WO2022139121A1 - Method for detecting target point mutation on basis of polymerase chain reaction - Google Patents

Abstract

The present invention relates to a method for detecting a target point mutation on the basis of a polymerase chain reaction.

Description

Detection method for target point mutations based on polymerase chain reaction

The present invention relates to a method for detecting a target point mutation based on a polymerase chain reaction.

Genetic variations that are abundant in genes are implicated in many parts of a variety of diseases, including cancer, multiple sclerosis, and autoimmune diseases. For this reason, gene polymorphism detection is essential for personalized medicine.

Genetic variation includes substitutions, deletions, insertions, and repetitions. Among them, a point mutation by SNP (Single Nucleotide Polymorphism) accounts for the most part, and it occurs with a probability of 1 per 1000 base sequences.

In order to detect such a point mutation, a number of point mutation analysis methods have been devised in the past. Representatively, an allele-specific PCR method (eg, US Patent Nos. 5,639,611 and 5,496,699), a method using a Taqman™ probe (eg, See, for example, U.S. Patent Nos. 5,538,848 and 6,326,145) and next-generation sequencing methods (Ronaghi, Mostafa, et al. "Real-time DNA sequencing using detection of pyrophosphate release." Analytical biochemistry 242.1 (1996): 84-89) .) and so on.

However, the next-generation sequencing method, which is currently most used, has the advantage of being able to analyze a wide area at once, but has limitations in that it has low sensitivity in detecting a point mutant gene with a low frequency and a long test period and expensive cost. exist.

Therefore, a new point mutation analysis method with high specificity and sensitivity is continuously in demand.

On the other hand, SNP (Single Nucletide Polymorphism) is a genetic mutation in which one nucleotide sequence is substituted in a DNA nucleotide sequence, which causes individual differences in each person, such as the cause of a disease or response to a treatment. The detection and confirmation of single nucleotide sequence mutations is attracting attention because it is linked to the development of new drugs as well as customized medicines.

Therefore, various detection methods applying real-time PCR technology are used for rapid detection of single nucleotide sequence mutations. Representatively, a method using a DNA intercalating fluorescent material, a method using a DNA probe, or and a method using a PNA probe.

However, it has limitations in using DNA intercalating fluorescent materials, and has the disadvantage that a program to analyze the melting curve must be used (Kirk M. Ririe et al., Analytical Biochemistry 245:154, 1997; U. Hladnik). et al., Clin Exp Med, 2:105, 2002), there is still a continuous need for new point mutation analysis methods with high specificity and sensitivity.

Under this background, the present inventors can selectively amplify a target when using a hairpin-type primer that discriminates point mutations, and use a probe to identify only singles that match the target to selectively distinguish point mutations. With this in mind, this detection method improves the specificity and sensitivity compared to the conventional method, it can analyze point mutations quickly at lower cost, and furthermore, it is a reagent for distinguishing or diagnosing SNPs, or diagnosing various molecules such as cancer or viruses. By confirming that it can be effectively used in the market, the present invention has been completed.

One object of the present invention is (a) a sample comprising a target point mutation region; (b) a hairpin primer to discriminate point mutations; (c) a probe that binds to the target point mutation; (d) a linear primer that degrades the probe; And (d) to provide a composition for detecting a target point mutation comprising a DNA polymerase.

Another object of the present invention is (a) a sample comprising a target point mutation region; (b) a hairpin primer to discriminate point mutations; (c) a probe that binds to the target point mutation; (d) a linear primer that degrades the probe; And (d) to provide a kit for detecting a target point mutation comprising a DNA polymerase.

Another object of the present invention is to provide a composition for SNP classification or diagnosis, including the composition or kit.

Another object of the present invention is to react a composition comprising a sample including a target point mutation region, a hairpin primer for discriminating a point mutation, a probe binding to a target point mutation, a linear primer decomposing the probe, and a polymerase to amplify the sample containing the target point mutation region; and measuring the fluorescence level of the probe, to provide a method for detecting a target point mutation.

Each description and embodiment disclosed herein is also applicable to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

In addition, in the entire specification of the present application, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated. do.

Hereinafter, the present invention will be described in more detail.

