CN114592037A - Method for isothermal amplification of target nucleic acid sequence - Google Patents
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Abstract
The invention discloses a method for isothermal amplification of a target nucleic acid sequence, and relates to the technical field of nucleic acid amplification. It includes: designing forward amplification primers F1 and F2 respectively; blocking primers B1 and B2; the split primers D1 and D2 of the forward amplification primer; reverse amplification primers R1 and R2; and stripping primers RS1 and RS2 of the reverse amplification product. Isothermal and synchronous amplification of two target genes is completed by utilizing the interactive 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, biological safety and the like.
Description
Technical Field
The invention relates to the technical field of nucleic acid amplification, in particular to a method for isothermal amplification of a target nucleic acid sequence.
Background
With the development of molecular biology technology, especially the rapid development of PCR detection technology and various universal detection technologies depending on the development of PCR detection technology. The human disease detection, animal and plant inspection and quarantine and various scientific research technical fields are brought into the space-time of molecular detection. Meanwhile, the synchronous appearance of the PCR detection technology and the NGS detection technology brings revolutionary revolution to a plurality of fields, particularly the precise medical field, such as drug resistance detection of human infectious diseases, tumor mutation detection, concomitant detection of hypertension drugs, genetic disease detection and the like. In addition, with the rapid spread of new coronavirus, the PCR technology has been successful in providing new coronavirus nucleic acid detection field, and becomes the most important nucleic acid detection technology in the world. Although the PCR detection technology has obvious advantages, the PCR detection technology depends on a high-added-value fluorescent quantitative PCR instrument, professional supporting facilities and professional supporting personnel. The construction cost of the PCR detection technology platform is high, the flexibility of the application of the PCR detection technology is influenced, and the PCR detection technology is influenced to sink into community hospitals and families.
Isothermal amplification technology is a new nucleic acid amplification technology developed in recent years based on isothermal amplification. The technology can rapidly realize the functions of copying and detecting 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 fluorescence detection device. The detection can also be carried out by adopting a visual interpretation mode such as a colorimetric method, a colloidal gold method and the like. Therefore, once the technology is available, it is considered by many scholars as a detection method which is possibly comparable to PCR. In addition, with the continuous maturation of the CRISPR technology, the deep combination of the CRISPR technology and the isothermal amplification technology makes the isothermal amplification detection technology one of the most possible alternatives to the PCR technology. In conclusion, with the continuous evolution and upgrade iteration of isothermal nucleic acid amplification technology and the emerging of matched diagnostic instruments, isothermal amplification technology is believed to be an important branch technology of nucleic acid detection in the future nucleic acid detection field. The following is a summary of the major isothermal amplification techniques available.
1. Loop-mediated Isothermal Amplification (LAMP).
The loop-mediated isothermal amplification method was a novel isothermal nucleic acid amplification method developed by Rongshi, Japan, 2000.
Has the characteristics of simple and rapid operation, strong specificity, easy detection of products and the like. Mainly designs 4 specific primers aiming at 6 regions of a target gene, and then completes biochemical reactions such as gene template amplification, primer extension, single-strand displacement and the like under the conditions of Bst DNA polymerase and constant-temperature amplification at 60-65 ℃. The loop-mediated isothermal amplification method has very rapid reaction, and the nucleic acid amplification of 10^9 to 10^10 times can be realized by the template within 15 to 60 minutes. Compared with the PCR method, the method does not need complex processes such as thermal denaturation, temperature cycling, fluorescence detection and the like of the template. After the isothermal amplification reaction is finished, the pyrophosphate ions precipitated from dNTPs in the nucleic acid amplification process react with the magnesium ions in the reaction solution to generate magnesium pyrophosphate precipitate color reaction. Finally, whether amplification is carried out or not is identified by naked eyes through turbidity reaction, and detection is carried out without an instrument. The loop-mediated isothermal amplification technology has wide application prospect because a PCR instrument and expensive reagents are not needed.
