CN113186261A

CN113186261A - Application of single probe-based self-primer isothermal index amplification method in detection of long-chain nucleic acid

Info

Publication number: CN113186261A
Application number: CN202110376763.6A
Authority: CN
Inventors: 陈俊; 黄婷; 刘碧蓉; 杨子中; 孙蒙旭; 陈金香; 谢宝平; 段文军
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-07-30
Anticipated expiration: 2041-04-08
Also published as: CN113186261B

Abstract

The invention discloses an application of a single probe-based self-primer isothermal index amplification method in detection of long-chain nucleic acid, wherein the method comprises the following steps: (1) designing a self-primer amplification template hairpin; (2) carrying out reverse transcription on the RNA of a sample to be detected as DNA of the sample to be detected, carrying out isothermal amplification on the DNA of the sample to be detected and the hairpin of the self-primer amplification template by using the DNA of the sample to be detected as the template, and calculating the content of long-chain nucleic acid in the sample to be detected according to the intensity of a fluorescence signal of an amplification product; wherein the long-chain nucleic acid is a nucleic acid sequence with the length of more than 1000 bases. Compared with the traditional EXPAR, the detection method disclosed by the invention can realize high-sensitivity and high-specificity detection of long-chain nucleic acid without additionally introducing a new primer probe, greatly simplifies the complexity of the method and reduces non-specific amplification.

Description

Application of single probe-based self-primer isothermal index amplification method in detection of long-chain nucleic acid

Technical Field

The invention belongs to the field of gene detection, and particularly relates to an application of a single probe-based self-primer isothermal index amplification method in detection of long-chain nucleic acid.

Background

Isothermal nucleic acid amplification techniques are a class of techniques for amplifying nucleic acids at constant temperature. Compared with the PCR technology, the constant temperature nucleic acid amplification does not need a complicated temperature changing step, has the amplification efficiency equivalent to that of the PCR, and can be widely used for detecting miRNA, DNA, mRNA, virus RNA and protein.

Isothermal nucleic acid amplification can be divided into amplification without participation of biological enzymes and amplification reactions based on biological enzymes. Reactions without participation of biological enzymes include hybrid chain reactions and catalytic hairpin self-assembly reactions, and although the reactions do not need participation of biological enzymes and reduce the cost to a certain extent, the method has low amplification efficiency and long reaction time. While isothermal amplification based on participation of biological enzymes, such as Rolling Circle Amplification (RCA), loop-mediated exponential amplification (LAMP) and isothermal exponential amplification (EXPAR), can realize high amplification efficiency and high amplification rate under the assistance of biological enzymes such as polymerase or nickase, the method has low specificity for the amplification reaction of a single probe triggered based on a target object, has poor amplification effect and efficiency for a long-chain sample, and cannot be effectively popularized and used.

Therefore, the development of a self-primer isothermal index amplification method which can be based on a single probe and has higher specificity has great significance for long-chain nucleic acid detection.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a single-probe-based self-primer isothermal index amplification method for long-chain nucleic acids, which can overcome the problem that the 3' end of long-chain nucleic acids (such as mRNA and viral RNA) cannot be directly used as a primer to trigger amplification when fragments with the length of dozens of bases are in the middle conserved regions of the long-chain nucleic acids in the traditional method, realizes amplification on the basis of no need of additionally adding primer probes, reduces the problem of nonspecific amplification caused by introduction of additional primer probes, simplifies a detection method, and realizes high-sensitivity and high-specificity detection of the long-chain nucleic acids.

In a first aspect of the present invention, there is provided a method for detecting a long-chain nucleic acid, comprising the steps of:

(1) designing a self-primer amplification template hairpin, wherein the length of a neck sequence of the self-primer amplification template hairpin is 11-15 nt;

(2) and carrying out reverse transcription on the RNA of the sample to be detected as the DNA of the sample to be detected, carrying out isothermal amplification on the DNA of the sample to be detected and the hairpin of the self-primer amplification template by using the DNA of the sample to be detected as the template, and calculating the content of the long-chain nucleic acid in the sample to be detected according to the intensity of a fluorescence signal of an amplification product.

According to a first aspect of the invention, in some embodiments of the invention, the long-chain nucleic acid is a nucleic acid sequence with a base length of greater than 1000.

In some preferred embodiments of the invention, the long-chain nucleic acids are mRNA, lncRNA, RNA viral genome and RNA fragments with a base length of greater than 1000.

When a conventional detection method, such as a qPCR method, is used for detecting long-chain nucleic acid, in the detection process, when fragments with the length of dozens of basic groups in a middle conserved region of the long-chain nucleic acid (such as mRNA and viral RNA) are faced, the 3' end cannot be directly used as a primer to trigger amplification, amplification can be realized only by adding an additional primer probe, and the introduction of the additional primer probe easily causes non-specific amplification and leads the detection method to be complicated, so that the method is not beneficial to popularization. For Rolling Circle Amplification (RCA), loop-mediated exponential amplification (LAMP) and isothermal exponential amplification (EXPAR), although the method has the characteristics of high amplification efficiency and high amplification rate, the method has low specificity for the amplification reaction of a single probe triggered based on a target object, and has poor amplification effect and efficiency for long-chain samples, so that the method cannot be effectively popularized and used.

Conventional isothermal exponential amplification is mainly used for short-chain nucleic acids (e.g., nucleic acid sequences such as miRNA, etc., generally having a length of about 20 bases), and when a primer is complementarily paired with a 3 'end sequence of an amplification template, the 3' segment sequence thereof will extend and amplify along the template under the action of polymerase (see fig. 1A). However, when detecting long-chain nucleic acids (e.g., mRNA, IncRNA, or viral RNA), the nucleic acid sequence of these targets is often several kilobases in length, in order to detect these targets, a region several tens of bases long as the conserved region is usually selected as the target recognition sequence, when the template is designed according to the idea of detecting short-chain nucleic acid, after the target recognition sequence of the target is combined with the amplification template probe, which is 3' suspended and cannot extend along the template (see FIG. 1B), so that an additional primer probe (primer probe) has to be designed as an auxiliary strand to simultaneously hybridize complementarily to the amplification template and the recognition sequence of the target substance to form a Y-type structure, then the 3' end of the primer probe extends and amplifies along the template to realize the detection of the long-chain nucleic acid (figure 1C), however, this clearly increases the probability of non-specific amplification and tends to complicate the detection method.

