CN114592037B - Method for isothermal amplification of target nucleic acid sequence - Google Patents

Abstract

The invention discloses a method for isothermal amplification of target nucleic acid sequences, and relates to the technical field of nucleic acid amplification. It comprises the following steps: respectively designing forward amplification primers F1 and F2; blocking primers B1 and B2; stripping primers D1 and D2 of the forward amplification primer; reverse amplification primers R1 and R2; the primers RS1 and RS2 were stripped from the amplified products. Isothermal synchronous amplification of two target genes is completed by utilizing interaction recognition of the two target genes. The primer of the invention has simple and reliable design, high sensitivity and high specificity. The isothermal amplification technology of the invention provides a new solution in the fields of medical detection, biological scientific research, agricultural scientific research, epidemic prevention and control, biosafety and the like.

Description

Method for isothermal amplification of target nucleic acid sequence

Technical Field

The invention relates to the technical field of nucleic acid amplification, in particular to a method for isothermal amplification of target nucleic acid sequences.

Background

With the development of molecular biology technology, in particular, the rapid development of PCR detection technology and various general detection technologies developed by depending on the PCR detection technology. The method brings the fields of human disease detection, animal and plant inspection and quarantine and various scientific research technology into the space-time of molecular detection. Meanwhile, the synchronous appearance of the PCR detection technology and the NGS detection technology brings revolutionary reform for a plurality of fields such as drug resistance detection of human infectious diseases, tumor mutation detection, hypertension drug concomitant detection, hereditary disease detection and the like, in particular to the precise medical field. In addition, with the rapid spread of new coronaviruses, PCR technology has become the most prominent nucleic acid detection technology worldwide in providing a new coronavirus nucleic acid detection field brilliant. Although the advantages of the PCR detection technology are obvious, the PCR detection technology depends on high-added-value fluorescent quantitative PCR instruments, professional supporting facilities and professional supporting staff. The construction cost of the PCR detection technology platform is high, the application flexibility of the PCR detection technology is affected, and the PCR detection technology is affected to sink into community hospitals and families.

Isothermal amplification techniques have been developed in recent years, and are novel nucleic acid amplification techniques based on isothermal amplification. The technology can rapidly realize the copy and detection functions of nucleic acid under the condition of constant temperature. The technology has very simple requirements on detection instruments, and can realize the detection function only by a constant temperature facility or a small fluorescent detection device. Or detecting by adopting a colorimetric method, a colloidal gold method and other naked eye judging modes. So that this technique, once developed, is considered by many scholars to be a detection method that is likely to be comparable to PCR. In addition, as CRISPR technology continues to mature, the combination of CRISPR technology with the depth of isothermal amplification technology makes isothermal amplification detection technology one of the most likely alternatives to PCR technology. In summary, with the continuous evolution and upgrading iteration of isothermal nucleic acid amplification technology and the obsolete and new of matched diagnostic instruments, it is believed that isothermal amplification technology will become an important branching technology for nucleic acid detection in the future nucleic acid detection field. The following outlines the major isothermal amplification techniques that exist.

1. Loop-mediated isothermal amplification (Loop-mediated Isothermal Amplification, LAMP).

The loop-mediated isothermal amplification method is a novel isothermal nucleic acid amplification method developed by Rongyan corporation in Japan in 2000.

Has the characteristics of simple and quick operation, strong specificity, easy detection of products and the like. The method mainly comprises the steps of designing 4 specific primers aiming at 6 regions of a target gene, and completing biochemical reactions such as gene template amplification, primer extension, single-strand displacement and the like under the conditions of Bst DNA polymerase and isothermal amplification at 60-65 ℃. The loop-mediated isothermal amplification method has very rapid reaction, and the template can realize 10-10 times of nucleic acid amplification within 15-60 minutes. Compared with the PCR method, the method does not need complex processes such as thermal denaturation, temperature circulation, fluorescence detection and the like of the template. After the isothermal amplification reaction is finished, the magnesium pyrophosphate precipitation color reaction can be generated by reacting pyrophosphate ions precipitated in dNTPs in the nucleic acid amplification process with magnesium ions in the reaction solution. Finally, the turbidity reaction is adopted to identify whether the amplification is carried out or not by naked eyes, and no instrument is needed for detection. The loop-mediated isothermal amplification technology does not need a PCR instrument and expensive reagents, so that the method has wide application prospect.

