JP2012200223A - Method for quantitatively determining nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and quantitatively determining nucleic acid.SOLUTION: This method for quantitatively determining the nucleic acid in the embodiment is as follows. (a) A specimen is stepwisely diluted with diluting liquid to obtain a plurality of stepwisely diluted liquid. (b) For each of the stepwisely diluted liquid having different concentration, a primer set containing tag sequence-introduced primers containing different tag sequences for amplifying a target nucleic acid is prepared. (c) In an independent reaction system for each of the stepwisely diluted liquid, a template nucleic acid is amplified by using the primer set to obtain an amplified product introduced with the tag sequence. (d) The amplified products obtained for each of the dilution liquid are mixed into one. (e) A nucleic acid probe which has a sequence for detecting a target sequence containing the tag sequence and a sequence originated from the template sequence, and immobilized on a substrate material is reacted with the amplified products mixed in the process (d). (f) The amount of the nucleic acid in the specimen is quantitatively determined by detecting the amount of hybridization produced in the process (e).

Description

  Embodiments of the present invention generally relate to nucleic acid quantification methods.

  Analyzing gene expression levels, gene mutations and polymorphisms can be done by discovering genes related to disease efficacy, side effects, etc., as well as diagnosing individual constitution and susceptibility to disease, tailor-made medicine tailored to each individual Is important to realize. Furthermore, it is important to analyze the infectious amount of bacteria and viruses that cause infectious diseases, gene mutations, and polymorphisms in order to perform appropriate diagnosis and treatment.

  The currently used methods for quantifying gene expression levels and bacterial / virus levels are determined by whether hybridization with a probe such as real-time PCR or DNA chip is possible. The real-time PCR method is simple and can test in a relatively short time. However, when it is desired to test a plurality of genes at the same time, the tests must be performed for each gene, which is inconvenient. The method using a DNA chip is mainly a fluorescent labeling method, requires a complicated operation and requires an expensive apparatus, and is limited to research use.

  The problem to be solved by the present invention is to provide a simple method for quantifying nucleic acids.

The nucleic acid quantification method of the embodiment includes:
(a) serially diluting the specimen with a diluent to obtain a plurality of serially diluted solutions;
(b) preparing a primer set for amplifying a target nucleic acid, including a tag sequence-introducing primer containing a different tag sequence for each of the serial dilutions having different concentrations;
Here, the tag sequence is designed to loop out when a primer containing it is hybridized to the template sequence in the template nucleic acid for each specimen;
(c) amplifying a template nucleic acid using the primer set in an independent reaction system for each of the serial dilutions to obtain an amplification product into which the tag sequence has been introduced;
(d) mixing the amplification products obtained for each of the serial dilutions into one;
(e) a nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence and immobilized on a substrate, and the amplification mixed in (d) Reacting with the product;
(f) quantifying the amount of nucleic acid in the sample by detecting the amount of hybridization produced in (e);
It comprises.

Design method for tag introduction primer and probe. The figure which shows the principle figure (basic) of a quantitative method. The figure which shows the principle figure (multiple nucleic acid multi-amplification) of a quantitative method. The figure which shows the principle figure (multiple sample simultaneous detection) of a quantitative method. The figure which shows the principle figure (quantitative analysis + gene mutation and polymorphism analysis separate amplification) of a quantitative method. The figure which shows the principle figure (quantitative + gene mutation and polymorphism analysis simultaneous amplification) of a quantitative method. The figure which shows the principle diagram (quantitative analysis + gene mutation and polymorphism analysis same target) of a quantitative method. The figure which shows the basic primer design of LAMP. The figure which shows the intermediate product of LAMP. . The figure which shows the principle figure (when LAMP amplification method is used) of a quantification method. The figure which shows the design method (in the case of LAMP method) of a tag introduction | transduction primer probe. FIG. 1 is a plan view showing a schematic configuration of a probe nucleic acid-immobilized substrate. FIG. 2 is a plan view showing a schematic configuration of a probe nucleic acid-immobilized substrate. The figure of Example 1. FIG. The figure of Example 1. FIG. The figure of Example 1. FIG. The figure of Example 1. FIG. The figure of Example 1. FIG. The figure of Example 1. FIG. The figure of Example 2. FIG.

[Definition]
“Nucleic acid” is a general term for substances that can represent a part of the structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo.

  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 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, in which case it may be referred to as a “test liquid”. Therefore, the “test solution” may be understood as a solution in which a nucleic acid or a template nucleic acid may exist.

  The nucleic acid contained in the sample is referred to as “sample nucleic acid”. Among the sample nucleic acids, the sequence to be amplified by the primer of this embodiment is referred to as “template sequence”. A nucleic acid containing a template sequence is referred to as a “template nucleic acid” or “template”.

  “Target nucleic acid” refers to an amplification product obtained by amplifying a template nucleic acid or template sequence using a forward primer and a reverse primer according to the present embodiment. The target nucleic acid includes a target sequence in a part thereof.

  A “target sequence” is used to detect a target nucleic acid using a nucleic acid probe.

  A “nucleic acid probe” is a nucleic acid containing a sequence complementary to a target sequence. The nucleic acid probe is used by being immobilized on a solid phase such as a substrate and forms a hybrid with an amplification product containing a region derived from the template sequence.

  The “region derived from the template sequence” means a region in which the template sequence is reflected in a region other than the region to which the primer binds among the regions amplified by the primer. When detecting gene polymorphisms or gene mutations, the sites are designed to fit within this region.

  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.

  The items to be analyzed include, 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, etc. Good. However, the present invention is not limited to this.