The invention idea (see fig. 2) of the invention mainly comprises: by designing a metastable hairpin amplification template (amplification template HP), the 5' end of the HP is a target recognition sequence (black part + green part), wherein a part of the sequence (green part) is complementary with a part of the base sequence near the 3' end, and the 3' end sequence (orange part) of the HP is complementary with the base sequence of the loop part. The loop portion sequence is designed as a nicking enzyme recognition sequence. Since the hairpin structure formed by the target recognition sequence at the 5 'end of the amplification template HP being complementary to part of the base sequence near the 3' end is more stable, the amplification template HP will preferentially form such a structure. Only when the target is present, the target undergoes a Toehold strand displacement reaction with the 5' end sequence of the amplification template HP to form a double strand, the hairpin is opened, and the HP 3' end sequence hybridizes with the loop sequence to form a new hairpin, and the 3' end of the new hairpin can be extended along the template and amplified by the polymerase.

According to the first aspect of the present invention, in some embodiments of the present invention, the reaction system of isothermal amplification in step (2) is:

in some preferred embodiments of the invention, the 10 xnt.aiwi endonuclease buffer contains 50 mchh₃COOK, 20mM Tris-acetate, 10mM Mg (CH)₃COO)₂100. mu.g/mL Bovine Serum Albumin (BSA), pH 7.9;

in some preferred embodiments of the invention, the Klenow fragment (3'→ 5' exo-) polymerase buffer contains 10mM Tris-HCl, 50mM NaCl, 10mM MgCl₂1mM dithiothreitol, pH 7.9.

In some preferred embodiments of the present invention, the reaction system is formulated by: respectively preparing a solution A and a solution B in a low-temperature environment (0-4 ℃) or on ice, and mixing after the preparation to obtain a reaction system.

The system of solution a was:

solution A component	Volume of dose
		2.5mM dNTPs premix	2μL
1 μ M self-primed amplification template HP	2μL
		Sample to be tested	2.μL
10 XNt. AiwI endonuclease buffer solution	1μL
		10U/. mu.LRNase inhibitors	1.6μL
DEPC water	0.4μL
		Total of	10μL

The system of solution B was:

according to a first aspect of the present invention, in some embodiments of the present invention, the reaction procedure of isothermal amplification in step (2) is: amplifying for 60min at 36-38 ℃.

According to a first aspect of the invention, in some embodiments of the invention, the long-chain nucleic acid is a classical swine fever virus genome and/or an RNA fragment thereof.

In a second aspect of the invention, there is provided a set of self-primer amplification template hairpins, wherein the nucleotide sequence of the self-primer amplification template hairpins is:

HP-11nt：5'-CGTCCACATAGCATCTCGAGGTGGGATAGATCCGCGTAGGTACTAACCCACCTCGAGTATTCCTACGC-3'(SEQ ID NO.7)；

HP-12nt：5'-CGTCCACATAGCATCTCGAGGTGGGATAGATCCGCGTAGGTACTAACCCACCTCGAGTATTCCTACGC-3'(SEQ ID NO.8)；

HP-13nt：5'-CGTCCACATAGCATCTCGAGGTGGGACGAGACGCGTAGGTACTAACCCACCTCGAGATCCTACGC-3'(SEQ ID NO.9)；

HP-14nt：5'-CGTCCACATAGCATCTCGAGGTGGGATAGATCCGCGTAGGTACTAACCCACCTCGAGATTTCCTACGC-3'(SEQ ID NO.10)；

HP-15nt：5'-CGTCCACATAGCATCTCGAGGTGGGATAGATCCGCGTAGGTACTAACCCACCTCGAGATGCCCTACGC-3'(SEQ ID NO.11)。

according to the second aspect of the present invention, in some embodiments of the present invention, the template hairpin amplified from the primer is designed based on the nucleotide sequence (SEQ ID NO.2) between 200-224 of the RNA full-length sequence (SEQ ID NO.1) of CSFV as the target recognition sequence: the template hairpin amplified from the primer is required to undergo configuration conversion of the template HP only in the presence of the target.

In a third aspect of the invention, there is provided a test preparation comprising the self-primer amplification template hairpin according to the second aspect of the invention.

According to a third aspect of the invention, in some embodiments of the invention, the detection reagent comprises the sequence shown in SEQ ID No. 9.

In a fourth aspect of the present invention, there is provided a use of the detection reagent according to the third aspect of the present invention in the preparation of a hog cholera virus detection kit.

According to a fourth aspect of the invention, in some embodiments of the invention, the classical swine fever virus is a genomic or RNA fragment of a classical swine fever virus comprising the sequence shown in SEQ ID No. 2.

The invention has the beneficial effects that:

1. compared with the traditional EXPAR, the self-primer isothermal index amplification method based on the single probe can realize the high-sensitivity and high-specificity detection of the long-chain nucleic acid intermediate target recognition sequence by designing a metastable self-primer amplification template HP without additionally introducing a new primer probe, greatly simplifies the complexity of the method and reduces the non-specific amplification.

2. The invention takes the classical swine fever virus as an example, the designed self-primer amplification template HP has high sensitivity and high specificity, can effectively distinguish sequences with single base difference, has short detection time and simple detection method, ensures that the relative error value of the measured effective data and the traditional qPCR is within 15 percent, and has extremely high application value.

Drawings

FIG. 1 is a schematic diagram of the principle of detecting nucleic acid by conventional exponential amplification;

FIG. 2 is a schematic diagram illustrating the principle of a single probe-based isothermal exponential amplification method using self-primers in an embodiment of the present invention;

FIG. 3 is a feasibility verification result of the single-probe-based self-primer isothermal index amplification method in the embodiment of the present invention, wherein A is a real-time fluorescence amplification curve under different conditions, and B is a denaturing polyacrylamide gel imaging graph;

FIG. 4 is a graph showing the relationship between neck length POI and template HP amplified from the primers in the examples of the present invention;

FIG. 5 is a graph showing the relationship between the amount of Klenow fragment (3'→ 5' EXO-) polymerase and the amplification effect in examples of the present invention;

FIG. 6 is a graph showing the relationship between the content of Nt.AiwI endonuclease and the amplification effect in the examples of the present invention;

FIG. 7 is a graph showing the relationship between the HP-13nt content and the amplification effect in the example of the present invention;

FIG. 8 is an amplification standard curve in an embodiment of the present invention, in which A is an amplification curve of RNAs to be detected at different concentrations, and B is a curve fitted to POI by the RNAs to be detected at different concentrations;

FIG. 9 shows the result of the specificity verification test in the example of the present invention, in which A is the amplification curve of different mismatch probes and B is the method interference degree evaluation result.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Self-primer constant temperature index amplification method based on single probe

The single probe-based self-primer isothermal index amplification method in this embodiment is directed to detection of long-chain nucleic acids, and the "long-chain nucleic acids" in this embodiment refer to nucleic acid sequences with longer length that are different from short-chain RNAs (e.g., mirnas) with a base length of only 20 to 30, and specifically refer to long-chain nucleic acids with a base length greater than 1000, such as mrnas, lncrnas, and full-length RNAs or RNA fragments of viruses.