2. Recombinant polymerase amplification techniques (Recombinase Polymerase Amplification, RPA).

The recombinant polymerase amplification technique was a proprietary technology developed successfully in 1999 by TwistDx, uk. Is praised as a revolutionary innovation in the field of DNA diagnosis. The recombinase polymerase amplification technique can realize the nucleic acid detection of trace nucleic acid molecules under the condition of lower constant temperature for only 10-20 minutes. The technology has low requirements on environment and hardware facilities, and has good application prospects in aspects of biological protection, water body inspection, food inspection, medical diagnosis, microfluidics, veterinarian and the like.

Recombinant enzyme polymerase amplification techniques rely primarily on three enzymes: recombinant enzymes capable of binding single stranded nucleic acids, single stranded DNA binding proteins and strand displacing DNA polymerases. The main principle of the recombinase polymerase amplification technology comprises that firstly, a protein-DNA complex is formed by combining a recombinase and a primer, then the complex can search a homologous sequence in a target gene, and once the homologous sequence is found, a chain exchange reaction occurs and DNA replication is carried out, wherein the replication mode adopts exponential amplification. At the same time, the replaced DNA single strand binds to SSB, preventing further substitution. In the systems of recombinase polymerase amplification techniques, the overall reaction is very rapid, and detectable levels of amplification product are typically obtained within 10-20 minutes. Meanwhile, the method can also be combined with a PCR related probe detection technology to finish the detection of the target gene.

3. Rolling circle amplification technology (Rolling Circle Amplification, RCA).

The rolling circle amplification technology is 90 s, and scientists refer to the principle of rolling circle replication of cyclic pathogenic microorganism DNA molecules in nature to establish a nucleic acid isothermal amplification technology mode. The technology is an isothermal nucleic acid amplification method based on ligase ligation, primer extension, and strand displacement amplification reactions. The technology mainly relies on a circular DNA molecule as a template, dNTPs are converted into single-stranded DNA by a short DNA primer complementary with the circular template under the catalysis of phi29DNA polymerase, and the single-stranded DNA consists of hundreds of repeated template complementary fragments and can be directly combined with a probe to realize signal amplification and detection. The detection technology can directly amplify DNA and RNA, has high amplification efficiency and high sensitivity reaching a very high level, and has very great application value and potential in the field of nucleic acid molecule detection.

4. Cross primer amplification techniques (Cross Priming Amplification, CPA).

The cross primer amplification technology is a novel nucleic acid isothermal amplification technology which is independently developed and successfully developed by Hangzhou Yongda company in 2008, and is also a first nucleic acid amplification technology with independent intellectual property rights in China. The technology can realize the efficient, rapid and high-specificity amplification of the nucleic acid template. The main principle of the cross primer amplification technology is that 4 or 5 specific primers are designed aiming at 4 or 5 regions of a target gene by means of the characteristics of Bst DNA polymerase with strand displacement characteristics, wherein the specific primers comprise 1 or 2 cross primers, and isothermal amplification reaction is carried out at the temperature of about 63-65 ℃. In the amplification process, the cross primer is first combined with template strand complementarily and then extended, the stripping primer strips off the newly synthesized single strand under the action of Bst DNA polymerase, and finally the cross primer synthesizes great amount of target fragments with the newly generated single strand as template under the action of Bst DNA polymerase.

5. SDA Strand Displacement Amplification technology (STRAND DISPLACEMENT Amplification, SDA).

The strand displacement amplification technology is an in-vitro isothermal amplification technology of DNA based on enzymatic reaction, and the main principle is that a restriction enzyme is used for cutting a DNA recognition site, then a DNA polymerase is used for extending to 3' at a cut and displacing a downstream sequence, and the amplification is carried out under isothermal conditions. The whole process comprises 3 steps of preparing a single-stranded DNA template, generating a target DNA fragment with enzyme cutting sites at two ends and performing SDA cyclic amplification.