  “Amplification” refers to amplifying a target nucleic acid using a primer set, and includes PCR and LAMP. The “LAMP method” includes the LAMP method and the RT-LAMP method. The “RT-LAMP method” is one type of LAMP method that is an isothermal gene amplification method. The RT-LAMP method is a method of performing LAMP amplification using RNA as a template by simultaneously performing a reverse transcription reaction and a LAMP reaction. Here, when it is simply described as “LAMP method”, it may be understood that both “LAMP method” and “RT-LAMP method” are referred to unless otherwise specified. The same primer can be used for the LAMP method and the RT-LAMP method.

[Embodiment]
Hereinafter, this embodiment will be described.

  In the quantification method of the embodiment, the specimen is diluted stepwise, and for each of the obtained multiple dilutions, whether or not the target sequence is detected is detected, and the dilution factor of the dilution in which the target sequence is detected; Using the principle of measuring the amount of nucleic acid contained in the sample that is the original solution, using the dilution factor of the undetected diluent, that is, the detection limit of the target sequence by dilution as an index, and using different tag sequences By using the target nucleic acid contained in the specimen, it becomes possible to simultaneously analyze nucleic acids containing multiple specimens, multiple genes, gene mutations and / or gene polymorphisms, and conveniently and inexpensively. The determination of the amount of nucleic acid is achieved.

<Method for obtaining diluted solution>
The diluted sample solution may be prepared by serial dilution using a manual operation or an automatic apparatus using a micropipette. In addition, a known gradient liquid feeding device technique may be used. Diluted solutions with different concentrations that are serially diluted are selected according to the accuracy of quantification, the state of the sample, etc., such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more or 9 or more Well, further, the degree of serial dilution, that is, the degree of dilution in one stage of dilution is 2 times dilution, 3 times dilution, 4 times dilution, 5 times dilution, 6 times dilution, 10 times dilution, 30 times dilution, 100 times. Dilution, 100-fold dilution, etc. may be selected as desired. Further, more detailed quantification may be achieved by repeating this embodiment.

<Primer for quantitative analysis>
As exemplified by the tag introduction F primer of FIG. 1, a sequence that binds to the primer binding site of the template nucleic acid and a tag sequence for analyzing the sample diluent are introduced into the primer. Alternatively, the tag sequence is also used to detect multiple specimens as necessary.

  The tag sequence is different depending on each serial dilution. When a plurality of specimens are detected simultaneously with a DNA chip, different tag sequences that can be distinguished from each other are prepared for each specimen diluent and for each specimen. The tag sequence may have 3 to 7 bases. The tag sequence is designed such that when the primer containing it binds to the template nucleic acid, the tag sequence portion does not bind to the template nucleic acid and loops out. The tag sequence is also simply referred to as “tag”.

  The length of the primer may be 13-40 bases, for example 15-30 bases. In the case of a primer containing a tag sequence (hereinafter also referred to as “tag sequence-containing primer” or “tag-containing primer”), the length may be 16 to 47 bases, for example, 18 to 37 bases.

  The following is important for the location where the tag sequence is inserted into the primer. That is, the primer needs to bind to the template by looping out the tag sequence portion. Furthermore, the nucleic acid probe needs to contain both a tag sequence and a sequence derived from the template sequence. Therefore, in the case of PCR amplification, the tag sequence may be inserted from the 6th base to the 12th base from the 3 'end side of the Forward primer (F primer) and / or the Reverse primer (R primer). In the case of LAMP amplification, it may be inserted into the 6th to 12th bases from the 3 ′ end of the F2 region and / or B2 region. Specifically, it may be inserted into the 6th to 12th bases from the 3 ′ end of the F2 sequence of the FIP primer and / or the 6th to 12th bases of the B2 sequence of the BIP primer. This provides good amplification and detection characteristics.

  The type of nucleic acid constituting the tag sequence is not particularly limited as long as it is a substance capable of representing a partial structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo.

<Primers for mutation and polymorphism analysis>
When it is desired to perform mutation, polymorphism or type discrimination in the template nucleic acid in addition to nucleic acid quantification, a primer set for mutation, polymorphism and / or type discrimination may be prepared. The primer set is designed to include a mutation, polymorphism or type in the amplification product, and mutation, polymorphism or type discrimination can be performed by detecting this region with a nucleic acid probe. The primer set may be the same as the primer set including the tag introduction primer for quantification, or a different primer set may be prepared for each primer set. Moreover, when preparing separately, you may carry out multi-amplification with the primer set prepared for fixed_quantity | quantitative_assay.

<Nucleic acid probe for quantitative analysis>
The nucleic acid probe is designed to include a sequence complementary to the tag sequence in order to detect the tag sequence contained in the target nucleic acid amplified for quantification. Moreover, as shown in FIG. 1, it is designed to include a sequence complementary to a region derived from the template sequence.

  The chain length of the nucleic acid probe according to the present embodiment is not particularly limited, but is preferably in the range of 5 to 50 bases, more preferably in the range of 10 to 40 bases, and still more preferably in the range of 15 to 35 bases.

  The nucleic acid probe may be modified with a reactive functional group such as an amino group, a carboxyl group, a hydrosyl group, a thiol group or a sulfone group, or a substance such as avidin or biotin in order to be immobilized on a substrate. It is also possible to introduce a spacer between the functional group and the nucleotide. As the spacer, for example, an alkane skeleton, an ethylene glycol skeleton or the like may be used.

  The solid phase for immobilizing the nucleic acid probe may be any substrate generally used as a solid phase for a DNA chip. Such a substrate may be composed of glass, silicon, nitrocellulose film, nylon film, microtiter plate, electrode, magnet, bead, plastic, latex, synthetic resin, natural resin, or optical fiber, but is not limited thereto. Not. A DNA chip may be constructed by immobilizing a plurality of types of nucleic acid probes on these substrates.

