WO2005005664A1

WO2005005664A1 - Method of detecting and method of identifying nucleic acid

Info

Publication number: WO2005005664A1
Application number: PCT/JP2004/010170
Authority: WO
Inventors: Ken Nemoto; Joji Oshima
Original assignee: G & G Science Co., Ltd.
Priority date: 2003-07-15
Filing date: 2004-07-09
Publication date: 2005-01-20
Also published as: JPWO2005005664A1

Abstract

The method in which single-stranded nucleic acids are synthesized with 1-primer and nucleic acid detection and identification are carried out on the basis of dissociation curve waveform pattern of mutual interference product thereof is advantageous but has had a problem of detection sensitivity because of low synthesis efficiency of single-stranded synthetic nucleic acid. For resolving the problem, there is realized a method comprising synthesizing by low-temperature annealing a single-stranded nucleic acid of low specificity for enhancing detection sensitivity together with a target single-stranded nucleic acid of high specificity; synthesizing by high-temperature annealing only a target single-stranded nucleic acid of high specificity and forming a single-stranded nucleic acid mutual interference product in the presence of both thereof; and carrying out high-sensitivity specific nucleic acid detection and identification on the basis of dissociation curve waveform pattern thereof. Further, there is realized a method comprising adding a synthetic single-stranded nucleic acid for enhancing detection sensitivity; forming a synthetic single-stranded nucleic acid mutual interference product in the presence of both the same and a target single-stranded nucleic acid of high specificity synthesized by high-temperature annealing; and carrying out high-sensitivity specific nucleic acid detection and identification.

Description

Method for detecting and identifying nucleic acid

The present invention provides a method for synthesizing a single-stranded nucleic acid having relatively low specificity under low-temperature annealing conditions to detect and identify a nucleic acid having an arbitrary base sequence with high sensitivity, and under high-temperature annealing conditions. Synthesize single-stranded nucleic acids with relatively high specificity, increase the physical quantity of single-stranded interfering substances by mixing the former and the latter, or by adding synthetic single-stranded nucleic acids to increase specificity and The present invention relates to a nucleic acid detection and identification method that achieves high sensitivity and implements them with one type of primer. Background art

Conventionally, nucleic acids have been amplified by using complementary specific primers that bind one end of one strand of a specific site of double-stranded DNA to the other end of the other strand using an enzyme DNA polymerase as a catalyst. The polymerase chain reaction (PCR) method for amplifying a target nucleic acid is widely used. The principle of the PCR method is to amplify a DNA fragment between primers by forming a double-stranded DNA into a 鍚 shape and repeating extension of the two primer strands sandwiching a specific region by DNA polymerase. Various DNA and RNA analysis techniques have been devised based on the method (see, for example, Japanese Patent Publication No. 46 7957 and Takeo Sekiya, Protein Nucleic Acid Enzyme, Vol. 41, No. 5, 415 (1996)). In this PCR method, only one PCR product is amplified to increase the specificity of the primer for type I DNA and to recognize only a specific site in the total DNA. In addition, in order to detect the presence of a specific sequence in a nucleic acid for use in genetic diagnosis of a hereditary disease, a cancerous disease, or an infectious disease, the base sequence of a target nucleic acid is slightly contained using a complementary primer. Technology to amplify and detect unidentified nucleic acid sequences The technique is disclosed (for example, see Japanese Patent Publication No. 4-67960). Disclosure of the invention

When a double-stranded nucleic acid or a double-stranded amplification product amplified by the PCR method is heated, it is dissociated into single strands each time the temperature is applied.The temperature point at which this rapid dissociation occurs is determined by Tm (Metting Temperature). That. In the patent specification and drawings filed earlier, the inventors of the present invention performed nucleic acid amplification by adding an intercalator dye such as cyber green to nucleic acid, and measured the Tm value and the melting curve (Me 1t Curve) or a dissociation curve waveform pattern such as a dissociation curve (Dissociation Curve) obtained by differentiating the dissolution curve has been proposed (Japanese Patent Application Laid-Open No. 2003-180374). ). Furthermore, we propose a method for synthesizing single-stranded nucleic acids simultaneously and in multiple regions using one type of primer, recognizing the dissociation curve of the mutual interference product as a dissociation curve waveform pattern, and detecting and identifying nucleic acids. (Japanese Patent Application Laid-Open No. 2003-334082).