6. HDA helicase-dependent amplification techniques.

The helicase-dependent amplification technique (HDA) was a novel nucleic acid isothermal amplification technique invented by NEB corporation in the united states, 2004. The technology utilizes the characteristic that helicase can unwind DNA double helix under the constant temperature condition, then SSB single-chain binding protein and the like are attached on a single chain to stabilize the unwound DNA single chain, thereby providing a replication template for the primer. Then, the complementary double strand is replicated by DNA polymerase. Repeating the cyclic reciprocating amplification process to finally realize the exponential growth of the target sequence.

At present, the technical problems of low sensitivity, poor specificity, single-target detection only and the like commonly exist in the nucleic acid isothermal amplification technology. In the isothermal amplification detection field, the problems of false negative and false positive result interpretation frequently caused by the problem and the difficult problem of multi-target synchronous real-time detection exist.

In view of this, the present invention has been made.

Disclosure of Invention

The present invention aims to provide a method for isothermal amplification of a target nucleic acid sequence. The method can solve the technical problems of low sensitivity, poor specificity, single-target detection only and the like commonly existing in the existing nucleic acid isothermal amplification technology in principle and path. Therefore, the problems of false negative and false positive result interpretation of frequent scaling-up and the difficult problem of multi-target synchronous real-time detection in the isothermal amplification detection field are solved.

The novel isothermal amplification method provided by the invention has the advantages of high detection sensitivity, strong specificity, good stability, simple and flexible operation, low threshold of automatic realization and the like. Meanwhile, the detection methodology of the invention can adopt a manual interpretation mode such as colloidal gold, turbidimetry and the like. Automatic result output can also be accomplished using small detection instruments, such as fluorescence detectors. And automatic detection can be finished by adopting a full-automatic microfluidic chip detection instrument. The invention is suitable for qualitative and quantitative detection in a plurality of fields such as medicine, genetics, inspection and quarantine science and the like.

The invention is realized in the following way:

The present invention provides a method for isothermal amplification of a target nucleic acid sequence, the method being for the diagnosis of a non-disease, comprising:

a. Designing forward amplification primers F1 and F2; it comprises the following steps:

the forward amplification primers F1 and F2 are respectively paired with a target sequence T1 and a target sequence T2 to extend amplification, reverse sequences RS2 and RS1 are respectively designed at the 5' ends of the forward amplification primers F1 and F2, and the RS2 is identical to the SD2 sequence of the target sequence T2; RS1 is identical to SD1 sequence of target sequence T1; SD2 and SD1 are located upstream of target sequence T2 and target sequence T1, respectively;

b. Designing blocking primers B1 and B2; it comprises the following steps:

Designing blocking primers B2 and B1 at the upstream of SD2 and SD1 respectively, and completing the first round of replication and amplification by forward amplification primers F1 and F2 and blocking primers B1 and B2 so that the nucleic acid amplification extension is finished at the downstream positions of SD1 and SD2 respectively (refer to the first-stage amplification schematic diagram shown in FIG. 1);

c. designing stripping primers D1 and D2 of the forward amplification primer; it comprises the following steps:

The stripping primers D1 and D2 of the forward amplification primers are respectively designed at the upstream of the forward amplification primers F1 and F2 and are used for displacing the first round amplification products to obtain single strands of the first round amplification products;

After the head and the tail of the first round of amplification products are complementarily hybridized, a Loop structure is formed, and templates are prepared for subsequent amplification (refer to the amplification schematic diagram of the second stage shown in FIG. 2);

d. reverse amplification primers R1 and R2 are designed according to the target sequence T1 and the target sequence T2, and a Loop structure formed by first round amplification is used as a template for amplification;

e. designing stripping primers RS1 and RS2 of the reverse amplification product, and replacing the amplification product in the step d to obtain a new single strand; after complementary hybridization of the head and the tail of the new amplification product, the Loop structure is formed again (refer to a third-stage amplification schematic diagram shown in FIG. 3);

f. The target sequence T1 and the target sequence T2 are amplified in a linear mode or a higher structure mode, and the two amplified target sequences are subjected to nucleic acid amplification synchronously through a mode of amplification extension, strand displacement, loop formation and amplification extension in a circulating mode.