A first aspect of the present invention for achieving the above object is (a) a sample comprising a target point mutation region; (b) a hairpin primer to discriminate point mutations; (c) a probe that binds to the target point mutation; (d) a linear primer that degrades the probe; And (d) it provides a composition for detecting a target point mutation comprising a DNA polymerase.

In addition, the second aspect of the present invention is (a) a sample comprising a target point mutation region; (b) a hairpin primer to discriminate point mutations; (c) a probe that binds to the target point mutation; (d) a linear primer that degrades the probe; And (d) it provides a kit for detecting a target point mutation comprising a DNA polymerase.

In addition, a third aspect of the present invention provides a composition for SNP classification or diagnosis, comprising the composition or kit.

As used herein, the term "sample including a target point mutation region" refers to a nucleic acid sequence to be detected, and refers to a nucleic acid sequence containing a point mutation region. The sample containing the target mutation region is not different from "target sequence", "target sequence", "target nucleic acid", "target nucleic acid sequence", "target sample" or "target nucleic acid" herein, can be mixed in

The sample may be a gDNA or cfDNA sequence, the gDNA may target a point mutation site in gDNA, and the cfDNA may target ctDNA in cfDNA, but if it is a sequence including a point mutation region, The sample may be included without limitation.

The "point mutation" refers to a mutation caused by a change of one base in a gene sequence, and may include a mutation in which a base of a nucleic acid of a target sample is substituted, deleted, or inserted.

As used herein, the term "primer" refers to a short gene sequence that serves as a starting point for making another polymer strand complementary to a template during DNA synthesis, that is, it consists of about 10 to 22 bases necessary for the initial stage of polymer synthesis. refers to a short section.

The composition of the present invention includes two primers for detection of point mutations in a target sample, one is a hairpin primer having a hairpin structure and the other is a linear primer in a linear form.

The "hairpin primer" refers to a primer having a hairpin structure, and the hairpin structure is, as can be inferred from the name, a stem portion formed by hydrogen bonding between complementary reverse repeats of single-stranded RNA or DNA. It refers to a structure with a ring portion in the form of a loop and a loop.

In the present invention, the hairpin primer is a primer that distinguishes point mutations in the target sample containing the point mutation. When a point mutation is present in the target sample, the stem portion of the hairpin structure is completely stretched and elongated, but the target sample It is characterized in that when no internal point mutation exists, the stem part of the hairpin structure is not fully stretched and thus elongation is not possible or very insignificant.

The hairpin primer may be any one selected from the group consisting of DNA, LNA and PNA, and specifically may be LNA, but is not limited thereto.

The LNA means Locked Nucleic Acid, and it increases its own melting point by creating a methylene bridge at the 2nd oxygen and 5th carbon in the DNA pentose, and increases specificity even in short oligo sequences. It is characterized in that it can be, and specifically has the following Structural Formula 1.

[Structural Formula 1]

In addition, the PNA refers to Peptide Nucleic Acid, and by substituting an amino group for the phosphate ribose backbone of DNA, PNA has the following structural formula 2, characterized in that it has chemical, biological and thermal stability compared to DNA.

[Structural Formula 2]

The "linear primer" is a general linear type primer, not a specific structure, as the term, referring to a primer that rejoins the new strand generated by extension of the new strand by the hairpin primer, and at this time, the probe is decomposed. , more specifically, it is characterized in that the probe decomposes as elongation proceeds after binding to a new strand.

As used herein, the term "probe" is a fragment or fragment complementary to a specific nucleotide sequence of DNA or RNA, and refers to a DNA or RNA fragment having a terminal base labeled with a radioactive element or fluorescence, also referred to as a nucleic acid probe. It usually varies in length from 10 to 1000 bp and has a complementary sequence to find a specific nucleotide sequence in a DNA or RNA sample.

In the present invention, the probe binds to a complementary sequence capable of binding to the point mutation sequence in the target sample. After the sample containing the point mutation in the target sample is extended by the hairpin primer, the linear primer is then added to the new strand. Upon binding, the probes are bound together, and as they are extended, the probe is degraded by DNA polymerase and the fluorescence value can be measured.