2. Recombinase Polymerase Amplification (RPA).
The recombinase polymerase amplification technique was a proprietary technique developed successfully in 1999 by twist dx corporation of uk. Is praised as a revolutionary innovation in the field of DNA diagnosis. The recombinase polymerase amplification technology can realize the nucleic acid detection of trace nucleic acid molecules by only using 10-20 minutes under the condition of lower constant temperature. The technology has low requirements on environment and hardware facilities, and has good application prospects in the aspects of biological protection, water body inspection, food inspection, medical diagnosis, microfluidics, veterinarian and the like.
Recombinase polymerase amplification techniques rely primarily on three enzymes: recombinases capable of binding single-stranded nucleic acids, single-stranded DNA binding proteins, and strand displacing DNA polymerases. The principle of recombinase polymerase amplification technology includes that a recombinase is combined with a primer to form a protein-DNA complex, the protein-DNA complex can search for a homologous sequence in a target gene, and once the homologous sequence is found, a strand exchange reaction occurs and DNA replication is carried out, wherein the replication mode adopts exponential amplification. At the same time, the displaced DNA single strand binds to SSB, preventing further displacement. In the recombinase polymerase amplification technology system, the whole reaction process is very rapid, and detectable level of amplification products can be obtained within 10-20 minutes. Meanwhile, the methodology can also be combined with a probe detection technology related to PCR to finish the detection of the target gene.
3. Rolling Circle Amplification (RCA).
The rolling circle amplification technology is a nucleic acid isothermal amplification technology mode established by scientists by taking the reference of the rolling circle replication principle of circular pathogenic microorganism DNA molecules in nature in the 90 s. The technology is an isothermal nucleic acid amplification method based on ligase connection, primer extension and strand displacement amplification reaction. The technology mainly depends on a circular DNA molecule as a template, and dNTPs are converted into single-stranded DNA through a short DNA primer which is complementary with the circular template under the catalysis of phi29DNA polymerase, wherein 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, and has great application value and potential in the field of nucleic acid molecule detection.
4. Cross Primer Amplification (CPA).
The cross primer amplification technology is a novel nucleic acid isothermal amplification technology which is successfully independently developed by Hangzhou Yosida company in 2008 and is also the 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 relying on 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 of the cross primers, the cross primers are firstly complementarily combined with a template strand respectively and then extended, then the stripping primers strip a newly synthesized single strand under the action of Bst DNA polymerase, and finally the cross primers synthesize a large number of target fragments by taking the newly synthesized single strand as the template under the action of the Bst DNA polymerase.
5. SDA Strand Displacement Amplification (SDA).
The strand displacement amplification technology is a DNA in vitro isothermal amplification technology based on enzymatic reaction, and the main principle is that firstly, restriction enzyme is utilized to cut a DNA recognition site, then DNA polymerase is utilized to extend to 3' at the cut and displace a downstream sequence, and amplification is carried out under isothermal condition. 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 circularly amplifying SDA.
6. HDA helicase-dependent amplification techniques.
Helicase-dependent amplification (HDA) was invented in 2004 by NEB, USA as a novel isothermal amplification technique for nucleic acids. The technology utilizes the characteristic that helicase can unwind double helix of DNA under the condition of constant temperature, and then SSB single-strand binding protein is attached to the single strand to stabilize the unwound single strand of DNA, thereby providing a copy template for a primer. Then, the complementary double strand is replicated and synthesized by the action of DNA polymerase. Repeating the cyclic 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 and the like generally exist in the nucleic acid isothermal amplification technology. In the field of isothermal amplification detection, the method has the problems of frequently encountered false negative and false positive results and the difficulty of synchronous real-time detection of multiple targets.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The object of the present invention is 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 and the like universally existing in the existing nucleic acid isothermal amplification technology on the principle and path. Therefore, the problem that in the field of isothermal amplification detection, false negative results and false positive results which are frequently subjected to fouling are identified is solved, and the problem of synchronous real-time detection of multiple targets is 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 for automatic realization and the like. Meanwhile, the detection methodology of the invention can adopt artificial interpretation methods such as colloidal gold, turbidimetry and the like. Automatic result output can also be accomplished using small detection instruments, such as fluorescence detectors. And a full-automatic micro-fluidic chip detection instrument can be adopted to complete automatic detection and the like. The invention is suitable for qualitative and quantitative detection in the fields of medicine, genetics, inspection and epidemic detection and the like.