The isothermal amplification method provided by the invention is also called sequence interaction mediated (Sequence Interaction mediated Amplification, SIA) isothermal amplification.

The invention is based on two target positions of a target gene, and firstly two initial amplification primers, two stripping primers and a pair of blocking primers are designed. The replication and stripping of the first round (namely the first strand) are completed under the action of enzyme, and the head and tail are complemented to form the Loop similar to a Chinese lantern. Then, two reverse primers and two stripping primers are designed for the single-stranded region of Loop to complete the replication of the complementary strand, forming double-stranded DNA. The double-stranded DNA forms Loop again, and the amplification cycle is carried out repeatedly, so that the two amplification targets synchronously amplify the nucleic acid.

The method is suitable for nucleic acid amplification in the fields of medical detection, biological scientific research, agricultural scientific research, prevention and control epidemic prevention, biological safety and the like. Based on the method, corresponding products such as detection reagents, kits, chips, detection systems and the like can be developed. In general, the method provided by the invention has the advantages of low precision requirement on equipment, low detection cost, high reagent sensitivity, high reagent specificity, capability of simultaneously aiming at multi-target detection and the like.

The enzyme used for amplification in the above method is selected from the group consisting of DNA polymerase and/or reverse transcriptase. For example, a single enzyme system selected from the group consisting of DNA polymerase or a single enzyme system selected from the group consisting of reverse transcriptase, or reverse transcriptase.

Preferably, one reaction system contains 2 to 6,2 to 5,2 to 4,2 to 3 or more preferably 2 reverse transcriptases. One or more DNA polymerases may also be included in the same or different systems.

The enzymes (reverse transcriptase and/or DNA polymerase) in the system are present in working concentration.

In a preferred embodiment of the present invention, the DNA polymerase used in the above method for amplification includes, but is not limited to, at least one of the following enzymes: bst DNA polymerase, klenow DNA polymerase, T4DNA polymerase, taq DNA polymerase, DNA polymerase I, vent DNA polymerase, phi29 DNA polymerase, tneDNA polymerase, tma DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, tfl DNA polymerase, tth DNA polymerase, stofel fragment DNA polymerase and DEEPVENT DNA polymerase and mutants, variants or derivatives thereof.

In a preferred embodiment of the present invention, the reverse transcriptase employed in the above method for amplification includes, but is not limited to, at least one of the following enzymes:

M-MLV reverse transcriptase, AMV reverse transcriptase, RSV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase and HIV reverse transcriptase and mutants, variants or derivatives thereof.

In a preferred embodiment of the invention, the enzyme used for amplification in the above method is selected from the group consisting of:

Bst DNA polymerase and M-MLV reverse transcriptase.

In a preferred embodiment of the invention, the amplification reaction in the method is isothermal amplification;

In an alternative embodiment, the amplification is at 55-72 ℃; in an alternative embodiment, the amplification time is 15-90min.

The amplification temperature is about 55 ℃ or higher, about 56 ℃ or higher, about 57 ℃ or higher, about 58 ℃ or higher, about 59 ℃ or higher, about 60 ℃ or higher, about 61 ℃ or higher, about 62 ℃ or higher, about 63 ℃ or higher, about 64 ℃ or higher, about 65 ℃ or higher, about 66 ℃ or higher, about 67 ℃ or higher, about 68 ℃ or higher, about 69 ℃ or higher, about 70 ℃ or higher, about 71 ℃ or higher, about 72 ℃; or the following temperature ranges: about 55 ℃ to about 72 ℃, about 56 ℃ to about 71 ℃, about 56 ℃ to about 65 ℃, about 56 ℃ to about 64 ℃, about 56 ℃ to about 62 ℃, about 58-62 ℃, about 60-72 ℃, or about 65-70 ℃.

The amplification time was in the following time range: 15-90min,15-60min,30-65min or 20-80min.