The specific detection steps of the single probe-based self-primer isothermal index amplification method in the embodiment comprise:

(1) design of template hairpin (hairpin RNA, HP) from primer amplification:

and designing a series of self-primer amplification templates HP with different neck hybridization numbers according to the RNA of the target to be detected for subsequent optimal combination screening.

(2) The method comprises the following steps:

respectively verifying whether the amplification reaction can be continued and the amplification condition of the amplification product when the target RNA to be detected or the HP polymerase as the self-primer amplification template is lacked by the methods of electrophoresis and fluorescence labeling, and judging whether the designed reaction system is effective and the method is feasible by comparing the experimental results.

If the electrophoresis and the fluorescence labeling method can not generate amplification reaction, the reaction system is failed to design, and needs to be redesigned until the electrophoresis and the fluorescence labeling method can display the successful amplification.

(3) Screening of the best self-primer amplification template HP:

and (2) respectively adding target RNA to be detected into the self-primer amplification template HP obtained by the design in the step (1) at a low temperature (0-4 ℃) or on ice, respectively supplementing equivalent substances required by amplification reaction to form a complete reaction system (the reaction can be selectively carried out on a PCR reaction plate), carrying out real-time PCR amplification reaction, and calculating the POI (time corresponding to the position with the maximum slope of the amplification curve) value of each amplification curve. So as not to contain the target RNA to be detected (adding equal amount of ddH)₂O or buffer as an alternative) as a voidWhite control group. And selecting a self-primer amplification template HP which enables the difference value between the amplification curve POI value with the target RNA to be detected and the blank group amplification curve POI value of the blank control group to be maximum as an optimal reaction HP for practical detection.

(4) Reaction condition optimization:

and (4) selecting the optimal reaction HP screened in the step (3) as an amplification template, and optimizing the determination conditions of the target RNA to be detected by a single-factor alternation method.

Optimizing the addition amount of the HP polymerase serving as a template amplified by a primer:

in order to explore the optimal addition amount of the self-primer amplification template HP polymerase in a reaction system and the influence of the self-primer amplification template HP polymerase on an actual detection result, under the condition that the content of other reagents in the reaction system is not changed (other reagents are quantified), self-primer amplification template HP polymerases with different content gradients are respectively added for testing, a fluorescence quantitative PCR curve of a target RNA to be tested of the self-primer amplification template HP polymerase with different content gradients and a blank control is observed, the difference value of an amplification curve POI of the target RNA to be tested and the blank control is calculated, and the self-primer amplification template HP polymerase content with the maximum POI difference value is selected as the optimal self-primer amplification template HP polymerase content.

Addition amount optimization of endonuclease:

in order to explore the optimal addition amount of the endonuclease in a reaction system and the influence of the endonuclease on an actual detection result, the endonucleases with different content gradients are respectively added to test under the condition that the content of other reagents in the reaction system is not changed (other reagents are quantified), the fluorescence quantitative PCR curves of the target RNA to be detected of the endonucleases with different content gradients and a blank control are observed, the difference value of the amplification curves POI of the target RNA to be detected and the blank control is calculated, and the endonuclease with the largest difference value of the POI is selected as the optimal endonuclease content.

③ optimizing the addition amount of the self-primer amplification template HP:

in order to explore the optimal addition amount of the self-primer amplification template HP in a reaction system and the influence of the self-primer amplification template HP on an actual detection result, under the condition that the content of other reagents in the reaction system is not changed (other reagents are quantified), self-primer amplification template HP with different content gradients is respectively added for testing, a target RNA to be tested of the self-primer amplification template HP with different content gradients and a fluorescence quantitative PCR curve of a blank control are observed, the difference value of a POI (point of interest) of the target RNA to be tested and the POI of the amplification curve of the blank control is calculated, and the content of the self-primer amplification template HP with the largest POI difference value is selected as the optimal content of the self-primer amplification template HP.

(5) Drawing a standard curve:

preparing target RNA solutions to be detected with different concentration gradients, and detecting in the optimal reaction system obtained in the step (4), wherein the specific steps are as follows: preparing a solution A and a solution B in a low-temperature environment (0-4 ℃) or on ice, wherein the solution A contains an endonuclease buffer solution, a self-primer amplification template HP, dNTP, an RNase inhibitor and a sample standard substance to be detected, and the solution B contains a self-primer amplification template HP polymerase, a polymerase buffer solution, endonuclease and SYBR Green I fluorescent dye. And (3) after the solution A and the solution B are uniformly mixed, immediately carrying out amplification on a PCR instrument to obtain an amplification curve, calculating a POI value, and carrying out statistics to obtain a target RNA concentration standard curve to be detected.

(6) And (3) specific determination:

according to the step in the step (5), replacing the standard sample to be detected with a test solution with different determined concentrations of one or more bases in the nucleic acid sequence, detecting by using the same reaction system, observing whether an amplification curve is generated, if not, indicating that the amplification curve meets the requirement of specificity, redesigning the self-primer amplification template HP, and repeating the steps (1) - (5).

(7) And detecting the actual sample by using the optimal reaction system, and obtaining the target concentration to be detected in the sample through a standard curve according to the detection value.

The reaction principle in this example can be seen in FIG. 2. A hairpin probe HP in a metastable state is designed, a hanging extension sequence (orange region) with a certain base length is designed at the 3' end of the HP, the extension sequence is complementary with a partial region with the length of 6 bases of the loop of the HP, and a nicking enzyme recognition sequence (yellow) is simultaneously designed at the loop of the HP. A part of segment of a conservative sequence of a target long-chain RNA is selected as a target recognition sequence, and when no target RNA exists, HP is in a metastable state, 3' of HP is still suspended, and amplification reaction cannot occur. When target RNA exists, the RNA and the hairpin 5 'end sequence carry out a Toehold strand displacement reaction, the HP is opened, the 3' end sequence of the HP forms a new hairpin structure on the HP (step I in figure 2), the 3 'end of the HP forms a double-stranded hybridization structure, the 3' end extends along the template under the action of polymerase and displaces the target RNA (step II in figure 2), and the displaced target RNA can react with the new HP again to initiate new amplification. The double strand extended along the template generates a nick under the recognition of the nicking enzyme (step three in fig. 2), the polymerase continues to extend along the template under the nick and replaces the amplification product strand (DNA amplics strand) and repeats the steps of cutting and extending (step (r) -c in fig. 2), the released DNA amplification strand further undergoes Toehold strand displacement with the 5' segment sequence of new HP (step (c) in fig. 2) and initiates a new extension reaction (step (c) in fig. 2) and generates a DNA amplification strand (step (r) -c in fig. 2). The circulation is repeated, and finally, the exponential growth of the DNA amplification strand is realized along with the time.