Accordingly, the probe of the present invention also has a terminal base labeled with a radioactive element or fluorescence, and is specifically labeled with a radioactive isotope or a chemical such as biotinylated uridine, so that the probe attached to the target base sequence can be detected. Biotin-labeled probes can be detected by indirect immunofluorescence or enzyme immunoassay method after reacting with avidin or streptavidin molecules labeled with a fluorescent dye or enzyme.

As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes new DNA while linking nucleotides to a DNA chain, and the enzyme that synthesizes DNA using DNA as a template refers to a DNA-dependent DNA polymerase .

The DNA polymerase is not particularly limited, but for example, may have exonuclease so that a fluorescence level can be measured while decomposing the probe when the probe binds to a new strand together with a linear primer.

Also, as another example, the DNA polymerase may not have terminal-degrading activity when using intercalating fluorescence.

The composition for detecting a target point mutation may further include tetramethylammonium chloride (TMAC).

As used herein, the term "Tetramethylammonium chloride (TMAC)" is tetramethylammonium chloride, and has the following structural formula 3, the molecular formula is C ₄ H ₁₂ ClN, the molecular weight is 110, and the density is 1.17 g/cm ³ Having white crystals say material. The TMAC is not particularly limited, but may be used at a concentration of 0 to 100 mM.

[Structural Formula 3]

In a specific embodiment of the present invention, the mechanism of the amplification product according to the presence or absence of a point mutation was confirmed. In particular, when there is a point mutation in a target sample, (a) a sample including a target point mutation region; (b) a hairpin primer to discriminate point mutations; (c) a probe that binds to the target point mutation; (d) a linear primer that degrades the probe; And (d) it was confirmed that if a composition including all of the DNA polymerase was used, the presence or absence of a point mutation could be rapidly and accurately detected only with the composition without additional experimentation. In particular, the presence or absence of point mutations in the hairpin primer LNA was confirmed more accurately and quickly than other types of hairpin primers.

In addition, in another specific embodiment of the present invention, when TMAC is additionally included in the composition, when the TMAC concentration is increased to 5 to 20 mM, the Ct values of mutants and wild-types increase as the concentration increases. As it was confirmed that it increased more, it was confirmed that the ΔCt value, which is a measurement standard of specificity, became larger.

A fourth aspect of the present invention for achieving the above object includes a sample including a target point mutation region, a hairpin primer for discriminating point mutations, a probe binding to the target point mutation, a linear primer degrading the probe, and a polymerase reacting the composition to amplify a sample containing the target point mutation region; And it provides a target point mutation detection method comprising the step of measuring the fluorescence level of the probe.

Specifically, the detection method is a method based on polymerase chain reaction (PCR), and more specifically,

(a) denaturing the sample containing the target point mutation region (denaturation); (b) binding and extending the hairpin primer to the point mutation in the sample; (c) denaturing the sample to bind the linear primer and the probe; (d) degradation of the bound probe by a polymerase; and (e) measuring the fluorescence level of the probe.

The "sample containing the target point mutation region", "point mutation", "hairpin primer", "linear primer", "probe" and "polymerase" are the same as described above.

As used herein, the term "polymerase chain reaction" refers to a method that can amplify a large amount of the DNA part sandwiched between two primers in vitro. DNA polymerase requires primers for DNA synthesis. , using the DNA synthesis in the 5′ → 3′ direction from this primer, ① DNA denaturation to single strand → ② Primer binding → ③ Complementary DNA synthesis by polymerase → ① denaturation → ② Primer binding It refers to a method in which only the desired gene region is propagated in vitro by repeating the circuit.

The detection method of the present invention is based on the polymerase chain reaction, and the sample containing the target point mutation region is denatured, then the hairpin primer is bound and extended, and then denatured again after the linear primer and the probe are bound. The point mutation in the target sample can be detected by the principle that the probe is decomposed and the fluorescence level is measured as it is elongated.

The denaturation is similar to the conditions used in a general PCR method, and specifically, it may be performed at 90 to 99° C. for 10 seconds to 3 minutes, but is not limited thereto.

In addition, the specific conditions for binding and extending the primer are also similar to the conditions used in the PCR method, specifically, at 55 to 70 ° C. for 10 seconds to 60 seconds, more specifically at 55 to 65 ° C. for 10 seconds to 40 seconds. It may be performed, but is not limited thereto.