<Nucleic acid probe for mutation and polymorphism analysis>
When it is desired to analyze the genetic variation and / or polymorphism of the nucleic acid in the sample simultaneously with the quantitative analysis of the nucleic acid, a nucleic acid probe for detecting the mutation and / or polymorphism is prepared. For example, using a wild-type detection nucleic acid probe and a mutant-type detection nucleic acid probe, respectively, comparing the amount of hybridization between the wild-type detection nucleic acid probe and the target nucleic acid, and between the mutation-type detection nucleic acid probe and the target nucleic acid. Thus, the genotype of the specimen can be determined at the same time.

  When performing type discrimination or species discrimination of a gene in a specimen, a nucleic acid probe for detecting the type and / or species to be detected is prepared. For example, when a plurality of species classified into the same genus of bacteria are identified, for example, a primer set that amplifies in common with the genus is prepared and amplified. If a tag is introduced into a primer set common to this genus and detected with a tag-introduced nucleic acid probe as described above, the amount of bacterial nucleic acid can be quantified. Furthermore, a region characteristic of each species and specific to other species is set as a probe region. By comparing the amount of hybridization between the nucleic acid probe for identifying the species and the target nucleic acid having a characteristic sequence for each species, the nucleic acid probe for the negative control indicating the sequence independent of the species, and the target nucleic acid. Species identification can be performed simultaneously with quantification (see Example 2).

<Method>
Next, a nucleic acid quantification method using a primer having a tag sequence and a DNA chip will be described.

As shown in FIG. 2, initially, a different tag sequence (tag sequence 1, tag sequence 2, the tag sequence 3 ...) introduced primers (10 ⁰ dilution primer of sequence for each sample diluent, 10 x ¹ Prepare primers for dilution, primers for 10 ² fold dilution ...), and perform amplification in a separate reaction system for each sample dilution. The reaction system independent for each specimen dilution solution may be a reaction system in which each dilution solution is not mixed. For example, amplification may be performed in a separate tube for each diluted solution. The “reaction system” refers to a reaction field for performing a reaction there, and may be a container such as a tube and a well, for example.

  After amplification, amplification products having partially different sequences for each diluted solution are obtained. When the template amount of the diluted solution is below the minimum detection concentration, amplification does not occur and no amplification product is obtained. The amplification product obtained in each reaction system contains a tag sequence and a region derived from the template nucleic acid that are different for each dilution. Therefore, the amount of nucleic acid in the stock solution can be quantified by identifying the tag sequence contained in the amplification product and specifying which stage of the diluted solution has been amplified.

  This amplified product is mixed and hybridized with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on a substrate as shown in FIG. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.

Figure 2 In 10 ⁰ fold, 10 x ^1, an example of a 10 ^two-fold dilutions, for dilution number, it is clear that the invention is not limited thereto.

  Furthermore, as shown in FIG. 3, it is also possible to multi-amplify a plurality of template sequences having different sequences within one reaction system. After amplification, nucleic acid quantification can be performed for a plurality of template sequences by detecting the DNA chip for the amplification product of the specimen. Detection is performed by hybridization with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on the substrate. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method. Although FIG. 3 shows an example of two template sequences, it is clear that the number of template nucleic acids is not limited to this.

  Furthermore, as shown in FIG. 4, it is possible to simultaneously detect a plurality of samples by preparing different tags for each diluted solution and for each sample. After amplification, nucleic acid quantification can be performed for a plurality of specimens by performing DNA chip detection on the amplification product of the specimen. Although FIG. 4 shows an example of three specimens, it is clear that the number of specimens is not limited to this.

  Furthermore, as shown in FIGS. 5, 6 and 7, if a primer set for performing mutation, polymorphism and / or type analysis is prepared, quantitative and mutation, polymorphism and / or type analysis can be performed simultaneously. Can do. The primer set for performing mutation, polymorphism and / or type analysis may be amplified independently of the primer set for quantification as shown in FIG. 5 or multi-amplification as shown in FIG. May be. Furthermore, as shown in FIG. 7, the primer set for quantification and the primer set for mutation, polymorphism and / or type analysis may be the same (Example 2 is that of FIG. 7). Although FIG. 5, FIG. 6, and FIG. 7 show examples of one mutation, polymorphism and / or type analysis primer set, it is clear that the number of primer sets is not limited to this.

  The detection target in the present embodiment includes, for example, an individual's genomic DNA, genomic RNA, mRNA, and the like. Individuals include, but are not limited to, humans, non-human animals, plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas.

  These nucleic acids are extracted from samples collected from individuals, such as blood, serum, leukocytes, urine, stool, semen, saliva, tissue, biopsy, oral mucosa, cultured cells, sputum and the like. Alternatively, it is extracted directly from the microorganism. Nucleic acid extraction can be performed using, but not limited to, commercially available nucleic acid extraction kits such as QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals), and the like. A solution containing nucleic acid extracted from an individual sample or a microorganism is used as a test solution.

  The specimen is amplified by the amplification method according to the method of the present embodiment. When the detection target is RNA, it can be converted into complementary strand DNA by, for example, reverse transcriptase before amplification. Both reverse transcriptase and DNA polymerase may be added in the same tube, and reverse transcription and DNA amplification may be performed simultaneously.

  For amplification methods, for example, Polymerase chain reaction method (PCR method), Loop mediated isothermal amplification method (LAMP method), Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN method), SMAP method, Nucleic acid sequence-based amplification Methods (NASBA method), Strand displacement amplification (SDA method), Ligase chain reaction (LCR method), Rolling Circle Amplification method (RCA method), etc. can be used. The obtained amplification product is fragmented or single-stranded as necessary. As a means for forming a single strand, for example, methods known per se such as heat denaturation, a method using beads or an enzyme, a method of performing a transcription reaction using T7 RNA polymerase may be used. When amplified by the LAMP method, ICAN method or the like and a single-stranded region is present in the product and this single-stranded region is used as a target sequence, it can be directly subjected to a hybridization step.

  Since this single-stranded process is not necessary, the target nucleic acid obtained by amplification preferably has a stem-loop structure. The amplification product having a stem-loop structure can conveniently use the sequence of the single-stranded loop portion for the reaction with the probe.