Effect of practical application

This example illustrates Classical Swine Fever Virus (CSFV) to show the practical effect of the single probe-based isothermal exponential amplification method in the above examples.

(1) Experimental materials:

the identification number of the classical swine fever virus database in this example is: NCBI number: NC _ 002657.1. The total length of the RNA of the classical swine fever virus is 12301 basic groups, and the sequence of the RNA is shown as SEQ ID NO. 1.

Selecting a base sequence between the 200 th and 224 th positions of the sequence shown in SEQ ID NO.1 as a target recognition sequence, wherein the specific sequence is as follows:

5'-CCCACCUCGAGAUGCUAUGUGGACG-3'(SEQ ID NO.2)。

based on the sequence shown in SEQ ID NO.2, a series of mismatched probe sequences were designed to test the specificity of the single probe-based self-primer isothermal exponential amplification method described in the above examples.

The mismatch probe sequence is specifically:

mismatch-free probe sequence: 5' -GAGCAGAAGCCCACCUCGAGAUGCUAUGUGGACGAGGGCAUGC-3'(SEQ ID NO.3)；

Mismatch G probe sequence: 5' -GAGCAGAAGCCCGCCUCGAGAUGCUAUGUGGACGAGGGCAUGC-3'(SEQ ID NO.4)；

Mismatch C probe sequence: 5' -GAGCAGAAGCCCACCUCGAGAUCCUAUGUGGACGAGGGCAUGC-3'(SEQ ID NO.5)；

Mismatch a probe sequence: 5' -GAGCAGAAGCCCACCUCGAGAUGCUAUGUGAACGAGGGCAUGC-3'(SEQ ID NO.6)；

Wherein, the underlined part of the mismatch probe sequence is a part corresponding to the sequence shown in SEQ ID NO. 2.

(2) Design of template HP amplified from primers:

designing a self-primer amplification template HP according to a sequence shown in SEQ ID NO.1, wherein the design principle is as follows: the primer template undergoes a configuration transition only in the presence of the target.

The designed self-primer amplification template HP sequence is as follows:

the designed self-primer amplification template HP sequence is denatured for 5min at 95 ℃, naturally cooled at room temperature to form a hairpin structure, and then placed at-20 ℃ for later use.

(3) The method comprises the following steps:

a reaction system (shown in table 1) is prepared in a low-temperature environment (0-4 ℃) or on ice, and then real-time exponential amplification is carried out at 37 ℃. The amplification curve was monitored with a StepOne Plus real-time fluorescent quantitative PCR instrument.

TABLE 1 amplification reaction System

Wherein the Nt.AiwI endonuclease buffer is purchased from NEB and has the component of 50mM CH₃COOK, 20mM Tris-acetate, 10mM Mg (CH)₃COO)₂100. mu.g/mL BSA, pH 7.9. RNase inhibitors were purchased from Shanghai. Klenow fragment (3'→ 5' exo-) polymerase was purchased from NEB. Klenow fragment (3'→ 5' exo-) polymerase buffer from NEB, 10mM Tris-HCl, 50mM NaCl, 10mM MgCl₂1mM dithiothreitol, pH 7.9.

And setting control groups, namely a sample deletion group to be detected (a sample to be detected is not added in a reaction system), a polymerase deletion group (Klenow fragment (3'→ 5' exo-) polymerase is not added in the reaction system) and an HP deletion group (a self-primer amplification template HP is not added in the reaction system).

The results are shown in FIG. 3.

As shown in the figure, curve A is an amplification curve under the condition that the reaction system shown in Table 1 has no deletion, curve B is an amplification curve lacking the sample to be detected, curve C is an amplification curve lacking polymerase, and curve D is an amplification curve lacking the self-primer amplification template HP. The occurrence of the experimental amplification curve of the group A is obviously earlier than that of the other three groups, but after the sample to be detected is lacked, obvious amplification occurs within about 80min, and no amplification occurs when polymerase or a self-primer amplification template HP does not exist. The above data indicate that exponential amplification based on the sample to be tested is feasible.

Further, the amplification products obtained from the 4 groups of experiments were verified by denaturing PAGE gel experiments, and the results are shown in FIG. 4.

As shown in FIG. 4, in the presence of the test sample, a distinct amplified band (band 4) appears, whereas in the absence of the test sample, only a weak background amplification (band 2) appears. In the absence of template HP amplified from the primer (lane 1) or polymerase (lane 3), no amplification was seen.

(4) Reaction condition optimization:

screening of an optimal self-primer amplification template HP:

according to the method shown in the step (3), the reaction systems shown in Table 1 are adopted to respectively test the amplification effects of the HP sequences HP-11nt, HP-12nt, HP-13nt, HP-14nt and HP-15nt of the self-primer amplification templates.

The results are shown in FIG. 5.

As shown in FIG. 5, the neck length of the template HP amplified by the self-primer has an influence on the POI, wherein when the neck length is 13 bases (i.e., HP-13nt), the difference between the POI value of the amplification curve and the POI value of the blank group is the largest, and the detection effect is the best, therefore, HP-13nt is selected as the best template HP amplified by the self-primer.

(ii) optimization of the amount of KF (3'→ 5' exo-) polymerase added:

according to the method shown in the step (3), the amplification effect of Klenow fragment (3'→ 5' exo-) polymerase was measured at the concentrations of 0.05U/. mu.L, 0.1U/. mu.L, 0.15U/. mu.L and 0.2U/. mu.L in the same volume using the reaction system shown in Table 1.

The results are shown in FIG. 6.

As shown in FIG. 6, the addition amount of Klenow fragment (3'→ 5' EXO-) polymerase has an influence on the POI, wherein when the concentration of Klenow fragment (3'→ 5' EXO-) polymerase is 0.1U/. mu.L, the difference between the amplification curve POI and the POI value of the blank group is the largest and the background value is the smallest, and therefore, 0.1U/. mu.L is selected as the optimum concentration addition amount of KF (3'→ 5' EXO-) polymerase.