The invention is realized by the following steps:
the present invention provides a method for isothermal amplification of a target nucleic acid sequence, which method is aimed at non-disease diagnosis, comprising:
a. designing forward amplification primers F1 and F2; it includes:
forward amplification primers F1 and F2 are respectively paired with a target sequence T1 and a target sequence T2 to carry out extension amplification, reverse sequences RS2 and RS1 are respectively designed at the 5' ends of the forward amplification primers F1 and F2, and RS2 is complementary to the SD2 sequence of the target sequence T2; RS1 is complementary to the 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 includes:
designing blocking primers B2 and B1 at the upstream of SD2 and SD1 respectively, and completing first round replication amplification by finishing extension when nucleic acid amplification extension is carried out at the downstream positions of SD1 and SD2 respectively through forward amplification primers F1 and F2 and blocking primers B1 and B2 (refer to the schematic diagram of first-stage amplification shown in FIG. 1);
c. designing stripping primers D1 and D2 of the forward amplification primer; it includes:
respectively designing stripping primers D1 and D2 of the forward amplification primers at the upstream of the forward amplification primers F1 and F2, and using the stripping primers to replace the first round amplification product to obtain a single strand of the first round amplification product;
after head-to-tail complementary hybridization of the first round amplification product, a Loop structure is formed, and a template is prepared for subsequent amplification (refer to a schematic diagram of second-stage amplification 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. d, 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; so that a Loop structure is formed again after head-to-tail complementary hybridization of the new amplification product (refer to a third-stage amplification schematic diagram shown in fig. 3);
f. amplifying the target sequence T1 and the target sequence T2 in a linear mode or a higher-order structure mode, forming an amplification extension mode through amplification extension-strand displacement-Loop, and circulating to make the two amplification target sequences synchronously carry out nucleic acid amplification.
The isothermal Amplification method provided by the present invention is also referred to as Sequence Interaction mediated (SIA) isothermal Amplification.
The invention is specifically based on two target positions of a target gene, and firstly designs two initial amplification primers, two stripping primers and a pair of blocking primers. The first round (namely the first chain) is copied and stripped under the action of enzyme, and the heads and the tails are complemented to form Loop similar to Chinese lantern. Then, aiming at the single-stranded region of Loop, two reverse primers and two stripping primers are designed to complete the copying of a complementary strand, and a double-stranded DNA is formed. And forming Loop again by the double-stranded DNA, and performing amplification circularly to ensure that the two amplification targets synchronously perform nucleic acid amplification.
The method is suitable for nucleic acid amplification in the fields of medical detection, biological scientific research, agricultural scientific research, epidemic prevention and control, 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. Generally, the method provided by the invention has the advantages of low precision requirement on equipment, low detection cost, high reagent sensitivity, high reagent specificity, multi-target detection and the like.
The enzyme used for amplification in the above method is selected from a DNA polymerase and/or a reverse transcriptase. For example, a single enzyme system selected from a DNA polymerase or a reverse transcriptase is used for amplification or reverse transcription, and a combined enzyme system selected from a DNA polymerase and a reverse transcriptase is used for reverse transcription amplification.
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 contained in the same or different systems.
The enzymes (reverse transcriptase and/or DNA polymerase) in the system are present at working concentrations.
In a preferred embodiment of the present invention, the DNA polymerase used for amplification in the above method includes at least one of the following enzymes: bst DNA polymerase, Klenow DNA polymerase, T4DNA polymerase, Taq DNA polymerase, DNA polymerase I, Vent DNA polymerase, Phi29DNA polymerase, TneDNA polymerase, Tma DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Stoffel fragment DNA polymerase and DEEPVENT DNA polymerase and mutants, variants or derivatives thereof.
In a preferred embodiment of the present invention, the reverse transcriptase used for amplification in the above method includes 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 present 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 present invention, the amplification reaction in the above method is isothermal amplification;
in an alternative embodiment, amplification is performed at 55-72 ℃; in an alternative embodiment, the amplification time is 15-90 min.