The above-mentioned method for synthesizing single-stranded nucleic acids using one type of primer and recognizing the dissociation curve of the mutual interference product as a dissociation curve waveform pattern to detect and identify nucleic acids is easy and diverse. Although useful as a method for analysis, there is room for improvement in the low efficiency of single-stranded nucleic acid synthesis and the resulting low detection sensitivity. In other words, in order to improve the low detection sensitivity due to low synthesis efficiency, a method of increasing the nucleic acid synthesis efficiency by adapting the low annealing temperature can be considered, but this method has a specificity for the detection and identification of the target base sequence. Causes a decrease in However, if a high annealing temperature is applied to improve specificity, the efficiency of nucleic acid synthesis is further reduced and the detection sensitivity is deteriorated. Solving such inconsistencies involves a method for synthesizing single-stranded nucleic acid using one type of primer, recognizing the dissociation curve of the mutual interference product as a dissociation curve waveform pattern, and detecting and identifying the nucleic acid. It was an issue.

The present invention has been proposed as a result of earnest research in view of the conventional situation, An object of the present invention is to provide a method for improving the detection sensitivity and at the same time maintaining the specificity of a nucleic acid detection and identification method utilizing single-stranded nucleic acid synthesis, which is more effective. A method for changing the annealing temperature according to claim 1 according to claim 1, that is, a method combining low-temperature annealing and high-temperature annealing, and a method similar to the relatively low-specificity single-stranded nucleic acid according to claim 2. A method for adding a synthetic nucleic acid or a chemical substance having a function was used as a means for solving the problem.

The present invention uses a single type of primer having specific complementarity to any specific nucleotide sequence region on a nucleic acid, and a single strand having a high relative specificity to the specific nucleotide sequence region under high temperature annealing conditions. The present invention has realized a method for synthesizing a nucleic acid, detecting and identifying the nucleic acid, and improving the reduction in sensitivity due to the low efficiency of single-stranded nucleic acid synthesis. Specifically, (i) at the beginning of the synthesis cycle, set low-temperature annealing conditions and

A method of simultaneously forming a plurality of single-stranded nucleic acids having low relative specificity to a specific base sequence region by forming an incomplete complementary chain even in a similar base sequence region having no complete complementarity at the 5 ′ side, (ϋ) A method of adding a separately synthesized single-stranded nucleic acid was devised. All of these methods are applicable to single-stranded synthetic nucleic acids that are synthesized under high-temperature annealing conditions and have relatively high specificity, but are insufficient in quantity and do not provide sufficient sensitivity in the detection step. By forming a partially complementary strand with a single-stranded nucleic acid, the physical quantity of hydrogen bonds is increased, and the effect of improving the sensitivity in the detection step is obtained.

Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings. This embodiment uses a single type of primer that amplifies only a single strand in response to a specific base sequence of a target double-stranded nucleic acid, and improves the sensitivity while improving the specificity. Is explained. Here, “anneal” means that a nucleotide chain forms a double-stranded structure by base pair bonding based on the Watson-Crick law. In the present invention, in order to increase the detection sensitivity that cannot be obtained only with a synthetic single-stranded nucleic acid having an increased specificity, (i) the annealing temperature is changed between a low temperature and a high temperature; Less likely 1 Synthesize single-stranded nucleic acids together with target single-stranded nucleic acids with high specificity.In high-temperature annealing, synthesize only single-stranded target nucleic acids with high specificity, and form a single-stranded nucleic acid mutual interference product in the presence of both. A method for detecting and identifying a nucleic acid with high sensitivity and specificity from its dissociation curve waveform pattern. (Ii) Synthesis for increasing the detection sensitivity.Synthesis by high-temperature annealing by adding single-stranded nucleic acid. Highly specific purpose Synthesize single-stranded nucleic acid mutual interference products in the presence of both single-stranded nucleic acids and realize highly sensitive and specific nucleic acid detection and identification from its dissociation curve waveform pattern The method was used.