③ optimizing the addition amount of Nt.AiwI endonuclease:

according to the method shown in the step (3), the reaction systems shown in Table 1 are adopted to respectively test the amplification effects of Nt.AiwI endonuclease under the same volume condition at the concentrations of 0.1U/. mu.L, 0.2U/. mu.L, 0.3U/. mu.L and 0.4U/. mu.L.

The results are shown in FIG. 7.

As shown in fig. 7, the addition amount of nt.aiwi endonuclease had an influence on POI, wherein when the concentration of nt.aiwi endonuclease was 0.2U/. mu.l, the difference between the amplification curve POI and the POI value of blank set was the largest and the influence on background was the smallest, so 0.2U/. mu.l was selected as the optimum concentration addition amount of nt.aiwi endonuclease.

Optimizing the addition amount of HP-13 nt:

according to the method shown in the step (3), the reaction systems shown in Table 1 were used to test the amplification effect of HP-13nt at concentrations of 50nM, 100nM, 150nM and 200nM, respectively, in the same volume.

The results are shown in FIG. 8.

As shown in FIG. 8, the addition amount of HP-13nt had an influence on POI, wherein the difference between the amplification curve POI and the POI value of the blank set was the largest and the background influence was the smallest when the concentration of HP-13nt was 100nM, and therefore, the optimal concentration addition amount of the Nt.AiwI endonuclease was selected at 100 nM.

(5) Drawing a standard curve:

according to the method shown in the step (3), the optimized reaction system (table 2) is adopted, and the sequence shown in SEQ ID NO.2 is used as a standard substance (the concentration is from 100aM to 1nM, specifically from 1nM to 100aM by 10-fold dilution gradient, and 8 groups are used for detection).

TABLE 2 optimized amplification reaction System

The buffer solution component of the Nt.AiwI endonuclease is 50mMCH₃COOK, 20mM Tris-acetate, 10mM Mg (CH)₃COO)₂100. mu.g/ml BSA, pH 7.9.

The Klenow fragment (3'→ 5' exo-) polymerase buffer contained 10mM Tris-HCl, 50mM NaCl, 10mM MgCl₂1mM dithiothreitol, pH 7.9.

The results are shown in FIG. 9.

As shown in FIG. 8, the different concentrations of the standard produced completely different amplification curves with a minimum detection value of 100 aM. By sorting the amplification curves, the standard amplification equation (FIG. 9B) is obtained, i.e., between 100aM and 1nM, and is:

POI＝9.2x-71.2；

R＝0.9991。

(6) and (3) specific determination:

and (4) respectively taking SEQ ID NO. 3-6 with the same concentration and volume as the sample to be detected according to the optimal reaction system shown in the step (5), and respectively detecting the amplification conditions of the sample to be detected.

The results are shown in FIG. 9.

As shown in fig. 9, the amplification curves of the samples to be detected have significant differences, and the differences of the samples to be detected and the results of the amplification curves are further compared by an interference degree evaluation method, so that the optimal reaction system in this embodiment can well distinguish sequences with single base mismatch, the effective detection sensitivity can reach a single base, and the high selectivity is achieved.

(7) Actual sample detection effect:

selecting 5 verified virus samples numbered as 1-5 virus samples (CSFV 1-5), and detecting by adopting the optimal reaction system shown in the step (5), wherein the results are shown in Table 3.

TABLE 3 actual assay of different virus samples

As shown in Table 3, compared with Rt-qPCR, the self-primer isothermal index amplification method based on a single probe in the above embodiment has good actual detection effect, and the relative error value of the measured effective data and the traditional qPCR is within 15%, which indicates that the self-primer isothermal index amplification method based on a single probe has high accuracy.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> southern medical university

<120> application of single probe-based self-primer isothermal index amplification method in detection of long-chain nucleic acid