Amplification temperatures are 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 20-80 min.
In a preferred embodiment of the present invention, the blocking primers B1 and B2 are further provided with blocking groups at the 3' ends;
in an alternative embodiment, the blocking group includes, but is not limited to, any of C3Spacer, C6 Spacer, C12Spacer, Spacer18, amino, dideoxy, base inversion, BHQ1, BHQ2, and MGB.
In a preferred embodiment of the present invention, the number of the 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 high-level structure is: amplification is performed in a secondary structure, or in a mixture of a linear structure and a secondary structure.
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 fungus 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 for detecting a pathogen of an infectious disease of a human, animal or plant.
In another embodiment, the invention provides the use of a method for amplifying a target nucleic acid sequence for detecting an infectious agent in a food or biological weapon.
In another embodiment, the present invention provides the use of a method for amplifying a target nucleic acid sequence for the detection of a human genetic disease or health risk gene and the evaluation thereof.
The invention has the following beneficial effects:
the method for isothermal amplification of the target nucleic acid sequence has the advantages of simple and easy design, high detection sensitivity, strong specificity, good stability and high repeatability, and can be used for qualitative analysis 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 and control 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 needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of a first stage amplification;
FIG. 2 is a schematic diagram of a second stage amplification;
FIG. 3 is a schematic diagram of a third stage amplification;
FIG. 4 shows the site and design of tuberculosis primer design;
FIG. 5 is the electrophoresis result of the lowest detection limit reference of Mycobacterium tuberculosis (lanes 1-5 are negative controls, respectively, lanes 1.0E +03, 1.0E +02, 1.0E +01, 1.0E +00, and 1.0E + 00);
FIG. 6 is a diagram showing the result of electrophoresis of a repetitive reference sample of Mycobacterium tuberculosis (lanes 1-2 are negative controls, 3-5 are 3 repetitions of 1.0E +02 bacteria/ml sample, respectively);
FIG. 7 shows the design site and design diagram of primer for hepatitis C;
FIG. 8 shows the electrophoresis results of the reference samples for hepatitis C, and lanes 1-10 show the detection results of the reference samples for negative samples N1-N10;
FIG. 9 shows the results of electrophoresis of positive reference samples of hepatitis C, lanes 1-7 show the results of detection of positive reference samples P1-P7, respectively;
FIG. 10 shows the electrophoresis results of the repetitive reference samples of hepatitis C, lanes 1-3 are the repetitive reference samples for 3 times, and lane 4 is the negative control result;
FIG. 11 shows the results of electrophoresis of a sensitivity reference for hepatitis C, lanes 1-5 are the results of 5 repeated detections of the sensitivity reference, and lanes 6-8 are the results of negative controls.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a method for the qualitative detection of Mycobacterium tuberculosis nucleic acid.
1. Preparation of tuberculosis nucleic acid samples
Taking a tubercle midge reference plate as a sample to be detected (a minimum detection limit reference substance: 1.0E +03 bacteria/ml, 1.0E +02 bacteria/ml, 1.0E +01 bacteria/ml, 1.0E +00 bacteria/ml, a repeatability reference substance: 1.0E +02 bacteria/ml). Then, nucleic acid purification was carried out using a "magnetic bead method universal genomic DNA extraction kit" manufactured by Tiangen Biochemical technology (Beijing) Ltd. The specific operation is carried out by referring to the product specification.
2. Tuberculosis isothermal primer design
The homology alignment was performed with "insertion sequence: IS6110" of the tuberculosis genome (Genbank No: Y14048) to determine the primer homology design region, referring to the primer design site shown in FIG. 4. Then, isothermal amplification primers were designed according to the primer design method of the present invention, and the primer sequences are shown in table 1.