In a preferred embodiment of the present invention, the 3' ends of the blocking primers B1 and B2 in the above method are further provided with a blocking group;

in an alternative embodiment, the blocking group includes, but is not limited to, any of C3 Spacer, C6Spacer, C12 Spacer, spacer18, amino, dideoxy, base inversion, BHQ1, BHQ2, and MGB.

In a preferred embodiment of the present invention, the number of target genes is a multiple of 2.

In an alternative embodiment, the number of target genes is 2, 4, 6, 8, 10 or 12.

In a preferred embodiment of the present invention, the above-mentioned high-level structure means: amplification is performed in a secondary structure, or in a mixture of linear and secondary structures.

In a preferred embodiment of the present invention, the nucleic acid of the target gene is DNA or RNA.

The amplification method can be used for detecting pathogenic microorganisms (plant pathogenic microorganisms, animal pathogenic microorganisms and fungal pathogenic microorganisms), environmental microorganisms, microorganism typing, archaebacteria identification, transgenic variety identification and biological invasion.

In another embodiment, the invention provides the use of a method for amplifying a target nucleic acid sequence in the detection of a pathogen of infectious disease in humans, animals or plants.

In another embodiment, the invention provides the use of a method for amplifying a target nucleic acid sequence for detecting infectious disease pathogens in food products or biological weapons.

In another embodiment, the invention provides the use of a method for amplifying a target nucleic acid sequence for detecting a human genetic disease or health risk gene and its assessment.

The invention has the following beneficial effects:

The isothermal amplification method of the target nucleic acid sequence has the advantages of simple and feasible design, high detection sensitivity, strong specificity, good stability and high repeatability, and can be used for qualitative and quantitative analysis. Can be used for qualitative and quantitative detection in a plurality of fields such as medical detection, biological scientific research, agricultural scientific research, prevention, control and epidemic prevention, biological safety and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic of a first stage amplification;

FIG. 2 is a schematic diagram of a second stage amplification;

FIG. 3 is a schematic of a third stage amplification;

FIG. 4 is a diagram of a tuberculosis primer design site and design;

FIG. 5 is a graph showing the electrophoresis results of the lowest limit of detection reference of Mycobacterium tuberculosis (lanes 1-5 are 1.0E+03 bacteria/ml, 1.0E+02 bacteria/ml, 1.0E+01 bacteria/ml, 1.0E+00 bacteria/ml. Lanes 6-7 are negative controls respectively.);

FIG. 6 is a graph of the results of electrophoresis of repetitive references of Mycobacterium tuberculosis (lanes 1-2 are negative controls, 3-5 are 3 replicates of 1.0E+02 bacteria/ml sample, respectively);

FIG. 7 is a design site and a design diagram of a hepatitis C primer;

FIG. 8 shows electrophoresis results of negative reference samples of hepatitis C, and lanes 1-10 show detection results of negative reference samples of N1-N10, respectively;

FIG. 9 shows the electrophoresis results of the positive reference sample of the liver C, lanes 1-7 are the detection results of the positive reference sample of P1-P7, respectively;

FIG. 10 shows the electrophoresis results of a repetitive reference of hepatitis C, lanes 1-3 show the results of 3 repetitive detection of the repetitive reference, and lane 4 shows the results of a negative control;

FIG. 11 shows the electrophoresis results of the sensitive reference of hepatitis C, lanes 1-5 show the results of 5 repeated detection of the sensitive reference, and lanes 6-8 show the negative control results, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

This example provides a qualitative detection method for nucleic acid of Mycobacterium tuberculosis.

1. Preparation of tuberculosis nucleic acid samples

The hospital reference plate in tuberculosis is taken as a sample to be tested (minimum detection limit reference: 1.0E+03 bacteria/ml, 1.0E+02 bacteria/ml, 1.0E+01 bacteria/ml, 1.0E+00 bacteria/ml; repetitive reference: 1.0E+02 bacteria/ml). Then, the nucleic acid purification work was performed using "magnetic bead method general genomic DNA extraction kit" manufactured by Tiangen Biochemical technology (Beijing) Co., ltd. Specific operations are performed with reference to the product specifications.