<130>

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 9590

<212> RNA

<213> classical swine fever viru

<400> 1

gaacgaggag caccgagcag aggacaaaca aaacaaggca gggccccccc agcgacggcc 60

gaacgggcag ccagcccaca gaggacagca aacggaggga cagccgaggg cgagccccgg 120

gggcaagccg agacaggaca gcgcagagcg acggagcaga agcccacccg agagcaggga 180

cgagggcagc ccaagacaca ccaacccagc gggggcgcag gggaaacaca ccacggaggg 240

agacgaccga agggcgcgca gaggcccaca aggcagaaaa aaccgcgaca ggcacaggag 300

gaacagaaca acaaaacaaa caaacaaaaa ccaagggagg gaggaaccgg aacgagccac 360

ggggaggcca gggagacccg aggaggacac ccacaacaac acgaagcacc acagaagggg 420

gagaggaaca caaaacaaca cgaagaacca ccaggaaagg cgacgcagga gggcaaccac 480

aggcccggag gggaaagaaa gcccggcccg cacaggacac agggcccggc accaagagcc 540

cccagagaac gaagcgcagg cgagggacca aaaggaagga ggggacagga ggacggaaag 600

caccaaaagg gcacgagggc aacgcgaagc agccaagagg gacgagccaa gaacccgaag 660

ggaagaaaca ccgacgccag gggaccaggc cgagaggcgc aagggaagaa agagaagaag 720

ccagaaggac aacaaaggca aaaaaaaagc cccaaaagag cagagaagga cagcagaaca 780

agccaccgac gcacgagagg gaaggagaaa aaccaggaaa aagaagggaa agaaaggaaa 840

gagacccaag acggccgacc acaacaagaa aaaccaccag aacaggaaga aaagaaaaag 900

cccaggcagg gcggaaagca aaggaccaac caggaagccg aaaaaaacca aggaaccgag 960

gacaacggca caaggaccag cagcagacca gaggggagca ggagcgcagg gacggccgga 1020

aaaaaagcaa aggagcccca ccaccggcca cagacacgga acgaaagaaa acagggaaga 1080

ggagccagcg aggggacaaa caacggcgaa gacagagaca gaaggaacaa acaggaggga 1140

acggacaaaa gaccccggaa caggagaaag aacccaagca aacggcagaa ggcccccggc 1200

caaggaggcg cggacgcagg acgaaaagag cgacacaacg ggcacccagg ccagaaacag 1260

gccaacaacc cgaccgggca agaaaggaaa aaacgcggga cagaagaggg cccagaacaa 1320

gccgggagga acgaggggac agaggcggca ggccaggaca cggccgacca gggaggaaga 1380

ccaacacaag agaagccaga cagggagcag cgagggaaca cggccgggag gcaaccagca 1440

cgccgggaag aggggaggga gaagcaaaac cgggggccag cccacgccac gaagaacaag 1500

caaaaaggga caaggacaca acaacgcacc ccggcgcccc ccaaaaaaca aagaaaaggc 1560

cccggaaaag acacaacgcg gaagacggaa agacccagag aggggggcac cacagaacgc 1620

gccccgggcg cgaccgcccc gaaacagcca gcgcgaaccc agcacacaga cccaacccag 1680

aagaaccgaa ggcgcgacac aaaccagcga acaacaggga accaggacga agacgaaacc 1740

gcacagcgga agggcaaaag gggagaccag acggggccaa gaaaccaagg ggcgagaaga 1800

ggcaggacag gcgaaaaagc cacgagcgcg agggaaacca gggcggaaag cgcacaacca 1860

cggcacccac gcgaaaaaga aagaggacag acggcaaggg gaaggcgcac agaacggggc 1920

acaaggccgg cagccgcaag gaagaacagg acgcaaacac gaccaagaga agggcaccgg 1980

ggccgaaggc caccaccacc ggaaagaaac aaccacgagc aacgaagacg ggaccgaagg 2040

ccagcgggca ggccaaagca agcacaaggg cagaggagga ggcacagcaa aggaggccac 2100

cacccggaca gagcccgcga cgggaccaac ccacaacgag gaaagggaga gaccgggcgg 2160

gcggcccgcg aacgagccgg caagggaaag acaaacaacc ggaacggagg ccacgcgccc 2220

aaagggggac ggggcaagag gcacagcagg agcccaacaa ccgagaacag aagggaaaga 2280

cccaggagag acaagccccc gcacagaagg aggcgaccac cacagggaaa aggagaacac 2340

gaaggggggg caacggacag ggaaagggaa ccagggcaca cgggggggca gaaaacaagc 2400

agagggggcc gaccaagagc ccgacggacc ccgcacaccc caaggaaggc acggaaagag 2460

acaggacaga aagagacaac ggacgaacag agaggcggaa cagcacagag ggagcagagg 2520

cgacggaaca caacgcaagg gcagcacaga gaaagacggg cccagccagc agacccaaag 2580

agagccaggc aggaccgaag gaaaacccga cacaacacgc aaaaacgaag aacaagacag 2640

agcccaggga cagcacccag caaaagcaag ggcgagacag acgggaccgg acggacgacc 2700

gccaccagaa ccgcagaagc gcggagggag cacgaggagg aagaagccgg gcaaaggacc 2760

acaagcaaca gaacaaccgc cgcggaccag ggccagggga ggagggaagg gaacaaaccc 2820

acacagacag aggcgagaac acaccaggca gagggagagc caaaagaaag gaacgcgcgc 2880

cagcagacaa caaccagcaa gaccaaacag ggcagcagga ggggggccaa gggggaaaga 2940

agacggcggg gcagcggcgc cagagaccag cgacaccaac cgcgcgacag aagagcgcgg 3000

agacggcaaa gagagaccaa cacgccccgg aaacagggca acccgagaac ggcaagagac 3060

aaggacagca cggaaagcca agcacaggca acagcggcaa ccggaccaca aggacaaaga 3120

acaagacggc acagaccaag cacaggacag gaccaaaagg gacgaaggga aagggaggga 3180

acacacccaa ccgccacaca gaccccccca ccggacccac cacgcaggga acaagaggaa 3240

cggacaagcc ggagcggcag ggcccaaccc gaggacgagg ggcagacacc acccgaccca 3300

acgcccacac gagcaacaaa acaaacccaa ggaaggaaga cggggcagaa aagggcggag 3360

gaagaccaac caagagggaa acgacaaacg aaggaccaac gggaagggga cccccgcaaa 3420

acaaaagaca agcaaaacag gaccaggcca gacaaagcca accacagcgc acagaaaagg 3480

gcagcaaaca gacgaagaag gcacacccca caagaagaca agagaaaagc aggagggacc 3540

aaccaccaag acgagccgcg acgaagccaa gggccgacaa cgaagaagag gggaaagaag 3600

cccggcagag ggaaagaacg acacaaacac aaaggaggaa gaagaaggcc acgggggacg 3660

aagaggaggg agccaaaggg ggcagcaagg cagcaacaga gaaaaaaaac agaggcaccg 3720

cggaagacag agagggagag gagaaaccgc ccaaaagcgg gcggggccac caagaccggg 3780

agacccagcc gacgaagaaa aacaaaagag gacagagagg acaacagaag ggccggagag 3840

aggagacgca gggacgcaaa agagccagag ggcaaaccga ggaaccccgg gcagcaacaa 3900

aagcaagagc ccggcggaaa cgggacggag ggggagacgg aacaccggcg ggccaggggg 3960

ccgccggcaa gaaggaccga acagagaaag caccacacca aaggacaaag acgccgggag 4020

ccaaggggca ccacaccaga gccccggaga ccccaccccc aaagaaagaa gggggggaaa 4080

cggcgggcga cacacaccaa ggggcaagca gggaccagca cggggaaaga cgcggaggac 4140