TABLE.1: tuberculosis primer sequence
| Primer name | Sequence (5 '-3') |
| F1/RS2 | TGGCTGCGCGGAGACGtccgaatcgtgctgaccg |
| F2/RS1 | GTACCCGCCGGAGCTGCGtcgtggtcccgggccgtg |
| R1 | GTACCCGCCGGAGCTGCGTGAGCGGGCGGTGCGG |
| R2 | TGGCTGCGCGGAGACGGTGCGTAAGTGGGTGC |
| B1 | cctcgatgaaccacctga(C3Spacer) |
| B2 | caccaagtagacgggcga(C3Spacer) |
| D1 | tcactgatcgctgcccac |
| D2 | tcagcggattcttcgg |
| RS1 | cgcagctccggcgggtac |
| RS2 | cgtctccgcgcagcca |
3. Preparation of isothermal amplification reaction solution
The preparation of the components of the isothermal amplification reaction solution was completed as shown in 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
| Composition of | Volume of |
| 10×Buffer | 2.5μl |
| dNTPs (containing dATP, dCTP, dGTP, dTTP, each 2mM) | 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 application and amplification
Adding 5 μ l of the extracted nucleic acid sample into a PCR reaction tube containing the isothermal amplification reaction solution, covering the tube cover tightly, and placing the tube cover in a PCR instrument or a water bath for amplification. The amplification conditions were 63 ℃ for 60 min. After amplification was complete, electrophoresis was then performed on a 1.5% agarose gel for 20min at 120V and 90A. And finally, carrying out imaging detection by using a gel imager.
The experimental results were analyzed as follows:
reference product of lowest detection limit: the invention is used for detecting the minimum detection limit reference substances (1.0E +03 bacteria/ml, 1.0E +02 bacteria/ml, 1.0E +01 bacteria/ml and 1.0E +00 bacteria/ml) in the check hospital, wherein 1.0E +00 bacteria/ml samples are repeated once. And finally, detecting by means of 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.
(II) good sample repeatability: the invention is used for carrying out three times of repeated detection on the repeated reference substance (1.0E +02 bacteria/ml) of the tuberculosis country, and finally, the detection is carried out in an agarose gel electrophoresis mode. 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 hepatitis C nucleic acid qualitative detection method.
1. Preparation of hepatitis C nucleic acid sample
Taking the hepatitis C midwifery reference plate as a sample to be detected (negative and positive reference substance: N1-N10, P1-P10; minimum detection limit reference substance: 3.0 × 10)6IU/branch). Then, nucleic acid purification was carried out using a "magnetic bead method universal genomic DNA extraction kit" manufactured by Tiangen Biochemical technology (Beijing) Ltd. The specific operation is carried out by referring to the product specification.
2. Hepatitis C isothermal amplification primer design
The 5' UTR gene of hepatitis C genome (Genbank No: EU255989) was subjected to homology alignment to determine the primer homology design region, as shown in FIG. 7. Then, isothermal amplification primers were designed according to the primer design method of the present invention, 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(C3Spacer) |
| B2 | tctccaggcattgagcg(C3Spacer) |
| 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 isothermal amplification reaction solution for hepatitis C.
| Composition of | Volume of |
| 5×RT Buffer | 5.0μl |
| dNTPs (containing dATP, dCTP, dGTP, dTTP, each 2mM) | 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 application and amplification
Adding 5 μ l of the extracted nucleic acid sample into a PCR reaction tube containing the isothermal amplification reaction solution, tightly covering the tube cover, and placing in a PCR instrument or a water bath for amplification. The amplification conditions were 60 ℃ for 60 min. After amplification was complete, electrophoresis was then performed on a 1.5% agarose gel for 20min at 120V and 90A. And finally, carrying out imaging detection by using a gel imager.
The results of the experiment were analyzed as follows:
one) negative and positive reference: the invention is used for detecting the positive and negative reference substance (N1-N10, P1-P7) of the hepatitis C, and finally, the detection is carried out by an agarose gel electrophoresis mode. The detection results are shown in FIGS. 8-9. The experimental result shows that all the positive reference products of the hepatitis C are detected, and all the negative reference products are negative results. The detection method provided by the embodiment has good detection sensitivity.