The concept of the above (i) is schematically shown in FIG. Figure 1 (A) shows the nucleic acid synthesis status during low-temperature annealing, where a single type of primer is used to increase the detection sensitivity for a large amount of low-specificity single-stranded nucleic acid, and the target base sequence X Both a synthetic nucleic acid of interest, a small amount of a target single-stranded nucleic acid with high specificity, and a synthetic nucleic acid of the target base sequence X are synthesized. FIG. 1 (B) shows the synthesis status of nucleic acids in high-temperature annealing, in which only a single-stranded target nucleic acid having high specificity and a synthetic nucleic acid derived from the target base sequence X are synthesized with one kind of primer. In low-temperature annealing, annealing occurs even if there is no perfect complementarity between the primer and type I nucleic acid, and nucleic acid synthesis proceeds, whereas in high-temperature annealing, annealing occurs when there is no perfect complementarity between the primer and type II nucleic acid. This indicates that only a specific target nucleic acid is synthesized because no reaction is performed.

FIG. 2 schematically shows the state of incomplete complementary annealing between the primer and the type I nucleic acid during low-temperature annealing, and the mutual interaction in the case where various single-stranded synthetic nucleic acids synthesized by the above-mentioned temperature change annealing are mixed. Figure 3 shows a schematic diagram of the interference products. A single-stranded nucleic acid with low specificity to increase detection sensitivity coexists with the target single-stranded nucleic acid with high specificity, causing a reaction between the two and increasing the physical quantity of hydrogen bond. The amount of intercalator increases, resulting in high sensitivity. When there is no single-stranded nucleic acid with low specificity, the hydrogen-bonding site is present when the target single-stranded nucleic acid with high specificity is present at the time of self-loop or hairpin structure formation by itself, or only at the time of dimer formation. The physical quantity is also small, resulting in lower sensitivity. The concept of the method for adding a synthetic single-stranded nucleic acid is the same as that in Fig. 3, and by separately synthesizing and adding a synthetic nucleic acid derived from outside the target base sequence X (synthetic nucleic acid for increasing the fluorescence intensity) in the same figure. An effect similar to the above-described method of performing low-temperature annealing and high-temperature annealing continuously or discontinuously is obtained.

In order to increase the detection sensitivity of the target nucleic acid, the method of performing low-temperature annealing and high-temperature annealing continuously or discontinuously, and the method of adding a single-stranded nucleic acid, use primers in the presence of the enzyme DNA polymerase. In order to stabilize annealing, it is useful to add a surfactant such as a nonionic type or a sulfuric acid compound such as sodium sulfate or sodium hydrogen sulfate, and it is particularly effective at low temperature annealing. Brief Description of Drawings

FIG. 1 is a diagram schematically illustrating nucleic acid synthesis according to an embodiment of the present invention. (A) shows a state of nucleic acid synthesis in low-temperature annealing of the present invention, and (B) shows a state of nucleic acid synthesis in high-temperature annealing. It is shown.

FIG. 2 is a diagram schematically illustrating annealing between a primer and a type III nucleic acid in low-temperature annealing.

FIG. 3 is a diagram schematically illustrating a mutual interference product of a nucleic acid synthesis product according to the embodiment of the present invention.

FIG. 4 is a dissociation curve waveform pattern obtained by the protocol of the present invention shown in Table 2. Figure 5 shows the dissociation curve waveform patterns obtained with the comparative protocols (A) and (B) shown in Table 2.

FIG. 6 is an electrophoresis photograph of the present invention and a comparative protocol.

FIG. 7 shows a dissociation curve waveform pattern when a synthetic nucleic acid was added.

FIG. 8 is a dissociation curve waveform pattern when no synthetic nucleic acid was added. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to Examples. However, it is needless to say that the present invention is not limited to the following Examples, and various changes may be made without departing from the gist of the present invention. It is possible.