  The LAMP method is suitably used for amplification of the target nucleic acid. The LAMP method is a rapid and simple gene amplification method, and the amplification product has a stem-loop structure.

  FIG. 8 is a diagram showing an example of basic primer design used in the LAMP method. The principle of the LAMP method will be briefly described with reference to the schematic diagram of FIG. In the LAMP method, six types of primers that recognize a maximum of eight regions of a template nucleic acid and a strand displacement type DNA synthase are used. The template nucleic acid is amplified under isothermal (60-65 ° C.) conditions. The eight regions are defined as F3 region, F2 region, LF region, F1 region, B3c region, B2c region, LBc region, and B1c region in this order from the 5 'end side of the template nucleic acid. The F1c, F2c, F3c, B1, B2, and B3 regions indicate regions in the complementary strands of the F1, F2, F3, B1c, B2c, and B3c regions, respectively. 8 types of primers shown in Fig. 8 have FIP inner primer with the same sequence as F2 on the 5 'end side and the same sequence as F2 on the 3' end side, and the same sequence as B1c on the 5 'end side. 'BIP inner primer with sequence complementary to B2c on the terminal side, F3 primer with the same sequence as F3 region, B3 primer with sequence complementary to B3c region, LFc primer with sequence complementary to LF region, LBc primer having the same sequence as the LBc region. Indispensable for the amplification reaction are the FIP inner primer and the BIP inner primer, and the F3 primer, B3 primer, LF primer and LB primer are added to increase the amplification efficiency.

  A loop structure as shown in FIG. 9 is formed in the amplification product by the LAMP method, between the F2 region and the F1 region, between the F2c region and the F1c region, between the B2 region and the B1 region, and between the B2c region and the B1c region. Is a single-stranded region. For this reason, if the target sequence is designed in this region, the target nucleic acid can be detected easily and with high sensitivity.

  When the target sequence overlaps with the LF primer and / or LB primer, it is preferable not to add the LF primer and / or LB primer.

  A nucleic acid quantification method using a tag sequence introduction primer and a DNA chip when the LAMP method is used will be described with reference to FIG.

  First, a primer having the tag sequence introduced into the F2 region and / or B2 region is prepared for each dilution.

Next, amplification is performed in the reaction system independent for each diluted solution using the primer. For example, amplification may be performed in a separate tube for each diluted solution. After amplification, amplification products having partially different sequences for each diluent are obtained in the single-stranded loop portion. When the template amount of the diluted solution is below the minimum detection concentration, amplification does not occur and no amplification product is obtained. This amplified product is mixed and hybridized with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on a substrate as shown in FIG. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method. The amplification product obtained in each reaction system contains a tag sequence and a region derived from the template nucleic acid that are different for each dilution. Therefore, the amount of nucleic acid in the stock solution can be quantified by identifying the tag sequence contained in the amplification product and specifying which stage of the diluted solution has been amplified. Figure 10 In 10 ⁰ fold, 10 x ^1, an example of a 10 ^two-fold dilutions, for dilution number, it is clear that the invention is not limited thereto.

  Furthermore, it is possible to multi-amplify a plurality of types of template sequences comprising different sequences within one reaction system. After amplification, nucleic acid quantification can be performed for a plurality of template sequences by performing DNA chip detection on the amplification product of each dilution. Detection is performed by hybridization with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on the substrate. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.

<DNA chip>
The DNA chip used in this embodiment may be provided with a substrate and a nucleic acid probe immobilized on the substrate. The substrate of the DNA chip 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.

  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 is shown in FIG. 12 as a schematic diagram, but is not limited thereto. The DNA chip has an immobilization region 2 on a substrate 1. The nucleic acid probe is immobilized on the immobilization region 2. Such a DNA chip 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 the arrangement of the immobilization regions 2 arranged on the substrate 1 as necessary. Such a DNA chip may be suitably used for a detection method using fluorescence.

  Another example of the DNA chip is shown in FIG. The DNA chip of FIG. 13 includes an electrode 12 on a base 11. The nucleic acid probe is immobilized on the electrode 12. The electrode 12 is connected to the pad 13. Electrical information from the electrode 12 is acquired via the pad 13. Such a DNA chip 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 electrodes 12 arranged on the substrate 11 as necessary. 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. Although an oxide or a compound can be used, it is not limited to them.

  The DNA chip as in this example may be suitably used for an electrochemical detection method.

<Hybridization conditions>
Hybridization may be performed under appropriate conditions that allow sufficient hybridization. Appropriate conditions vary depending on the type and structure of the target nucleic acid, the type of base contained in the target sequence, and the type of nucleic acid probe. For example, hybridization may be performed in a buffer solution having an ionic strength in the range of 0.01 to 5 and a pH in the range of 5 to 9. The reaction temperature may range from 10 ° C to 90 ° C. The reaction efficiency may be increased by stirring or shaking. In the reaction solution, a hydration accelerator such as dextran sulfate, salmon sperm DNA, and bovine thymus DNA, EDTA, or a surfactant may be added.

<Cleaning conditions>
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, Tris-HCl solution, Tween 20 solution, or SDS solution prepared using sodium chloride or sodium citrate 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. Alternatively, the DNA chip may be immersed in a cleaning solution. In this case, the cleaning liquid is preferably stored in a temperature-controllable container.

<Detection method>
The detection of the hybrid produced by the hybridization process can utilize a fluorescence detection method and an electrochemical detection method.

(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 detection device detects the labeled sequence or the label in the secondary probe. 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.

  Although the double-stranded recognition substance varies depending on the type, it 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-stranded recognition substance reacts electrochemically is applied, and the reaction current value derived from the double-stranded 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.