acagggccgg acaagggcgg ccaacaaaaa aagagacaga gagcgagagg agaaaacgac 4200

ccggagcccg gaaggagcag ggaggcaacc cagaggcaga acaacaggga caaaggagcc 4260

aggccacaca aaaaacggag gagaacaccg ggacagcaca ggaacccggc ccgaccaaga 4320

acccaaaggc ggcagggcac cgaagaggca caagggaagg gagcggcagg gcaaggcggg 4380

aagaagagga ccaaaccaac caagcagagg gaaacaaaca gcccaaaaga ccacagacga 4440

cagaaaggaa agaaaaaacg accagaacag gggagaacag acaaaaaccc gcacagggcc 4500

ggaaaaacca cggaacccca ggcagcaaga agagaaggga ggcaaagaga gcggcgaccc 4560

cgagggcggc agcagagcag aaccaaaaga gacaaaaaca ccaagcacgc aaaccgagga 4620

aggggagaga aggaagggga caggccacag ggaaaccagc caacggaccg cagagccaca 4680

accaaggcga gccgcgaggg agaccccaac gacgagacca cggccacccc agaacaaggc 4740

cacagggaaa gaccacagac agagaaccgc gggagagcca gaccgcaaca ccagcaggca 4800

cagaacaacc acagggcaga aacacccaag aagaacaagc cccagaagga gaaaggggaa 4860

gacaggccag agacggacag cggacaaaga accagagagg agagaagagc aacagcgggg 4920

cccacaggaa caggcgggga gacagcaaag aaagaaagca agggacaacc aggcacaaag 4980

ggagaggacc acaaccgagg gggaacgcgc agccccgacg ggggggcaac caacgcgaag 5040

aacagggacc cccggacgga ggggcgaaca gggcaaggga aaagagaaac ggcgcaccaa 5100

gagccccaag gacgggccga agagaaggcg cacgagggga acaagcccag agaaggggga 5160

gaggggagag aaagccggga gaacacagga gcaagaaacc ccgggcaaag aaccaagaca 5220

cgcaagcaca gaggacggag aagagggaaa acacaccaaa ccagagagag aacagaggag 5280

ccagaggagg acagcgagaa cacaaggaaa cccaaaagga acagaagaac accgaggcag 5340

aaaaaaaaag gccaggacga ccacccagaa ccaacagcgg cgacaacagc acgaaacaca 5400

aggccaggca cccaaaaaaa agaaggagag ggacgaagac gaaacaaccc ccaacgcaag 5460

aaaaggggga gagaccccca cggagccaca gaggagagga cagcggagag cacgggcaga 5520

cggccagacc cggaaaccaa ggaaccgaga ggcggcagag cacaaaacaa gagggcacaa 5580

cagcgagaag cccgagagcc acggcagagg aacaggcacc aaagaggcaa accagagcac 5640

agaaaacaag aagacacagg ggaagacacc acacaccaca gacgccccaa agcacaagac 5700

ggaggggaag gagacagaag aaggagcagc ccagggggag gcagagaggg gaagcagacc 5760

aaagcaagag agggaccaac agaagccagg cacgaaggga aagaaacccc cacacaaaga 5820

gacaagaaca cggacgacag aaagaaacag gaggcgcggc agacagaaag aagacacaag 5880

aagggggggg gacgcacaca gccaaaagag cacaggccag gcgggaggag acgcgcgcac 5940

ccggcggaag ggcggcaggg ggggaacaaa gcagaccagc aaacaagcgg ccacagacgg 6000

cgcacaacac aacagaccca gcccaggaga cacagagaca caacaggaag gaaggaaagg 6060

gccagccacg gccagccagc acacacaaca aaagcggaaa caaaacgcca agaaggaacc 6120

ggcggccacc gcccagccgc cacagcccaa acacgccccc accgaggaga gcggcaagag 6180

accgcaacac aagaccacca caacaggcgc ggaaaaagcg agggcaggca caggggaggc 6240

ggcaggagac agcacaaaac cagaccgggg caagcagcag caggggaggg gccgggcagc 6300

ccacaagcaa cgaggccagg agcagaagag aacacaccag aaaggaaaga accggaccaa 6360

gcagccacga gaaagcaagg agagccgaga aaaaaaaggc ggaagcaggc agacagcgga 6420

accccagaca gaaccaccag gagcaaaggg ggggaggcaa aagagggccc aaaggacagc 6480

cggaggaacc cacgaaagcg aggcgggaac acgggagaga aggaaggaaa gaccgccagc 6540

acaagaaaca acagagcccg aaagccggac agacaagcag cggagggaga ggcaacagcg 6600

ggccccgccc ccagcggagg acaccgacgg agacagaaag ggccccccaa gacaacccca 6660

agggagacga aagccccggg acaagagaag gcagaagaag gcggagagcg agaccggagg 6720

aggaaggcca ccgcagaaaa aacgggagag gcacggaaca caggggacaa aaacagagac 6780

aacacagaaa aaagccagga aagaaggaag ggcagggaac cacacaaggg agccaccaca 6840

aacggaccaa caacagaaaa caaacggcaa ccgaaaaggg agagacaccc accggcaggg 6900

gccaagaggc acacaggggc ggaacagggg caaccgggcg agaaaccgaa cacaaacacg 6960

aagagaggga cggcaaccac accaaagaaa gggccaaaag aagagagggg gcaacagaca 7020

ccccacaacc acccgacgag aaggacacaa gaaaacggaa gacaaagaga ccgcgacgga 7080

caaccggcgg cacacggaaa gaagaaaggg accaaaaacc agcccgggga gaaagaacac 7140

cggagagcag gaggaaaaac cgcagccgcg agggggaaca acgacggacc ggacgggagg 7200

ggaagcccca cagacacagg ggagacccga cagcgcacca gccaggcaga cccgaaaggc 7260

caacaagaaa acgggggagg gaaggccaaa ccccgggaca accacagagg gcaagccgca 7320

cgaagccaac aagggcagag agagaccccg ggcgaaaggg cgaaaagcca cccaaagagg 7380

aaaacgcaaa gaagaaagga acagaggcag ggacccacag aaggagagaa gagaggaggg 7440

gaaagaccgg caagcccgca ggggaaagcc agaaaggaca caaaggcacc caacagagag 7500

acccagaggc aaaggggagg ccaaaaagaa aaacaaacca aggcagaagc gcaggggcgg 7560

cccgaagacc aaaggaagag cacccgaggc gcagccgggg aacccacaga ggcaacaaaa 7620

cagacaggac cacggggggg aaagcaaaca aggaaaaagc caaacagggg agcacagacc 7680

cacaaagaac aaggaggggg caaaaggaca gaaacgagaa ggggagcaag aagaaaaaaa 7740

cagggcaacc gacccccgga agagcccgca acaggccacc cggaggccag aacaaaacgc 7800

acaaggccgc accaacagcc aagggaacgg agccaaccag cgccagaggg gaccaacgcc 7860

aggaggacca agacccacca agaagcaacg caagaaggga gggagaggaa cacaagagaa 7920

aacaggccgg acaagccggg aagcacaacg aaggaaagga aaacaaaacc agggaaaccg 7980

aggaccaaac acaggaaccc cggcaagggg cagagcaacg gcagagaggg acacagacac 8040

aaggaaacaa gacaaaggcc agaagacagc acggacaggg gagaaggccc gggagggccc 8100

agacagacac aaccaaccca ccaagcaaaa gggaaagaag acaaggaaga gaaccacaaa 8160

ccccgggaca aagaaacaag gaagcaagca gaaacgaccc gagagagccc cacgagccgg 8220

gaagggagga acggagagag gaaaaacagg aaggggcgcg gcgaacgcaa aaaaagggga 8280

aaaggacaga gaaaaacaaa gcgaagagaa gacaacgaaa aaaggcagaa acacaaaaca 8340