2. Isothermal primer design for tuberculosis

A primer homology design region was determined by homology alignment with "insertion sequence:IS6110" of tuberculosis genome (Genbank No: Y14048), and the primer design site shown in FIG. 4 was referred to. Then, according to the primer design method of the present invention, isothermal amplification primers were designed, and the primer sequences are shown in Table 1.

Table 1: tuberculosis primer sequences

Primer name	Sequence (5 '-3')
		F1/RS2	TGGCTGCGCGGAGACGtccgaatcgtgctgaccg
F2/RS1	GTACCCGCCGGAGCTGCGtcgtggtcccgggccgtg
		R1	GTACCCGCCGGAGCTGCGTGAGCGGGCGGTGCGG
R2	TGGCTGCGCGGAGACGGTGCGTAAGTGGGTGC
		B1	cctcgatgaaccacctga(C3 Spacer)
B2	caccaagtagacgggcga(C3 Spacer)
		D1	tcactgatcgctgcccac
D2	tcagcggattcttcgg
		RS1	GTACCCGCCGGAGCTGCG
RS2	TGGCTGCGCGGAGACG

3. Preparation of isothermal amplification reaction solution

The preparation of the components of the isothermal amplification reaction solution was completed according to table 2. Then, 20. Mu.l of the PCR reaction solution was dispensed into each PCR reaction tube.

TABLE 2 preparation of the components of the isothermal amplification reaction solution for tuberculosis

The components	Volume of
		10×Buffer	2.5μl
DNTPs (containing dATP, dCTP, dGTP, dTTP, 2mM each of the components)	3.0μl
		Bst DNA polymerase	1.0μl
RS2/F1(20μm)	0.5μl
		RS1/F2(20μm)	0.5μl
R1(20μm)	0.5μl
		R2(20μm)	0.5μl
B1(20μm)	0.2μl
		B2(20μm)	0.2μl
D1(20μm)	0.1μl
		D2(20μm)	0.1μl
RS1(20μm)	0.1μl
		RS2(20μm)	0.1μl
BSA(5mg/ml)	2.5μl
		RNase-free water	6.2μl

4. Sample addition and amplification

5 Μl of the extracted nucleic acid sample is added into a PCR reaction tube containing the isothermal amplification reaction solution, the tube cover is closed, and the tube cover is placed in a PCR instrument or a water bath for amplification. The amplification conditions were 63℃for 60min. After the amplification was completed, electrophoresis was performed on 1.5% agarose gel for 20min at a voltage of 120V at a current of 90A. And finally, performing imaging detection by using a gel imager.

The experimental results were analyzed as follows:

Minimum limit of detection reference: the invention is used for detecting the lowest detection limit reference (1.0E+03 bacteria/ml, 1.0E+02 bacteria/ml, 1.0E+01 bacteria/ml and 1.0E+00 bacteria/ml) of the hospital in tuberculosis, wherein 1.0E+00 bacteria/ml samples are repeated once. Finally, detection is carried out by agarose gel electrophoresis. The detection results are shown in FIG. 5. Experimental results show that the detection method provided by the embodiment has good detection sensitivity.

And (II) the repeatability of the sample is good: the application is used for carrying out three-time repeated detection on the repetitive reference (1.0E+02 bacteria/ml) of the nuclear country, and finally, the detection is carried out by agarose gel electrophoresis. The detection results are shown in FIG. 6. Experimental results show that the detection method provided by the application has good detection repeatability.

Example 2

The embodiment provides a qualitative detection method for hepatitis C nucleic acid.

1. Preparation of hepatitis C nucleic acid sample

The hospital reference plate in the hepatitis C is taken as a sample to be tested (negative and positive reference substances: N1-N10, P1-P10; minimum detection limit reference substance: 3.0X10 ⁶ IU/branch). Then, the nucleic acid purification work was performed using "magnetic bead method general genomic DNA extraction kit" manufactured by Tiangen Biochemical technology (Beijing) Co., ltd. Specific operations are performed with reference to the product specifications.