<Assay kit>
This embodiment may be in the form of an assay kit. The assay kit may include a primer set including a primer into which at least one tag sequence for nucleic acid quantification according to any of the above-described embodiments is introduced, and optionally, a diluent, various enzymes, a reaction container, and a handling kit. Instructions etc. may be included.

  Examples of primer sets included in the assay kit are described below.

When setting F3 region, F2 region, LF region, F1 region from the 5 ′ end side of the template sequence, and B3c region, B2c region, LBc region, B1c region from the 3 ′ end side,
(1) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, And a BIP primer having the same sequence as B1c at the 5 ′ end and a sequence complementary to B2c at the 3 ′ end;
(2) FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and the same sequence as B1c at the 5 'end and complementary to B2c at the 3' end A BIP primer having a typical sequence and having different tag sequences inserted into the B2c sequence with different concentrations of serial dilutions;
(3) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, And a BIP primer having the same sequence as B1c on the 5 ′ end side, a sequence complementary to B2c on the 3 ′ end side, and a different tag sequence inserted in the B2c sequence with different concentrations of serial dilutions;
(4) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, A BIP primer having the same sequence as B1c at the 5 ′ end and a sequence complementary to B2c at the 3 ′ end, an F3 primer having the same sequence as the F3 region, and a B3 primer having a sequence complementary to the B3c region;
(5) FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and the same sequence as B1c at the 5 'end and complementary to B2c at the 3' end A BIP primer having a unique sequence and a different tag sequence inserted into the B2c sequence with different concentrations of serial dilution, an F3 primer having the same sequence as the F3 region, and a B3 primer having a sequence complementary to the B3c region;
(6) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted into the F2 sequence with different concentrations of serial dilutions, A BIP primer having the same sequence as B1c on the 5 ′ end side, a sequence complementary to B2c on the 3 ′ end side, and a different tag sequence inserted into the B2c sequence with different concentrations of serial dilutions, and the F3 region An F3 primer having the same sequence, and a B3 primer having a sequence complementary to the B3c region;
(7) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, BIP primer having the same sequence as B1c on the 5 'end side and having a sequence complementary to B2c on the 3' end side, F3 primer having the same sequence as the F3 region, B3 primer having a sequence complementary to the B3c region, LF An LFc primer having a sequence complementary to the region, and an LBc primer having the same sequence as the LBc region;
(8) FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and the same sequence as B1c at the 5 'end and complementary to B2c at the 3' end BIP primer with different sequence and B2c sequence inserted by different serial dilutions at different concentrations, F3 primer with the same sequence as F3 region, B3 primer with sequence complementary to B3c region, and LF region An LFc primer having a complementary sequence, and an LBc primer having the same sequence as the LBc region;
(9) FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, A BIP primer having the same sequence as B1c on the 5 ′ end side, a sequence complementary to B2c on the 3 ′ end side, and a different tag sequence inserted into the B2c sequence with different concentrations of serial dilutions, and the F3 region F3 primer having the same sequence, B3 primer having a sequence complementary to the B3c region, LFc primer having a sequence complementary to the LF region, and LBc primer having the same sequence as the LBc region.

  However, various primer sets may be selected according to the above-described embodiment, and such a configuration is well understood by those skilled in the art, and those configurations are also included in the examples of the present embodiment.

<Minimum detection limit>
As the measurement method of the minimum detection limit that can be used in the present embodiment, any measurement method known per se may be used. For example, the measurement method of the minimum detection limit may be used as follows.

  First, (1) The weight of the standard mold is measured with an existing measuring instrument. Next, (2) the mass per copy is calculated from the length of the standard template, the type of base, etc. Thereafter, the number of copies of the standard template can be calculated by dividing the weight measured in (1) above by the mass per copy calculated in (2). Next, a standard template with a clear copy number is serially diluted and amplified with the designed primers. The composition of the amplification reaction solution and the reaction conditions are appropriately examined and optimized. The lowest detection limit can be calculated by examining up to which serial dilution was able to be amplified. The standard template may be a plasmid into which the region to be amplified is introduced, a synthesized RNA, or the whole genome of the species to be examined.

For example, when the standard template is a plasmid into which the region to be amplified is introduced, for example, when the length of the plasmid is 4000 bp and the length of the amplification region (insert) is 1000 bp, the total length of the plasmid into which the amplification region is introduced is 5000 bp. It becomes. Assume that the weight of the standard template was 20 ng / μL. The mass per copy of the 5000 bp plasmid is (5000 bp × 660) / (6.02 × 10 ²³ ). Therefore, the number of copies of the standard template is (20 × 6.02 × 10 ²³ ) / (5000 bp × 660 × 10 ⁹ ) = 3.69 × 10 ⁹ copies / μL. For the standard template whose copy number is known in this way, the minimum detection limit may be examined by serial dilution.

  The specific example of the detection by the method of this embodiment is shown.

  An embodiment showing a method for quantifying the amount of nucleic acid with a DNA chip using a primer into which a tag sequence has been introduced will be described in detail.

(1) Preparation of dilution solution of template nucleic acid The template nucleic acid used was a plasmid in which a 16S rRNA sequence of Helicobacter hepaticus belonging to the genus Helicobacter that infects mice was introduced. ^{10 6 ccopies / μL, 10 5} ccopies / μL, 10 4 ccopies / μL, 10 3 ccopies / μL, 10 2 ccopies / μL, respectively 10 ⁰ times the plasmid solution of 10 ¹ ccopies / μL in TE solution (stock solution), 10 ¹ fold, 10 ² fold, 10 ³ fold, and 10 ⁴ fold dilutions were made to make serial dilutions.

(2) Amplification of target nucleic acid 1 μL of the solution serially diluted in the above (1) was added to each LAMP reaction solution to perform LAMP amplification.