Measuring the fluorescence level refers to measuring the level of fluorescence emitted from the terminal base of the fluorescently-labeled probe. Only when there is a point mutation in the target sample, it is stretched by the hairpin primer to smoothly generate an amplification product, Fluorescence is measured. On the other hand, when there is no point mutation in the target sample, the hairpin primer is not fully extended, so that the amplification product is not smoothly generated.

In a specific embodiment of the present invention, the mechanism of the amplification product according to the presence or absence of a point mutation was confirmed. In particular, if there is a point mutation in the target sample, (a) denaturing the sample including the target point mutation region, The point mutation in the target sample is effectively detected by binding the hairpin primer to the point mutation and extending it, denaturing the sample again, binding the linear primer and the probe, and measuring the fluorescence level of the probe while the bound probe is decomposed by a polymerase confirmed that it can be done.

In the present invention, multiple targets can be analyzed simultaneously in one reaction using primers designed according to the type of each target point mutation, and the presence or absence of point mutations can be accurately and quickly confirmed only with the polymerase chain reaction composition without additional experiments. Therefore, it can be effectively used as a reagent for identification or diagnosis of SNPs, or in various molecular diagnostic markets such as cancer or viruses.

1 shows the composition of the polymerase chain reaction for the detection of a target point mutation of the present invention.

Figure 2 shows the mechanism of the amplification product according to the presence or absence of a target point mutation. Specifically, (a) shows a mechanism of an amplification product having a base complementary to a point mutation when there is a target point mutation target, and (b) shows a mechanism by which an amplification product is generated in the absence of a target point mutation target.

3 shows a detection method according to the presence or absence of a point mutation. Specifically, (a) shows that the probe is degraded with a polymerase exonuclease function, and (b) shows a method for discriminating point mutations.

4 shows the thermocycling process of the polymerase chain reaction, the temperature and time of each process can be adjusted to fit the optimal conditions.

5 shows the detection ability of the hairpin-type primer of the present invention. Specifically, (a) and (b) show the results of measuring the Ct value of a target without a point mutation using a non-hairpin type primer and a hairpin type primer, respectively.

6 shows the results of measuring Ct values according to the presence or absence of a hairpin type in the primer.

7 shows the results of measuring Ct values according to the types of hairpin primers.

8 shows the results of measuring Ct values in the mutant and wild type according to the increase in TMAC concentration.

9 shows an increase in the Ct value of the wild-type according to an increase in the TMAC concentration.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.

Example 1: Confirmation of the mechanism of generating amplification products according to the presence or absence of target point mutations

First, the mechanism of generating an amplification product according to the presence or absence of a target point mutation was checked, and the specific steps are shown in FIG. 2 .

Specifically, Fig. 2 (a) shows the mechanism of generating an amplification product when a point mutation is present, and the sample containing the point mutation (red) in 1 was denatured at 98 °C. Thereafter, primer 1 having a sequence complementary to the point mutation located in the denatured strand was bound at 2 to 64°C. In addition, the base (orange) complementary to the point mutation was extended after binding the primer at 3 to 64°C. After that, this strand was again denatured at 98°C. At 4 to 64 ° C, primer 2 is bound to the strand having a base complementary to the point mutation, and at 5 to 64 ° C, the strand is extended with primer 2 polymerase, and the target having a base complementary to the point mutation at 6 is was confirmed to be amplified.

Next, FIG. 2 (b) shows the mechanism of generating an amplification product in a wild-type target without a point mutation, where 1 represents a strand not including a point mutation, and this strand was denatured at 98°C. 2 represents the binding of the primer to the denatured strand at 64° C., and 3 represents the product extended by the primer bound at 64° C. After this product was denatured at 98 ° C again, primer 2 was bound to the strand produced in step 3 in step 4, and a target having no point mutation was generated by primer 2 at 5 to 64 ° C. After 6, it was confirmed that a target without a point mutation was generated.

Thus, it was possible to confirm the mechanism of how the amplification product was generated by the primer when and when the point mutation was not included.