2. Hepatitis C isothermal amplification primer design

The homology of the 5' UTR gene of the hepatitis C genome (Genbank No: EU 255989) was aligned to determine the primer homology design region as shown in FIG. 7. Then, according to the primer design method of the present invention, isothermal amplification primers were designed, and the primer sequences are shown in Table 3.

TABLE 3 hepatitis C primer sequences

Primer name	Sequence (5 '-3')
		F1/RS2	tgggcgtgcc cccgcaagacccggtcgtcctggc
F2/RS1	ccct cccgggagag ccaagcaccctatcaggcagtac
		R1	ccct cccgggagag ccatagtggt ctgcggaac
R2	tgggcgtgcc cccgcaagac tgctagccga gtagtg
		B1	gggtcctggaggctgcacg(C3 Spacer)
B2	tctccaggcattgagcg(C3 Spacer)
		D1	ttgatccaagaaagg
D2	tcccggggcactcgc
		RS1	ccct cccgggagag cca
RS2	tgggcgtgcc cccgcaag

3. Preparation of isothermal amplification reaction solution

The preparation of the components of the isothermal amplification reaction solution was completed according to Table 4. Then, 20. Mu.l of the PCR reaction solution was dispensed into each PCR reaction tube.

Table 4 preparation of the components of isothermal amplification reaction solution for hepatitis c.

The components	Volume of
		5×RT Buffer	5.0μl
DNTPs (containing dATP, dCTP, dGTP, dTTP, 2mM each of the components)	3.0μl
		Bst DNA polymerase	1.0μl
M-MLV reverse transcriptase	0.5μl
		RT primer	0.5μl
F1/RS2 primer (20 μm)	0.5μl
		F2/RS1 primer (20 μm)	0.5μl
R1 primer (20 μm)	0.5μl
		R2 primer (20 μm)	0.5μl
B1 primer (20 μm)	0.2μl
		B2 primer (20 μm)	0.2μl
D1 primer (20 μm)	0.1μl
		D2 primer (20 μm)	0.1μl
RS1 primer (20 μm)	0.1μl
		RS2 primer (20 μm)	0.1μl
BSA(5mg/ml)	2.5μl
		RNase-free water	3.2μl

4. Sample addition and amplification

5 Μl of the extracted nucleic acid sample is added into a PCR reaction tube containing the isothermal amplification reaction solution, the tube cover is closed, and the tube cover is placed in a PCR instrument or a water bath for amplification. The amplification conditions were 60℃for 60min. After the amplification was completed, electrophoresis was performed on 1.5% agarose gel for 20min at a voltage of 120V at a current of 90A. And finally, performing imaging detection by using a gel imager.

The experimental results were analyzed as follows:

Yin-yang reference: the invention is used for detecting yin and yang references (N1-N10, P1-P7) of the hepatitis C, and finally the detection is carried out by agarose gel electrophoresis. The detection results are shown in FIGS. 8-9. Experimental results show that all positive reference substances of the hepatitis C are detected, and all negative reference substances are negative results. The detection method provided by the embodiment has good detection sensitivity.

And two) the repeatability of the sample is good: the reference with the lowest detection limit (3.0X10 ⁶ IU/branch) is firstly diluted by calf serum to prepare a repetitive reference (1.0X10 ⁴ IU/ml), then the repetitive reference is repeatedly detected for three times by the method, and finally the detection is carried out by agarose gel electrophoresis. The detection results are shown in FIG. 10. Experimental results show that the detection method provided by the embodiment has good detection repeatability.