[Primer]
Primer sets including Helicobacter hepaticus, Helicobacter bilis, Helicobacter typhlonius, and Helicobacter pylori (sequences shown in Tables 1A and B) were used to amplify the Helicobacter genus in common. The synthetic DNA oligo primers used for amplification of the target nucleic acid are shown in Table 2. To cope with the mutation, two equivalent amounts of FIP primer were mixed. Tag 1 (CTG) introducing primers that amplify 10 ^0-fold dilutions in FIP primer, the tag 2 (GGA) introducing primers that amplify 10 ^1-fold dilution, the tag 3 for amplifying the 10 ^two-fold dilutions (CCT) introducing primer Five types of primers were introduced: tag 4 (TCC) -introduced primer for amplifying 10 ³ -fold diluted solution and tag 5 (ATC) -introduced primer for amplifying 10 ⁴ -fold diluted solution. The minimum detection limit of this primer set was 10 ² ccopies / reaction solution.

[LAMP reaction solution]
The composition of the reaction solution used in the LAMP method is shown in Table 3.

[Amplification of nucleic acid]
Nucleic acids were amplified by the LAMP method at 63 ° C. for 1 hour. In the negative control, sterilized water was used instead of the template. Amplification was performed with GeneAmp PCR System 9700 (Applied Biosystems).

(3) DNA chip detection of target nucleic acid Chip detection of the product amplified in (2) above was performed.

[Nucleic acid probe]
The synthetic DNA oligonucleic acid probes used for detection are shown in Table 4.

As a nucleic acid probe, tag 1 (10 ^0-fold dilution) for detecting the tag 2 (10 ^1-fold dilution) for detecting the tag 3 (10 ^two-fold dilutions) for detecting the tag 4 (10 ³ fold dilutions) Detection And 5 types for detecting tag 5 (10 ⁴ times diluted solution) were prepared. As negative controls, 5 types of negative probes having sequences unrelated to the Helicobacter gene sequence were mixed and used. The above probe was thiol-modified at the 3 ′ end for immobilization on a gold electrode.

[Probe immobilized electrode]
The above probe was immobilized on a gold electrode. The probe was immobilized using the strong covalent bond between thiol and gold. A probe solution containing 1 mM mercaptohexanol solution was spotted on a gold electrode and dried. The same probe was spotted on each of the four electrodes. The positions of the electrodes of each probe are shown below. After drying, it was washed with ultrapure water and air-dried to obtain a probe-immobilized electrode.

[Electrode arrangement]
The electrode arrangement was as follows:
1-4 electrode Negative probe
5-8 electrode Tag 1 detection probe
9-12 electrode Tag 2 detection probe
13-16 electrode Tag 3 detection probe
17-20 electrode Tag 4 detection probe
21-24 electrode Tag 5 detection probe
25-28 electrode negative probe.

[Hybridization]
The probe-immobilized electrode prepared as described above was immersed in a LAMP product to which 2 × SSC salt was added and allowed to stand at 64 ° C. for 10 minutes for hybridization. Thereafter, the probe-immobilized electrode was washed by immersing it in a 0.2 × SSC solution at 44 ° C. for 3 minutes. Next, the probe-immobilized electrode was immersed for 1 minute in a phosphate buffer containing 50 μM Hoechst 33258 solution as an intercalating agent. Thereafter, the oxidation current response of Hoechst 33258 solution was measured.

[result]
As shown in FIG. 14, for 10 ⁶ ccopies / reaction, 10 ⁰ dilution, 10 ¹ diluted, 10 ² dilution, 10 ³ diluted in ¹⁰⁴ dilution of all, the signal increase was detected. Up to 10 ³ dilution for 10 ⁵ copies / reaction up to 10 ² dilutions for 10 ⁴ copies / reaction, 10 ³ copies / to the reaction liquid 10 ¹ dilution for 10 ⁰ dilution for 10 ² copies / reaction An increase in signal was detected. For 10 ¹ copies / reaction, no signal increase was detected at all dilution steps. Thus, it was shown that the amount of nucleic acid in the sample can be quantified by using the tag introduction primer.

  Examples illustrating the method of simultaneously detecting both nucleic acid content and genotype with a DNA chip are described in detail.

(1) Preparation of dilution solution of template nucleic acid The same plasmid as in Example 1 was used as the template nucleic acid. 10 ⁰ times 10 ⁴ plasmid solution ccopies / μL with TE solution (stock solution), 10 x ^1, 10 ² times, 10 ³ fold, diluted 10 ⁴ fold, to prepare serial dilutions.

(2) Amplification of target nucleic acid It was carried out in the same manner as in Example 1. This primer set amplifies the Helicobacter genus in common, and the amplified template-derived region includes a region that can distinguish Helicobacter hepaticus, Helicobacter bilis, Helicobacter typhlonius, and Helicobacter pylori, and this region is probed. By detecting, species discrimination can also be performed.

(3) DNA chip detection of target nucleic acid [Nucleic acid probe]
The synthetic DNA oligonucleic acid probes used for detection are shown in Table 4.

As a nucleic acid probe to quantify the template nucleic acid amount, tag 1 (10 ^0-fold dilution) for detecting the tag 2 (10 ^1-fold dilution) for detecting the tag 3 (10 ^two-fold dilutions) for detecting the tag 4 Five types for detection (10 ³ times diluted solution) and for detection of tag 5 (10 ⁴ times diluted solution) were prepared. In addition, probes for detecting each of Helicobacter hepaticus, Helicobacter bilis, Helicobacter typhlonius, and Helicobacter pylori were prepared as nucleic acid probes for species discrimination. As in Example 1, five negative probes having sequences unrelated to the Helicobacter gene sequence were mixed and used as a negative control. The above probe was thiol-modified at the 3 ′ end for immobilization on a gold electrode.

[Probe immobilized electrode]
The same method as in Example 1 was performed.