Example 2: Confirmation of detection according to the presence or absence of a target point mutation using the composition of the present invention

First, the composition of the composition for detecting a target point mutation of the present invention is shown in FIG. 1 . Specifically, the composition of the present invention comprises (a) a sample comprising a target point mutation region; (b) a hairpin primer for discriminating point mutations; (c) a linear primer that degrades the probe; (d) a probe that binds to the target point mutation and (e) a polymerase.

Whether or not the target point mutation was detected using the composition was checked, and the specific process is shown in FIG. 3 .

Specifically, the method of confirming the detection according to the presence or absence of a target point mutation using the composition of the present invention is also based on Example 1, and among them, 1 and 2 of FIG. 2 (a) are the same. Thereafter, the linear primer and probe are bound to the strand having a base complementary to the point mutation generated in 3 of FIG. 3 (a), in 4 of FIG. 3 (a), and in 5 of FIG. 3 (a), the When the probe is decomposed by the exonuclease function of the polymerase and the probe is bound and then decomposed, as shown in 6 of FIG. 3(a), the fluorescence value of the probe can be confirmed.

On the other hand, when there is no point mutation, as can be seen in (b) of FIG. 3, as in (b) 3) of FIG. 3), the hairpin primer has a 3' end sequence of the primer and a sequence that does not have a point mutation It was not complementary to, and it was confirmed that the stem portion of the hairpin structure was not completely straightened, thereby not elongating.

However, even in this case, some targets are extended (refer to (b) 4 of FIG. 3), and as can be seen in 4 to 6 of FIG. 3 (b), some of the strands are stretched so that the linear primer binds but the probe does not. Therefore, even if a new strand is extended by the polymerase, the probe cannot bind, so the fluorescence value could not be measured.

Combining Examples 1 and 2, when using the composition of the present invention, when a point mutation is included in the target sample, the primer of the hairpin structure is completely stretched and extended to form a new strand, and after binding of the linear primer and the probe thereto Thereafter, by measuring the fluorescence value while the probe was decomposed by the polymerase, it was confirmed that the presence or absence of a point mutation in the target sample could be effectively and quickly detected using only one experiment.

Example 3: Confirmation of the thermocycling process of the polymerase chain reaction and the primer detection ability of the hairpin structure in the detection method of the present invention

In the detection method of the present invention, specific conditions of the thermocycling process of the polymerase chain reaction were established, and the ability to detect the primer of the hairpin structure was checked.

Specifically, in FIG. 4, the thermocycling process of the polymerase chain reaction of the present invention is shown based on temperature and time, denaturation is performed at 98 ° C. for 2 minutes, then 10 seconds are further performed, and then annealing and extension are It was confirmed that it was effective when it was carried out at 64 °C for about 20 seconds.

Next, it was confirmed whether there was any difference in the degree of point mutation confirmation when the primer was prepared in a hairpin structure and when it did not have such a structure.

Specifically, the primer used in (a) of FIG. 5 used a linear type primer that did not make a hairpin shape, and the primer used in FIG. After making it in the form, PCR experiments were carried out.

As a result, as can be seen in (a) of FIG. 5 , the Ct (threshold cycle, C(T), Ct) value measured as a target without a point mutation with a primer not prepared in the form of a hairpin was measured as 32. On the other hand, as shown in (b) of FIG. 5 , in the case of a hairpin primer prepared in the form of a hairpin to better distinguish a point mutation, the Ct value of the target without a point mutation was measured as 40. Could know. Since an increase in the Ct value in qPCR means that the number of cycles required for amplification increases, it can be interpreted that amplification is suppressed. Therefore, from the above results, if the hairpin form is actually created by adding the reverse repeat sequence to the primer, the Ct value increases by 8. Therefore, if the hairpin form is made using the reverse repeat sequence in the primer, amplification is suppressed by 256 ( ²⁸⁾ times. it can be seen that

Example 4: Confirmation of Ct value measurement results according to the presence or absence of hairpin form in the primer

In order to confirm the change in the Ct value according to the presence or absence of the hairpin type in the primer, the experiment was conducted by concentration with the wild type by dividing the primer into the presence and absence of the hairpin type. The specific experimental method is the same as in Example 3.

