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

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

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

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

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

(2) Array-type Primer Probe Chip Next, an example of the configuration and production method of an array-type primer probe chip in which a primer set and a probe nucleic acid are immobilized on the chip material 111 produced in (1) above is shown in FIG. This will be described with reference to FIG. 5A is a plan view of the array-type primer probe chip 1, and FIG. 5B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 5A.

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

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

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

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

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

(3) Array type primer probe chip at the time of use The usage method of the array type primer probe chip 1 produced in said (2) is demonstrated, referring FIG. 6 and FIG.

  6A is a plan view of the array-type primer probe chip 1 in use, and FIG. 6B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 6A. It is.

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

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

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

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

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

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

  FIG. 7A schematically shows a state in which an amplification product is formed in the region 401 of the reaction solution 302 that is a reaction field. 7A is a plan view of the array-type primer probe chip 1 in use, and FIG. 7B is a cross-sectional view taken along line BB of the array-type primer probe chip 1 in FIG. 7A. It is. As described above, the sample added in FIG. 6 contained a nucleic acid containing a sequence that can be bound by the second primer set 204b. Therefore, as shown in FIG. 7 (a) and FIG. 7 (b). The second primer set 204b is released and diffused in the region 401, and after encountering the template nucleic acid 303, an amplification reaction occurs and an amplification product is formed. The amplification product by the second primer set 204b diffuses around the second primer immobilization region 203b and reaches the second probe immobilization region 201b. When the reached amplification product includes the target sequence, the second probe nucleic acid 202b and the amplification product are hybridized to form a double-stranded nucleic acid. The intercalator contained in the reaction solution 302 is combined with this double-stranded nucleic acid to generate a hybrid signal.

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

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

  In such an array type primer probe chip, a salt coated with hardened oil or wax may be included in the reaction solution. Alternatively, salt may be fixed in the vicinity of the probe fixing region and primer fixing region of the chip material, and hardened oil or wax may be fixed so as to cover it. As a result, both the amplification reaction and the hybridization reaction can be started and proceeded under more suitable conditions.

4). Assay Kit This embodiment may be in the form of an assay kit. The assay kit may include the DNA chip of any of the above-described embodiments, and may optionally include an enzyme, a substrate, a buffer, an instruction manual, and the like.

Example Example 1
The salt concentration adjustment mechanism using hardened oil or wax was first verified using a tube.

<Coating of salt solution with hardened oil or wax>
1.5% agar and blue dye were added to the 20 × SSC solution, and 2.8 μL each was immobilized on the bottom of the tube. Thereafter, 4 μL each of rapeseed hydrogenated oil (melting point: 68 ° C.), rice wax (melting point: 73-77 ° C.), and candelilla wax (melting point: 68-72 ° C.) were added. As shown in a) and b) of FIG. 8, it was confirmed that the hardened oil or wax as the oil component was located in the upper layer and the 20 × SSC solution was located in the lower layer. The hardened oil is solidified at room temperature and liquefies at 80 ° C.

<Amplification>
The amplification reaction used the LAMP method. As the template and primer, Template A and Primer Set A described in Tables 1 and 2 and Tables 1 and 2 were used.

The composition of the amplification reaction solution is shown in Table 3.

  This amplification solution was poured into a tube to which hardened oil or wax was added, and an amplification reaction was performed at 62 ° C. for 1 hour. During the amplification, as shown in FIG. 8 c), the hardened oil and the wax were solidified, and the pigment did not leak out. After amplification, white turbidity associated with amplification was observed in all solutions. Thereafter, when heat was applied at 95 ° C. for 5 minutes, it was confirmed that the hardened oil and wax were liquefied and the retained salt was released as shown in FIG. 8 d). In order to confirm the presence or absence of amplification and detection inhibition of the hardened oil or wax, a sample obtained by amplifying only the amplification solution with a tube was also prepared as a positive control.

<Chip detection>
Probe DNA (A) was prepared for chip detection of the product amplified above. As a negative control, a nucleic acid probe showing a sequence unrelated to template A was prepared.