(1) Effect of annealing temperature change

Of the DNA sequence encoding bacterial 16S ribosomal RNA (rRNA), select 12 bases that are conserved in about 3000 bacterial species and have a low frequency of occurrence in non-bacterial DNA, and use this as a BSS primer. Then, the protocol of the present invention in which the annealing temperature was changed and the protocol in which the annealing was not changed were compared, and the effect of improving the sensitivity and specificity was performed as an example. Specifically, in both protocols, in addition to the sequence encoding bacterial 16 S ribosomal RNA (rRNA), a sequence having homology to 163 rRNA, such as 23 S rRNA and 8 S, is 1 and so on. Amplified at the same time. In the protocol using only low-temperature annealing conditions, the mutual interference products composed of synthetic single-stranded nucleic acids can be obtained in a sufficient physical quantity in the detection process, but have low relative specificity in many regions. Unnecessary presence of the single-stranded nucleic acid reduces specificity. Under high-temperature annealing conditions alone, the mutual interference products composed of synthetic single-stranded nucleic acids have high specificity because only single-stranded nucleic acids with high relative specificity to the target gene region are synthesized. However, it is difficult to ensure a sufficient physical quantity in the detection process. According to the protocol of the present invention, a plurality of single-stranded nucleic acids having low relative specificity in multiple regions are simultaneously and simultaneously synthesized from regions other than these target gene regions under low-temperature annealing conditions. Single-stranded nucleic acids with low relative specificity from regions other than the target gene region are not synthesized, and only single-stranded nucleic acids with high relative specificity to the target gene region are synthesized. By being mixed, the mutual interference products composed of these synthetic single-stranded nucleic acids are expected to secure sufficient physical quantity in the detection step and also obtain specificity. E. coli (E.co1i) was selected as the bacterium to be used, and DNA was extracted using ISOGEN-LS (manufactured by Nippon Gene Co., Ltd.) according to the manual. It was. Each sample DNA was adjusted to 30011 _§ / 51 (H ₂ O) and added to 501 reaction solution as shown in Table 1 below, and the total amount was converted to 55 / X 1 Carried out. In the examples, each was confirmed by Dup 1 icate (repeated experiment). Table 1 shows examples of composition liquid formulations for implementing the present invention.

table 1

In Table 1, the 10X PCR buffer was supplied with Taq DNA polymerase or Taq-REX (TAKARA), and the 5X Cyber Green dilution was prepared by diluting the stock solution with sterile distilled water. The one-fold dilution was used as a 5X Genopattern buffer, a product manufactured by Adgene, and a Taq DNA polymerase was used, Taq-REX (TAKARA). In addition, the BSS primer is composed of 5, 1 ATCGCTATGTGC 13 ′ (base sequence 1),

Was used. Table 2 shows the protocol of the present invention and the comparative protocol.

Table 2

Each of the above protocols was performed using i-cyc 1 eri Q (made of pioradne earth), and the effect of the invention was observed at a temperature range of 65 to 95 ° C and a temperature step of 0.1 ° C for 1 second at each temperature. As a result, the dissociation curve waveform pattern of the synthetic nucleic acid interference product was observed and judged.

Next, 5 μl of loading buffer was added to 20 μl of the sample after the completion of the reaction, and electrophoresis was performed at 100 V for 20 minutes using a 2% agarose gel. The synthetic nucleic acid was confirmed by staining with ゥ mbromide. In addition, 200 bpl ad d e r m a r k er was used as a molecular size marker. The dissociation curve waveform patterns of the present invention and comparative protocols A and B are shown in FIGS. 4 and 5 (A) and (B), respectively. As can be seen from FIG. 4, when the protocol of the present invention is used, each peak of the waveform pattern is sharp and clear, the difference in Dip i cate is small, and a sufficient amount of fluorescence is obtained. In contrast, when the comparative protocol A in Fig. 5 (A), that is, the protocol under only low-temperature annealing conditions, was used, the same amount of fluorescence was obtained as compared to Fig. 4, but the sharpness of each peak in the waveform pattern was clear. Although the synthetic nucleic acid can secure a sufficient amount of fluorescence in the detection step, the specificity is reduced by the presence of unnecessarily large single-stranded nucleic acid with low relative specificity in multiple regions. Show. When the comparative protocol B in Fig. 5 (B), that is, the protocol under only the high-temperature annealing condition, was used, a sufficient amount of fluorescence was not obtained as compared with Fig. 4, and the waveform pattern was obtained only with simple single-strand synthesis. This indicates that it is difficult to detect Based on the waveform patterns of the protocol of the present invention and the comparative protocol, the present protocol makes it possible to obtain a sufficient amount of detected fluorescence while increasing the specificity required for nucleic acid identification with only one primer for synthesizing a single-stranded nucleic acid. Was recognized. By using this protocol, highly sensitive nucleic acid detection that focuses on the presence of the target nucleotide sequence of nucleic acids, and various waveform patterns can be stably observed. Nucleic acid identification is achieved.