[Electrode arrangement]
The electrode arrangement was as follows:
1-4 electrode Negative probe
5-8 electrode Helicobacter hepaticus detection probe
9-12 electrode Helicobacter bilis detection probe
13-16 electrode Helicobacter typhlonius detection probe
17-20 electrode Helicobacter pylori detection probe
21-24 electrodes Tag 1 detection probe
25-28 electrode Tag 2 detection probe
29-32 electrode Tag 3 detection probe
33-36 electrode Tag 4 detection probe
37-40 electrode Tag 5 detection probe
41-44 electrodes Negative probe.

[Hybridization]
The same method as in Example 1 was performed.

[result]
As shown in FIG. 15, for the probe for quantification, signal increase was observed up to 10 ² dilution, indicating that the stock solution was 10 ⁴ copies / reaction solution. As for the type discrimination probe, an increase in signal was observed from the probe detecting Helicobacter hepaticus. From these results, it was shown that nucleic acid quantification and type discrimination can be analyzed simultaneously by using a tag introduction primer, a tag introduction nucleic acid probe, a type discrimination nucleic acid probe, and a DNA chip.

  As described above, according to the present embodiment, it is possible to simultaneously analyze nucleic acids containing a plurality of specimens, a plurality of genes, gene mutations and / or gene polymorphisms, and it is possible to easily and inexpensively determine the amount of nucleic acids. The

  DESCRIPTION OF SYMBOLS 1 ... Base | substrate, 2 ... Immobilization area | region, 11 ... Base | substrate, 12 ... Electrode, 13 ... Pad

Claims (8)