  A DNA chip for an electrochemical detection method as shown in FIG. 3 and more specifically in FIG. 4 was produced. Titanium and gold thin films were formed on the Pyrex glass surface by sputtering. Thereafter, titanium and gold electrodes were formed on the glass surface by etching treatment. Further, an insulating film was applied thereon, and a circular window and a rectangular window were opened in the insulating film by an etching process to expose the working electrode, the counter electrode, the reference electrode, and the prober contact portion. The working electrode was used as a probe immobilization region, and 100 nL of 3 μM probe DNA (A) was spotted thereon and dried. The same probe was spotted on 4 electrodes each. After drying, it was washed with ultrapure water and air-dried to obtain a probe-immobilized electrode. Next, a covering was attached to the DNA chip. The space constituted by the covering functions as a reaction vessel. Silicon rubber was used for the covering. The solution after LAMP amplification was poured into the reaction vessel from a hole provided in advance in the silicon rubber, and then the hole in the covering was covered. Hybridization was performed by allowing to stand at 55 ° C. for 10 minutes. Then, it was washed by dipping in a 0.2 × SSC solution at 25 ° C. for 1 minute. Next, the washing solution was removed, and a PIPES buffer containing 75 μM Hoechst 33258 solution as an intercalating agent was injected. Next, the potential was swept to the probe nucleic acid immobilization working electrode, and the oxidation current of Hoechst 33258 molecule specifically bound to the double strand formed by the probe DNA and the LAMP product was measured. The 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.

<Result>
In Test 1, when the LAMP amplification solution prepared as a positive control was detected without adding salt as it was, Test 2 was detected by adding 20 × SSC (final concentration 2 × SSC) later, and in Test 3 Detected when 20 × SSC was coated with rapeseed hydrogenated oil, rice wax and candelilla wax to adjust the salt concentration (final concentration 2 × SSC). The obtained results are shown in FIG.

  When detected without adding salt, the positive signal was low (1 in FIG. 9). The salt concentration was adjusted with 20x SSC added later (Fig. 9-2), rapeseed hydrogenated oil (Fig. 9-3), rice wax (Fig. 9-4), and candelilla wax (Fig. 9-5). A positive signal almost as high as that detected was detected. As a result, it was shown that the salt concentration can be adjusted with the hardened oil or wax, and that the hardened oil and wax do not hinder amplification and detection.

Example 2
From the amplification to the detection using the DNA chip integrated with the salt concentration control mechanism, the usefulness of the salt concentration control mechanism was verified. In the same manner as in Example 1, in addition to template A, primer set A, and probe DNA (A), template B, primer set B, and probe DNA (B) were prepared.

<Preparation of DNA chip integrated with salt concentration control mechanism>
A DNA chip for an electrochemical detection method as shown in FIG. 3 and more specifically in FIG. 4 was produced. Probe DNAs (A) and (B) were immobilized by spotting and drying in the same manner as in Example 1 to obtain probe-immobilized electrodes. Then, as shown in FIG. 5, 1.6 U enzyme, 200 μM FIP, BIP, F3, B3, LP primer were added at 0.1 μL, 0.1 μL, 0.0125 μL, 0.00125 μL and 0.05 μL, respectively. The solution containing it was spotted and fixed by a heat drying method. At this time, primer set A was immobilized in the vicinity of nucleic acid probe A, and primer set B was immobilized in the vicinity of nucleic acid probe B. Next, 20 × SSC was spotted in the vicinity of each nucleic acid probe and heat-dried. Thereafter, 20 × SSC was coated with candelilla wax. As a control, a similar chip without immobilizing 20 × SSC and candelilla wax was produced. Next, a covering was attached to the DNA chip. The space constituted by the covering functions as a reaction vessel. Silicon rubber was used for the covering.

<Amplification>
A primer, an amplification reagent other than an enzyme, and a template were poured into a reaction container from a hole provided in advance in the silicon rubber, and the hole of the cover was covered. Then, an amplification reaction was performed at 62 ° C. for 1 hour. The concentration of the template and the amplification reagent was prepared to be the same as in Example 1. Thereafter, the coated wax was dissolved by heating at 95 ° C. for 5 minutes to release (ie, elute) the salt.

<Detection>
Hybridization and detection were performed under the same conditions as in Example 1.