II) good repeatability of the sample: firstly, the lowest detection limit reference product (3.0 multiplied by 10) is used6IU/branch), diluted with calf serum to prepare repetitive reference substance (1.0 × 10)4IU/ml), then carrying out three repeated detections on the repeated reference substance by using the method, and finally carrying out detection by means of agarose gel electrophoresis. The results are shown in FIG. 10. The experimental result shows that the detection method provided by the embodiment has good detection repeatability.
Third) minimum detection limit reference: firstly, the lowest detection limit reference product (3.0 multiplied by 10) is used6IU/branch), diluting with calf serum to prepare a reference substance with the lowest detection limit (50IU/ml), performing five-time repeated detection by using the reference substance with the lowest detection limit (50IU/ml), and finally performing detection by means of 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 a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement 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 for the purpose of non-disease diagnosis, comprising:
a. designing forward amplification primers F1 and F2; it includes:
forward amplification primers F1 and F2 are respectively paired with a target sequence T1 and a target sequence T2 to carry out extension amplification, reverse sequences RS2 and RS1 are designed at the 5' ends of the forward amplification primers F1 and F2 respectively, and the RS2 is complementary to the SD2 sequence of the target sequence T2; the RS1 is complementary 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 includes:
designing blocking primers B2 and B1 at the upstream of the SD2 and the SD1 respectively, and completing first round replication amplification by forward amplification primers F1 and F2 and blocking primers B1 and B2 to enable nucleic acid amplification extension to be at the downstream positions of the SD1 and the SD2 respectively, wherein the extension is ended;
c. designing stripping primers D1 and D2 of the forward amplification primer; it comprises the following steps:
stripping primers D1 and D2 of the forward amplification primers are designed at the upstream of the forward amplification primers F1 and F2 respectively, and are used for replacing the first round amplification product to obtain a single strand of the first round amplification product;
after head-to-tail complementary hybridization of the first round amplification product, a Loop structure is formed, and a template is prepared for subsequent amplification;
d. reverse amplification primers R1 and R2 are designed according to the target sequence T1 and the target sequence T2, and amplification is carried out by taking a Loop structure formed by the first round of amplification as a template;
e. designing stripping primers RS1 and RS2 of reverse amplification products, and replacing the amplification products of the step d to obtain new single strands; after head-to-tail complementary hybridization of a new amplification product, a Loop structure is formed again;
f. and amplifying the target sequence T1 and the target sequence T2 in a linear mode or a higher-order structure mode, forming an amplification extension mode through amplification extension-strand displacement-Loop, and circulating to make the two amplification target sequences synchronously perform nucleic acid amplification.
2. The method according to claim 1, wherein the enzyme used for amplification in the method is selected from the group consisting of a DNA polymerase and/or a reverse transcriptase.
3. The method according to claim 2, wherein the DNA polymerase used for amplification in the method 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, Phi29DNA polymerase, TneDNA polymerase, Tma DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Stoffel fragment DNA polymerase and DEEPVENT DNA polymerase.
4. The method according to claim 2, wherein the reverse transcriptase used for amplification in the method is selected from 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.
5. The method according to claim 2, wherein the enzymes used for amplification in the method are selected from the group consisting of:
bst DNA polymerase and M-MLV reverse transcriptase.
6. The method according to any one of claims 1 to 5, wherein the amplification reaction in the method is isothermal amplification;
preferably, amplification is carried out at 55-72 ℃; preferably, the amplification time is 15-90 min.
7. The method of any one of claims 1 to 5, wherein the blocking primers B1 and B2 are further provided with a blocking group at the 3' end;
preferably, the blocking group is selected from any one of C3Spacer, C6 Spacer, C12Spacer, Spacer18, amino, dideoxy, base inversion, BHQ1, BHQ2 and MGB.
8. The method according to any one of claims 1 to 5, wherein the number of target genes is a multiple of 2;
preferably, the number of target genes is 2, 4 or 6.
9. The method of claim 1, wherein the high level structure is: amplification is performed in a secondary structure, or in a mixture of a linear structure and a secondary structure.
10. The method of any one of claims 1 to 5, wherein the nucleic acid of the target gene is DNA or RNA.
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