FIG. 6 shows electrophoresis photographs of the protocol of the present invention and the comparative protocol. here, Lane 1 is the protocol of the present invention, lane 2 is comparative protocol A, and lane 3 is comparative protocol B. As can be seen from FIG. 6, sufficient nucleic acid synthesis is performed by the protocol of the present invention and comparative protocol A, that is, the protocol using only low-temperature annealing conditions, but the detailed identification of nucleic acids must be performed by electrophoresis. And a step of detecting the dissociation curve waveform pattern is required. In addition, in the protocol only under the high-temperature annealing condition, the nucleic acid was not sufficiently synthesized, and unreacted primers were observed. In this method, stable nucleic acid identification is difficult even if the dissociation curve waveform pattern is detected. For this reason, it is difficult to identify nucleic acids stably even if the step of detecting the present pattern is performed. This confirmed the useful life of the present invention.

(2) Effect of added synthetic nucleic acid

Of the DNA sequence encoding the bacterial 16S ribosome RNA (rRNA), select 12 bases that are conserved in about 300 species and have a low frequency of appearance in non-bacterial DNA. It was used as a BSS primer, and reacted with single-stranded nucleic acid synthesized under these conditions to capture the drawback that it was difficult to ensure a sufficient amount of nucleic acid in the detection step only under high-temperature annealing conditions. The effect of improving the fluorescence intensity was performed as an example. Under high-temperature annealing conditions, single-stranded nucleic acids with high specificity are synthesized, but with this single-stranded nucleic acid alone, for example, when an interlator is used, hydrogen bonds are formed only in each self-loop or dimer form. Insufficient hydrogen bonding sites, which are the basis of fluorescence intensity, cause a decrease in fluorescence intensity. Therefore, by adding a nucleic acid providing a hydrogen bonding site to the synthesized single-stranded nucleic acid after the completion of the synthesis reaction, it is expected that this disadvantage will be solved. E. coli was selected as a bacterium to be used, and DNA was extracted using ISOGEN-LS (manufactured by Nippon Gene Co., Ltd.) according to the manual. Each sample D NA was adjusted to 3 0 0 ng / 5 μ 1 (Η 2 0), as shown in Table 1 below, 5 0 mu This was added to the reaction solution of No. 1 and performed as a composition solution having a total amount of 551. In addition, the examples were each performed in duplicate experiments. Table 3 shows examples of formulation of the composition for implementing the present invention.

Table 3

6 97

o 82

Seconds o o

Seconds seconds

In Table 3, 10 XP CR buffer was supplied with Taq DNA polymerase or Taq REX (TAKARA), and 5X Cyber Green diluent was prepared by diluting the stock solution with sterile distilled water. The one-fold dilution, 5 X Genopattern buffer, Adgene product, and Taq DNA polymerase, Taq-REX (TAKARA) were used. The BSS primers are 5, -ATCGCTATGTGC-3 '(base sequence 1),

Was used. Table 4 shows the protocol for implementing the present invention.

Table 4

Protocol 3

50 cycles The above protocol was performed using i-cyc 1 eri Q (manufactured by Piorad), and then 2 μl of various synthetic nucleic acids at a concentration of 100 pino 1 Z / 1 was added. The effect of the invention according to this embodiment is that the temperature range of 60 to 95 ° C and the temperature step of 0.1 ° C By observing at each temperature for 1 second under the conditions, the dissociation curve waveform pattern of the synthetic nucleic acid mutual interference product was observed and judged. Table 5 shows the base sequence of the added synthetic nucleic acid used in the experiment.

Table 5

The dissociation curve waveform patterns after addition of each of the synthetic nucleic acids A, B, and C are shown in FIGS. 7 (A), (B) and (C), respectively, and the dissociation curve waveform patterns without addition are shown in FIG. As can be seen from the comparison of FIGS. 7 (A), (B), (C) and FIG. 8, when the synthetic nucleic acid of the present invention is added, it becomes the basis of the fluorescence intensity which was insufficient under the high temperature annealing condition. It was possible to capture the hydrogen bonding site, and the effect of improving the nucleic acid detection sensitivity, which was lacking when no addition was performed, was recognized. At the same time, this example suggests that by devising the base sequence of the added synthetic nucleic acid, it is possible to adjust the dissociation curve waveform pattern of the detected nucleic acid, and it is easy to detect and identify various nucleic acids according to the purpose. Industrial applicability shown to be feasible

According to the present invention, any base sequence of a target nucleic acid can be specifically detected with high sensitivity and easily detected and identified. The advantage of using one type of primer is that by synthesizing any nucleotide sequence of the target nucleic acid widely including its related region, the information is reflected in the dissociation curve waveform pattern, which is extremely useful. Can be observed. Therefore, after using one type of primer, Combining the present invention as an improved method of low sensitivity due to the low synthesis efficiency of the problem can be used as an easy nucleic acid detection and identification method.