gaaaccgcga cccaaagaag agaagaggga cgcaagagac ggacgcggga ccgggaagag 8400

aagaaaccca gagcaacaaa cccgaagcaa aaacaaggcg gccacaccaa gggagaaagg 8460

gggaagcaga agccagagaa cccgggagaa gggaagacac ccaccaaaga caaagaaaga 8520

aggaagggac aaccaaaacc agggcaggag gacacaaggc ggggacaccc aggaaccaca 8580

aaagaggagg aaaaggacaa caaaagacac aagaagaaag gcaaaaagac acccgaccag 8640

cacagcagaa gacccgaaca ggcgagggga agaacaaagg aaagggcaaa gaggcaggga 8700

caaccgacac aagcgcaggc aaagcagcaa aggaacaaga acgcccgcga ggccacggga 8760

gacccacaag agccgacagg gggcaaaaac agggggggag aggccgacac agaaagagcc 8820

cgggagaaac gcgagaaggg agccagacca agaagcggga agccccagaa gacacgaagg 8880

ggacaagaga aagggccacc aagagaagag gccccaacac caaacaagaa ggggcagaaa 8940

caccagacag ccggggagaa aacaaccaca accggcaaaa ggccacaagg agaccagggg 9000

agaggggacc aagcaagaga aagcagagca cagcccgcga gacccggaac ccacaacaga 9060

aggacgcacg ggcacaacga acgcaaggaa accagggaag caaccacaca cagaagggga 9120

cccgaacgcc acaaggaagc acggccacaa cgacaagaga acaagccgag aagcggccaa 9180

gaaaccagca gcagaccgga gccggacaga cacaccagaa aagacacaca agacggcaag 9240

ggggaaagag ggcaacggca gaagcagaag acagaagagc aagacggaaa aggacaaccc 9300

ggagagggcc acacccgcaa gggagacaag aagaacgggg gcaagaaaac agacaacaac 9360

caagggaccg acaggacaac aggcccaaag caaagggaag gaggcgagag cagagagacc 9420

cgaagggaga ggggagagcg cgggaacccg ggacggaccc gccagagaac ccggagaaac 9480

acaaaaagaa acaaaaaaga agagaagaac ggacaaacac ccaagaccac acacaccaaa 9540

cagcacagcg gaaggaaaac cgacgccaca gggacaagga accaacggcc 9590

<210> 2

<211> 25

<212> RNA

<213> classical swine fever viru

<400> 2

cccaccucga gaugcuaugu ggacg 25

<210> 3

<211> 43

<212> RNA

<213> Artificial sequence

<400> 3

gagcagaagc ccaccucgag augcuaugug gacgagggca ugc 43

<210> 4

<211> 43

<212> RNA

<213> Artificial sequence

<400> 4

gagcagaagc ccgccucgag augcuaugug gacgagggca ugc 43

<210> 5

<211> 43

<212> RNA

<213> Artificial sequence

<400> 5

gagcagaagc ccaccucgag auccuaugug gacgagggca ugc 43

<210> 6

<211> 43

<212> RNA

<213> Artificial sequence

<400> 6

gagcagaagc ccaccucgag augcuaugug aacgagggca ugc 43

<210> 7

<211> 68

<212> DNA

<213> Artificial sequence

<400> 7

cgtccacata gcatctcgag gtgggataga tccgcgtagg tactaaccca cctcgagtat 60

tcctacgc 68

<210> 8

<211> 68

<212> DNA

<213> Artificial sequence

<400> 8

cgtccacata gcatctcgag gtgggataga tccgcgtagg tactaaccca cctcgagtat 60

tcctacgc 68

<210> 9

<211> 65

<212> DNA

<213> Artificial sequence

<400> 9

cgtccacata gcatctcgag gtgggacgag acgcgtaggt actaacccac ctcgagatcc 60

tacgc 65

<210> 10

<211> 68

<212> DNA

<213> Artificial sequence

<400> 10

cgtccacata gcatctcgag gtgggataga tccgcgtagg tactaaccca cctcgagatt 60

tcctacgc 68

<210> 11

<211> 68

<212> DNA

<213> Artificial sequence

<400> 11

cgtccacata gcatctcgag gtgggataga tccgcgtagg tactaaccca cctcgagatg 60

ccctacgc 68

Claims

1. A method for detecting a long-chain nucleic acid, comprising the steps of:

(2) carrying out reverse transcription on the RNA of a sample to be detected as DNA of the sample to be detected, carrying out isothermal amplification on the DNA of the sample to be detected and the hairpin of the self-primer amplification template by using the DNA of the sample to be detected as the template, and calculating the content of long-chain nucleic acid in the sample to be detected according to the intensity of a fluorescence signal of an amplification product;

wherein the long-chain nucleic acid is a nucleic acid sequence with the length of more than 1000 bases.

2. The method of claim 1, wherein the long-chain nucleic acid is mRNA, IncRNA, RNA viral genome, or RNA fragment having a base length of greater than 1000.

3. The detection method according to claim 1 or 2, wherein the reaction system of isothermal amplification in step (2) is:

wherein the 10 XNt.AiwI endonuclease buffer solution contains 50mM CH₃COOK, 20mM Tris-acetate, 10mM Mg (CH)₃COO)₂100. mu.g/mL BSA, pH 7.9;

the Klenow fragment (3'→ 5' exo-) polymerase buffer contains 10mM Tris-HCl, 50mM NaCl, 10mM MgCl₂1mM dithiothreitol, pH 7.9.

4. The detection method according to claim 1 or 2, wherein the reaction procedure of the isothermal amplification in step (2) is: amplifying for 60min at 36-38 ℃.

5. The detection method according to any one of claims 1 to 4, wherein the long-chain nucleic acid is a hog cholera virus genome and/or an RNA fragment thereof.

6. The group of self-primer amplification template hairpins are characterized in that the nucleotide sequence of the self-primer amplification template hairpins is as follows:

7. a test preparation comprising the self-primer amplification template hairpin of claim 6.

8. The assay preparation of claim 7, wherein the assay preparation comprises the sequence of SEQ ID No. 9.

9. Use of a detection formulation according to claim 7 or 8 in the preparation of a hog cholera virus detection kit.

10. The use according to claim 9, wherein the classical swine fever virus is a genomic or RNA fragment of a classical swine fever virus comprising the sequence shown in SEQ ID No. 2.