Three) minimum limit of detection reference: the reference with the lowest detection limit (3.0X10 ⁶ IU/branch) is firstly diluted by calf serum to prepare the reference with the lowest detection limit (50 IU/ml), then the reference with the lowest detection limit (50 IU/ml) is used for five times of repeated detection, and finally the detection is carried out by agarose gel electrophoresis. The detection results are shown in FIG. 11. Experimental results show that the detection method provided by the embodiment has good detection sensitivity.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for isothermal amplification of a target nucleic acid sequence, wherein said method is for the purpose of diagnosis of a non-disease, comprising:

a. Designing forward amplification primers F1 and F2; it comprises the following steps:

The forward amplification primers F1 and F2 are respectively paired with a target sequence T1 and a target sequence T2 to extend amplification, reverse sequences RS2 and RS1 are respectively designed at the 5' ends of the forward amplification primers F1 and F2, and the RS2 is identical to the SD2 sequence of the target sequence T2; the RS1 is identical to the SD1 sequence of the target sequence T1; the SD2 and SD1 are located upstream of the target sequence T2 and the target sequence T1, respectively;

b. Designing blocking primers B1 and B2; it comprises the following steps:

Designing blocking primers B2 and B1 at the upstream of the SD2 and the SD1 respectively, and completing first round replication and amplification by forward amplification primers F1 and F2 and blocking primers B1 and B2 so that nucleic acid amplification and extension are respectively at the downstream positions of the SD1 and the SD 2; the blocking primer B1 is complementary to the base of the target sequence T1, and the blocking primer B2 is complementary to the base of the target sequence T2;

c. designing stripping primers D1 and D2 of the forward amplification primer; it comprises the following steps:

Designing stripping primers D1 and D2 of the forward amplification primers on the upstream of the forward amplification primers F1 and F2 respectively, and obtaining single strands of the first round amplification products by displacement of the first round amplification products;

After the head and the tail of the first round of amplification products are complementarily hybridized, a Loop structure is formed, and a template is prepared for subsequent amplification;

d. Designing reverse amplification primers R1 and R2 according to the target sequence T1 and the target sequence T2, and performing amplification by taking a Loop structure formed by the first round of amplification as a template; the 5 'end of the reverse amplification primer R1 is an RS1 sequence, and the 5' end of the reverse amplification primer R2 is an RS2 sequence;

e. Designing stripping primers RS1 and RS2 of the reverse amplification product, and replacing the amplification product in the step d to obtain a new single strand; after complementary hybridization of the head and the tail of the new amplification product, a Loop structure is formed again;

f. Amplifying the target sequence T1 and the target sequence T2 in a linear mode or a higher structure mode, and circularly reciprocating the modes of amplification extension, strand displacement, loop formation and amplification extension to synchronously amplify the nucleic acid by the two amplified target sequences; the high-level structure refers to: amplification is performed in a secondary structure, or in a mixed manner of a linear structure and a secondary structure;

The enzyme employed for amplification in the method is selected from the group consisting of DNA polymerase and/or reverse transcriptase; and the enzyme is an enzyme having isothermal amplification and strand displacement activity; the amplification reaction in the method is isothermal amplification.

2. The method according to claim 1, wherein the DNA polymerase used in the method for amplification is selected from at least one of the following enzymes: bst DNA polymerase, klenow DNA polymerase, T4DNA polymerase, taq DNA polymerase, DNA polymerase I, vent DNA polymerase, phi29 DNA polymerase, tneDNA polymerase, tma DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, tfl DNA polymerase, tth DNA polymerase, stofel fragment DNA polymerase and DEEPVENT DNA polymerase.

3. The method according to claim 1, wherein the reverse transcriptase employed for amplification in the method is selected from at least one of the following enzymes:

M-MLV reverse transcriptase and AMV reverse transcriptase.

4. The method of claim 1, wherein the enzyme employed in the method for amplification is selected from the group consisting of:

Bst DNA polymerase and M-MLV reverse transcriptase.

5. The method of claim 1, wherein the amplification is performed at 55-72 ℃ for a period of 15-90 minutes.

6. The method according to any one of claims 1 to 5, wherein the 3' ends of the blocking primers B1 and B2 are further provided with a blocking group.

7. The method of claim 6, wherein the blocking group is selected from any one of C3 Spacer, C6 Spacer, C12 Spacer, spacer18, amino, dideoxy, base inversion, BHQ1, BHQ2, and MGB.

8. The method of any one of claims 1-5, wherein the number of target genes is a multiple of 2.

9. The method of claim 8, wherein the number of target genes is 2, 4 or 6.

10. The method of claim 8, wherein the nucleic acid of the target gene is DNA or RNA.