As a result, as can be seen in FIG. 6 , it was confirmed that the Ct value increased when the primer had a hairpin form, regardless of the wild-type concentration. Accordingly, it was confirmed that wild-type amplification could be effectively suppressed if the primer had a hairpin form.

In addition, the Ct value according to the type of hairpin primer was checked.

As a result, as can be seen in FIG. 7 , it was confirmed that the Ct value was increased in the hairpin primer LNA compared to the DNA in the wild type, the mutant type, and the negative type to more effectively inhibit amplification.

Example 5: Confirmation of Ct value measurement results when additionally including TMAC

When TMAC was additionally included in the composition for detecting a target point mutation of the present invention, the Ct values of the mutant and wild type were checked, respectively. The specific experimental method is the same as in Example 3.

As a result, as can be seen in FIGS. 8 and 9 , when the TMAC concentration was increased to 5 to 20 mM, the Ct value of the mutant and wild type increased as the concentration increased. It could be confirmed that the ΔCt value of phosphorus increased, and when only wild-type was compared separately, it was confirmed that the wild-type Ct value increased as the TMAC concentration increased.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (16)

1. (a) a sample comprising a target point mutation region;

   (b) a hairpin primer to discriminate point mutations;

   (c) a probe that binds to the target point mutation;

   (d) a linear primer that degrades the probe; and

   (d) comprising a DNA polymerase, a composition for detecting a target point mutation.
2. According to claim 1,

   Wherein the sample is gDNA or cfDNA, a composition for detecting a target point mutation.
3. 3. The method of claim 2,

   Wherein the composition targets a point mutation site in gDNA or ctDNA in cfDNA, a composition for detecting a target point mutation.
4. According to claim 1,

   Wherein the probe comprises a fluorescent material, a composition for detecting a target point mutation.
5. According to claim 1,

   The point mutation is a composition for detecting a target point mutation, including a mutation in which a base of a nucleic acid of a target sample is substituted, deleted, or inserted.
6. According to claim 1,

   In the hairpin primer, when a target point mutation is present in the sample, an amplification product is generated and the fluorescence value of the probe bound thereto can be measured,

   A composition for detecting a target point mutation, characterized in that, when the target point mutation is not present in the sample, the fluorescence value of the probe cannot be measured because the probe is not bound due to non-generation of the amplification product.
7. According to claim 1,

   The hairpin primer is any one selected from the group consisting of DNA, LNA and PNA, a composition for detecting a target point mutation.
8. According to claim 1,

   A composition for detecting a target point mutation that may further include TMAC (Tetramethylammonium chloride) in the composition for detecting a target point mutation.
9. (a) a sample comprising a target point mutation region;

   (b) a hairpin primer to discriminate point mutations;

   (c) a probe that binds to the target point mutation;

   (d) a linear primer that degrades the probe; and

   (d) a kit for detecting a target point mutation comprising a DNA polymerase.
10. A composition for classification or diagnosis of SNP, comprising the composition of claim 1 or the kit of claim 9.
11. A sample containing the target point mutation region, a hairpin primer for discriminating the point mutation, a probe binding to the target point mutation, a linear primer decomposing the probe, and a DNA polymerase are reacted with a composition comprising a target point mutation region to contain the target point mutation region amplifying the sample; and

    A method for detecting a target point mutation comprising the step of measuring the fluorescence level of the probe.
12. 12. The method of claim 11,

    The detection method is one that consists of the following steps:

    (a) denaturing the sample containing the target point mutation region (denaturation);

    (b) binding and extending the hairpin primer to the point mutation in the sample;

    (c) denaturing the sample to bind the linear primer and the probe;

    (d) degradation of the bound probe by a polymerase; and

    (e) measuring the fluorescence level of the probe.
13. 12. The method of claim 11,

    The sample is gDNA or cfDNA, the detection method.
14. 13. The method of claim 12,

    The denaturation of (a) will be carried out at 90 to 99 ℃ 10 seconds to 3 minutes, the detection method.
15. 13. The method of claim 12,

    Wherein the step (b) is carried out at 50 to 70 ℃ for 10 seconds to 60 seconds, the detection method.
16. 13. The method of claim 12,

    Wherein the step (e) is carried out for 10 seconds to 60 seconds at 50 to 70 ℃, the detection method.

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