The present invention makes it possible to detect and identify any base sequence of a target nucleic acid with high sensitivity and high specificity.The advantage of using one type of primer is that By widely synthesizing the nucleotide sequence including its related region, it is extremely useful to reflect the information derived from the nucleotide sequence on the waveform pattern of the dissociation curve, so that the properties of the nucleic acid can be widely observed. Therefore, it can be applied not only to identification of bacteria, but also to genetic analysis of humans, such as genetic diagnosis of hereditary diseases, cancerous diseases or infectious diseases, drug metabolism, and gender discrimination.

Claims

The scope of the claims

1. One base having specific complementarity to any specific base sequence region on nucleic acid, base sequence is selected to maintain high stability on the 3 'side of the base sequence, and low temperature Under the augmenting conditions, the primer not only forms a perfect complementary chain in any specific nucleotide sequence region, but also forms an incomplete complementary chain in a similar nucleotide sequence region without sufficient 5′-side perfect complementarity, In the presence of NA polymerase, multiple single-stranded nucleic acids with low relative specificity to a specific nucleotide sequence region are synthesized simultaneously and in multiple regions, and under high temperature annealing conditions, the primer can be used only in an arbitrary specific nucleotide sequence region. Form a completely complementary strand, synthesize a single-stranded nucleic acid with high relative specificity to a specific base sequence region in a single or a small number of regions in the presence of DNA polymerase, and execute both conditions continuously or discontinuously To do After mixing single-stranded nucleic acids with lower relative specificity and higher relative specificity, and forming single-stranded nucleic acids under appropriate temperature conditions, forming a mutual interference product of these single-stranded nucleic acids, This is a nucleic acid detection and identification method that observes the state of dissociation and denaturation due to physicochemical stimulation over time and detects and identifies nucleic acids based on the change. For targets that do not have sufficient sensitivity and specificity, mix single-stranded nucleic acids with relatively low specificity, and increase the detection sensitivity due to the increase in the physical quantity of interfering substances between the two. A method for detecting and identifying a nucleic acid, comprising:

2. The method for detecting and identifying a nucleic acid according to claim 1, wherein a low-temperature condition for forming a mutual interference with a single-stranded nucleic acid having relatively high specificity and increasing its sensitivity and specificity is provided. The feature is that the detection sensitivity is increased by adding a synthetic nucleic acid or chemical substance having the same function as a single-stranded nucleic acid with relatively low specificity synthesized by Nucleic acid detection and identification method.

3. The nucleic acid detection and identification method according to claim 1, wherein the nucleic acid synthesis efficiency, detection efficiency, identification efficiency, and the like are defined based on the frequency of occurrence of a specific base sequence in the target nucleic acid. Primers whose base sequence is the base sequence of the target nucleic acid and which simultaneously anneals to both the sense strand and the sense strand of the target nucleic acid and simultaneously synthesizes the nucleic acid, does not involve the generation of a completely complementary double-stranded nucleic acid. A method for detecting and identifying a nucleic acid, comprising using a primer.

4. The method for detecting and identifying a nucleic acid according to claim 1, wherein the frequency of occurrence in type 核酸 nucleic acid is searched for all base sequence combinations at an arbitrary primer base chain length, and the high frequency base sequence is detected. Use primers that are incorporated in the design concept as a base sequence that can have specificity representing the entire type II nucleic acid irrespective of the region, or as a base sequence that can have gene specificity in a base sequence that frequently appears in a specific gene A nucleic acid detection and identification method characterized by the above-mentioned.

5. The method for detecting and identifying a nucleic acid according to claim 1, wherein a surfactant and a Z or sulfate compound are added to stabilize annealing of the primer particularly under low-temperature annealing conditions. And identification methods.

6. The method for detecting and identifying a nucleic acid according to claim 1, wherein the temperature is set at 40 ° C. or less, particularly under low-temperature annealing conditions.

7. The method for detecting and identifying a nucleic acid according to claim 1, wherein the method for observing the state of dissociation and denaturation due to physicochemical stimulation over time after the formation of a mutual interference product of the single-stranded nucleic acid comprises physicochemical stimulation and denaturation A nucleic acid detection and identification method characterized in that the relation between dissociation states is drawn or graphed, and the recognition is performed from graphic recognition.