<Result>
As shown in FIG. 10, the DNA chip (2 in FIG. 10) having a mechanism for adjusting the salt concentration with 20 × SSC and candelilla wax is compared with the case where the salt concentration is not adjusted (1 in FIG. 10), A high positive signal was detected.

  It has been clarified that the use of wax enables the amplification reaction and the hybridization reaction to be continuously performed in the same reaction field.

Claims (12)

A nucleic acid analysis method comprising:
(A) preparing a specimen, a substrate, a primer set for amplifying a target nucleic acid, and a nucleic acid probe for detecting a product amplified using the primer set;
(B) coating the salt with hardened oil or wax to prepare a salt capsule;
(C) immobilizing the nucleic acid probe on at least one surface of the substrate;
(D) immobilizing a primer set at or near the same position as the nucleic acid probe;
(E) contacting the reaction solution containing the specimen and the salt capsule with the nucleic acid probe and the primer set on the substrate and performing an amplification reaction to amplify the target nucleic acid;
(F) after the amplification reaction, raising the temperature of the reaction solution to melt the hardened oil or wax and eluting the salt;
(G) the step of hybridizing the amplification product obtained by the amplification reaction with the nucleic acid probe, and (h) the presence or absence of the target nucleic acid in the sample by measuring the hybridization signal generated in step (g) Detecting the quantity;
A method comprising: A nucleic acid analysis method comprising:
(A) preparing a specimen, a substrate, a primer set for amplifying a target nucleic acid, and a nucleic acid probe for detecting a product amplified using the primer set;
(B) immobilizing the nucleic acid probe on at least one surface of the substrate;
(C) immobilizing a primer at or near the same position as the nucleic acid probe;
(D) a step of immobilizing a salt in the vicinity of the nucleic acid probe;
(E) laminating a hardened oil or wax on the immobilized salt to coat the salt;
(F) contacting the reaction solution containing the specimen with the nucleic acid probe, primer set and coated of the substrate, and performing an amplification reaction to amplify the target nucleic acid;
(G) after the amplification reaction, raising the temperature of the reaction solution to melt the hardened oil or wax and eluting salts;
(H) the step of hybridizing the amplified product with a nucleic acid probe, and (i) the presence or absence and / or amount of the target nucleic acid in the sample is detected by measuring the amount of hybridization generated in step (h) Process,
A method comprising:   The method according to claim 1 or 2, wherein the hardened oil or wax has a melting point higher than an amplification temperature.   The method according to any one of claims 1 to 3, wherein the salt is a salt necessary for a hybridization reaction between two nucleic acids.   5. The method according to claim 4, wherein the salt is a salt selected from the group consisting of sodium chloride, sodium citrate, potassium chloride, sodium acetate and combinations thereof.   The method according to any one of claims 1 to 5, wherein the substrate includes a flow path, and the inside of the flow path is used as a reaction part. A substrate;
A primer immobilization region disposed on at least one surface of the substrate;
A probe immobilization region disposed at the same position as or in the vicinity of the primer immobilization region;
A salt-immobilized region located near the primer-immobilized region and located at a position different from the probe-immobilized region;
A primer set releasably immobilized in the primer immobilization region and configured to amplify a target sequence;
A nucleic acid probe immobilized on the probe immobilization region and comprising a sequence complementary to at least a part of an amplification product generated by amplification of the target sequence;
A salt fixed in the salt fixing region and coated with hardened oil or wax;
A DNA chip comprising:   8. The DNA chip according to claim 7, wherein a melting point of the hardened oil or wax is higher than an amplification temperature.   The DNA chip according to claim 7 or 8, wherein the salt is a salt necessary for a hybridization reaction between two nucleic acids.   The DNA chip according to claim 9, wherein the salt is a salt selected from the group consisting of sodium chloride, sodium citrate, potassium chloride, sodium acetate, and combinations thereof.   The DNA chip according to any one of claims 7 to 10, wherein the substrate includes a flow path, and the inside of the flow path is used as a reaction part.   An assay kit for use in the nucleic acid analysis method according to any one of claims 1 to 6, wherein the DNA chip, enzyme, substrate and buffer according to any one of claims 7 to 11 are used. An assay kit comprising.

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