A method for quantifying the amount of nucleic acid,
(a) serially diluting the specimen with a diluent to obtain a plurality of serially diluted solutions;
(b) preparing a primer set for amplifying a target nucleic acid, including a tag sequence introduction primer containing a different tag sequence for each of the serial dilutions having different concentrations;
Here, any of the tag sequences is designed to loop out when a primer containing the tag sequence hybridizes to a template sequence in a template nucleic acid contained in the specimen;
(c) amplifying the template nucleic acid using the primer set in an independent reaction system for each of the serial dilutions to obtain an amplification product into which the tag sequence has been introduced;
(d) combining the amplification products obtained for each of the serial dilutions into one;
(e) a nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence and immobilized on a substrate, and the amplification mixed in (d) Reacting with the product;
(f) quantifying the amount of nucleic acid in the sample by detecting the amount of hybridization produced in (e);
An analysis method comprising: A method for quantifying the amount of nucleic acid of a plurality of genes,
(a) serially diluting the specimen with a diluent to obtain a plurality of serially diluted solutions;
(b) A primer set for amplifying a plurality of target nucleic acids, including a primer for introducing a tag sequence having a different tag sequence for each of the serial dilutions having different concentrations Preparing each set,
Here, any of the tag sequences is designed to loop out when a primer containing the tag sequence hybridizes to a template sequence in a template nucleic acid contained in the specimen;
(c) Multi-amplifying a template nucleic acid using a primer set including a plurality of primer introduction primers different from each other in an independent reaction system for each serial dilution solution, and obtaining an amplification product into which the tag sequence is introduced When,
(d) combining the amplification products obtained for each of the serial dilutions into one;
(e) a nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence of the template nucleic acid, immobilized on a substrate, and mixed in (d) Reacting with said amplified product;
(f) quantifying the amount of nucleic acid of a plurality of genes in a specimen by detecting the amount of hybridization produced in (e);
An analysis method comprising: A method for quantifying the amount of nucleic acid in a plurality of samples,
(a) serially diluting a plurality of specimens with a diluent to obtain a serially diluted solution;
(b) preparing a primer set for amplifying a target nucleic acid, including primers containing different tag sequences for each of the serial dilutions having different concentrations and for different samples;
Here, any of the tag sequences is designed to loop out when a primer containing the tag sequence hybridizes to a template sequence in a template nucleic acid contained in any of the specimens;
(c) Amplifying a template nucleic acid using a primer set containing a tag sequence introduction primer in a reaction system independent for each serial dilution and each sample, and obtaining an amplification product into which the tag sequence has been introduced;
(d) combining the amplification products obtained for each of the serial dilutions and the specimens into one;
(e) a nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence of the template nucleic acid and immobilized on a substrate, and obtained in (e) Reacting with said amplification product;
(f) quantifying the amount of nucleic acid in a plurality of samples by detecting the amount of hybridization produced in (e);
An analysis method comprising: A method for quantifying the amount of nucleic acid of a plurality of genes in a plurality of samples,
(a) serially diluting a plurality of specimens with a diluent to obtain a plurality of serially diluted solutions;
(b) preparing each primer set for amplifying a plurality of target nucleic acids;
Preparing a tag sequence-introducing primer containing a different tag sequence for each serial dilution having different concentrations and for each different sample;
Here, any of the tag sequences is designed to loop out when a primer containing the tag sequence hybridizes to a template sequence in a template nucleic acid contained in any of the specimens;
(c) Amplification in which the template nucleic acid is multi-amplified by using a primer set including a plurality of different primer sequences for introducing primer sequences in an independent reaction system for each of the serial dilutions and different samples, and the tag sequences are introduced Getting the product,
(d) combining the amplification products obtained for each of the serial dilutions and different samples;
(e) a nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence of the template nucleic acid, immobilized on a substrate, and mixed in (d) Reacting with said amplification product;
(f) quantifying the amount of nucleic acid of a plurality of genes in a plurality of samples by detecting the amount of hybridization produced in (e);
An analysis method comprising: A method for simultaneously quantifying nucleic acid content and detecting gene mutation / polymorphism,
(a) serially diluting the specimen with a diluent to obtain a plurality of serially diluted solutions;
(b) separately preparing primer sets for amplifying a target nucleic acid for quantitative analysis and a target nucleic acid for mutation / polymorphism detection;
Here, the primer set for quantitative analysis includes a primer containing a different tag sequence for each of the serial dilutions at different concentrations, and the tag sequence includes a primer in the template nucleic acid for each specimen. Designed to loop out when hybridized to a sequence;
(c) amplifying the template nucleic acid using a primer set containing a primer sequence introduction primer for quantitative analysis in an independent reaction system for each serial dilution, and obtaining an amplification product into which the tag sequence has been introduced; ,
(d) amplifying the template nucleic acid using the mutation / polymorphism detection primer set using either one of the serially diluted solution or the sample before dilution, and obtaining an amplification product;
(e) combining the amplification product for quantitative analysis and the amplification product for mutation / polymorphism detection obtained for each of the serial dilutions into one;
(f) A nucleic acid probe having a sequence for detecting a target sequence including the tag sequence and a sequence derived from the template sequence of the template nucleic acid, and detecting a gene mutation / polymorphism immobilized on the substrate Reacting the nucleic acid probe having a sequence to be immobilized on the substrate with the amplification product mixed in (e);
(g) quantifying the amount of nucleic acid in the sample and detecting gene mutation / polymorphism by detecting the amount of hybridization produced in (f);
An analysis method comprising: A method for simultaneously quantifying nucleic acid content and detecting gene mutation / polymorphism,
(a) serially diluting the specimen with a diluent to obtain a plurality of serially diluted solutions;
(b) preparing a common primer set for amplifying the target nucleic acid for quantitative analysis and the target nucleic acid for mutation / polymorphism detection;
Here, the primer set includes a primer for quantitative analysis and a primer for gene mutation / polymorphism detection includes a primer including a tag sequence that differs for each serial dilution having different concentrations, and the tag sequence includes a primer including the primer sequence. , Designed to loop out when hybridized to the template sequence in the template nucleic acid for each specimen;
(c) Amplifying the template nucleic acid using a primer set including a tag sequence introduction primer for quantitative analysis and gene mutation / polymorphism detection in an independent reaction system for each serial dilution, and the tag sequence Obtaining an amplified product in which
(d) combining the amplification product for quantitative analysis and the amplification product for mutation / polymorphism detection obtained for each of the serial dilutions into one;
(e) a nucleic acid probe having a sequence for detecting a target sequence for quantitative analysis including the tag sequence and a sequence derived from the template sequence of the template nucleic acid, immobilized on a substrate, and gene mutation / multiple Reacting a nucleic acid probe having a sequence for detecting a mold and immobilized on a substrate with the amplification product mixed in (d);
(f) quantifying the amount of nucleic acid in the sample and detecting gene mutation / polymorphism by detecting the amount of hybridization produced in (e);
An analysis method comprising:   The analysis method according to claim 1, wherein the gene amplification method is a LAMP method. An assay kit comprising a primer set for LAMP amplification for quantifying the amount of nucleic acids of a plurality of genes,
At least one primer set selected from the group consisting of:
When setting F3 region, F2 region, LF region, F1 region from the 5 ′ end side of the template sequence, and B3c region, B2c region, LBc region, B1c region from the 3 ′ end side,
1． FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted into the F2 sequence with different concentrations of serial dilution, and 5 ′ A BIP primer having the same sequence as B1c on the terminal side and a sequence complementary to B2c on the 3 ′ terminal side;
2． FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and a sequence complementary to B2c at the 3 'end with the same sequence as B1c at the 5' end A BIP primer having a B2c sequence and a tag sequence different from each other inserted by serial dilutions at different concentrations;
3． FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted into the F2 sequence with different concentrations of serial dilution, and 5 ′ A BIP primer having the same sequence as B1c on the terminal side, a sequence complementary to B2c on the 3 ′ terminal side, and a different tag sequence inserted into the B2c sequence with different concentrations of serial dilutions;
Four. FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, 5 ′ end A BIP primer having the same sequence as B1c on the side and a sequence complementary to B2c on the 3 ′ end side, an F3 primer having the same sequence as the F3 region, and a B3 primer having a sequence complementary to the B3c region;
Five. FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and a sequence complementary to B2c at the 3 'end with the same sequence as B1c at the 5' end And a BIP primer in which different tag sequences are inserted with different concentrations of serial dilutions in the B2c sequence, an F3 primer having the same sequence as the F3 region, and a B3 primer having a sequence complementary to the B3c region;
6． FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, 5 ′ end BIP primer with the same sequence as B1c on the side, a sequence complementary to B2c on the 3 'end side, and the same sequence as the F3 region, with different tag sequences inserted into the B2c sequence with different concentrations of serial dilutions An F3 primer with a B3 primer having a sequence complementary to the B3c region;
7． FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, 5 ′ end BIP primer with the same sequence as B1c on the side and complementary sequence to B2c on the 3 'end side, F3 primer with the same sequence as F3 region, B3 primer with a sequence complementary to B3c region, complementary to LF region An LFc primer having a typical sequence and an LBc primer having the same sequence as the LBc region;
8． FIP primer with a sequence complementary to F1 at the 5 'end and the same sequence as F2 at the 3' end, and a sequence complementary to B2c at the 3 'end with the same sequence as B1c at the 5' end In addition, BIP primers with different B2c sequences inserted by serial dilutions of different concentrations, F3 primers having the same sequence as the F3 region, B3 primers having a sequence complementary to the B3c region, and complementary to the LF region An LFc primer having a sequence, and an LBc primer having the same sequence as the LBc region;
9． FIP primer having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a different tag sequence inserted in the F2 sequence with different concentrations of serial dilutions, 5 ′ end BIP primer with the same sequence as B1c on the side, a sequence complementary to B2c on the 3 'end side, and a different sequence of the B2c sequence inserted by different serial dilutions, and the same sequence as the F3 region F3 primer having, B3 primer having a sequence complementary to the B3c region, LFc primer having a sequence complementary to the LF region, and LBc primer having the same sequence as the LBc region;
Assay kit.

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