A lambda exonuclease-mediated RPA amplification-detection integrated SNP detection method

Abstract

The application discloses a kind of based on lambda exonuclease mediated RPA amplification-detection integrated SNP detection method.The method innovatively constructs "amplification-detection synchronization" reaction system, mismatch recognition cutting characteristics of lambda exonuclease, the isothermal high-efficiency amplification capacity of RPA and the fluorescence signal response function of specific probe are organically fused, without step operation, only through single isothermal reaction can complete SNP site recognition, target amplification and signal detection.The method solves the problems of existing SNP detection, such as complicated operation, low reaction efficiency and easy pollution, without PCR instrument and step reagent addition, total detection time is less than or equal to 40 min, detection limit can reach 100 copies / μL, specificity is greater than or equal to 99%, and wild type and mutant SNP sites can be accurately distinguished.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically involving an integrated isothermal amplification-detection technology for single nucleotide polymorphisms (SNPs), and particularly a lambda exonuclease-mediated RPA amplification-detection integrated SNP detection method, which is particularly suitable for clinical diagnosis, molecular screening and on-site testing scenarios that require rapid, simple and accurate simultaneous amplification and detection. Background Technology

[0002] Single nucleotide polymorphisms (SNPs), as genetic markers formed by single nucleotide variations at the genomic level, are key evidence for disease diagnosis, treatment selection, and prognostic assessment. Their accurate and rapid detection is of great significance for clinical applications.

[0003] Existing SNP detection methods have significant limitations: direct sequencing is complex and time-consuming; PCR-RFLP lacks specificity and has a narrow range of applications; quantitative real-time PCR relies on thermal cycling instruments and requires step-by-step optimization of reaction conditions; conventional RPA detection methods mostly utilize CRISPR to achieve isothermal amplification of SNPs, but are limited by the constraints of protospacer adjacent motifs (PAM) or protospacer flanking sequences (PFS), and some optimization strategies also suffer from off-target effects or high costs. The recently reported SNP-SENSE system integrates recombinase polymerase amplification (RPA), Lambda exonuclease treatment, and LbuCas13a detection, but optimization strategies such as chemical modification of crRNA, although capable of improving performance, have not been applied in the platform due to cost issues, which may limit its large-scale promotion in some scenarios.

[0004] Lambda exonucleases exhibit high substrate specificity, recognizing and cleaving only perfectly complementary double-stranded DNA with 5' phosphorylation modification. They completely lose their cleavage activity against double-stranded DNA containing single-base mismatches (SNP sites). Based on this characteristic, integrating lambda exonucleases with RPA amplification and probe detection into a single reaction system, achieving simultaneous amplification and detection, could fundamentally simplify the operational process and improve detection specificity and efficiency. Summary of the Invention

[0005] To address the shortcomings of existing SNP detection methods, such as cumbersome step-by-step operations, insufficient specificity, and low detection efficiency, this invention provides an integrated SNP detection method based on lambda exonuclease-mediated RPA amplification and detection. This invention constructs a "simultaneous amplification-detection" reaction system, eliminating the need for step-by-step reagent addition and simplifying the operation process. Secondly, it utilizes the mismatch recognition characteristics of lambda exonuclease to accurately distinguish single-base mismatched SNP sites, improving detection specificity. It maintains the advantages of isothermal RPA amplification, eliminating the need for sophisticated instruments and enabling rapid detection. It achieves real-time fluorescence signal response, allowing direct determination of genotype based on the presence or absence of the signal, providing intuitive and reliable results. It is compatible with conventional nucleic acid extraction samples, meeting the diverse needs of clinical and field testing.

[0006] This invention provides an integrated SNP detection method based on lambda exonuclease-mediated RPA amplification and detection, comprising the following steps: Nucleic acid sample solution is added to the integrated reaction system to obtain integrated reaction solution. The integrated reaction solution is incubated in an isothermal environment of 37-42℃ for 15-30 min. The intensity of the associated fluorescence signal of the target SNP is monitored in real time during the reaction. If the intensity of the associated fluorescence signal of the target SNP is greater than the threshold and an obvious fluorescence growth curve appears, it is judged as positive; otherwise, it is negative. The integrated reaction system includes: a specific probe, RPA amplification primers, lambda exonuclease, and RPA reaction solution; The specific probe binds specifically to the target containing the target SNP, and a fluorescent group is modified on a T base below the 3' end of the corresponding SNP site. The RPA amplification primers include upstream and downstream primers, used to amplify the target containing the target SNP; The RPA reaction solution includes recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer.

[0007] The core of this invention lies in the construction of an "integrated reaction system," which simultaneously completes target amplification, SNP recognition, and signal detection under a single isothermal condition by mixing all reaction components at once. The RPA reaction system comprises recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer. It can achieve exponential amplification of the target sequence at 37-42°C and exhibits no mutual inhibition with lambda exonuclease or probes.

[0008] In some preferred embodiments, the specific probe is 20-35 bp in length, corresponding to the SNP site located at positions 10-15, with 5' end phosphorylation modification and 3' end labeled with a quenching group. This forms a fluorescence resonance energy transfer (FRET) system, where fluorescence is quenched when uncut and released when perfectly matched.

[0009] In some preferred embodiments, introducing a mutation site at the 3' end of the SNP site corresponding to the specific probe can reduce the fluorescence intensity of non-target samples and improve the signal-to-noise ratio.

[0010] The corresponding SNP site is located in the middle region of the probe (positions 10-15), ensuring that the stability of the double strand decreases significantly when there is a single base mismatch.

[0011] In some preferred embodiments, the fluorescent group is selected from FAM, HEX, and ROX.

[0012] In some preferred embodiments, the quenching group is selected from BHQ1 and BHQ2.

[0013] In some preferred embodiments, the upstream or downstream primer is 30-35 bp in length. The upstream or downstream primer avoids overlapping with the probe sequence or forming secondary structures, ensuring that the primer and probe can bind to the target sequence synchronously. The final primer concentration in the integrated reaction solution is 0.1-0.5 μM, with an optimal concentration of 0.2 μM, ensuring efficient RPA amplification.

[0014] In some preferred embodiments, the lambda exonuclease has an activity ≥5 U / μL, cleaves only perfectly complementary probe-target double strands, and has no cleavage activity against mismatched double strands. The final concentration of the lambda exonuclease in the reaction solution is 0.5-2 U / reaction.

[0015] In some preferred embodiments, each 50 μL integrated reaction solution includes nucleic acid sample solution, 1 μL lambda exonuclease, 31.9 μL RPA reaction solution, a specific probe, an RPA upstream primer, and an RPA downstream primer, with water added to bring the volume to 50 μL. The concentration of the specific probe in the integrated reaction solution is 0.1-0.5 μM, and the concentrations of the RPA upstream and downstream primers are 0.1-0.5 μM. The amount of nucleic acid sample added can be adjusted according to actual conditions, and the integrated reaction solution can be brought to 50 μL by adding water.

[0016] In some preferred embodiments, the target nucleic acid sample solution has a concentration ≥10 copies / μL and a purity OD260 / OD280 of 1.8-2.0. The target nucleic acid sample can be derived from various sample types such as swabs, blood, saliva, and tissue.

[0017] In some preferred embodiments, the incubation temperature is 38°C and the time is 20 minutes.

[0018] In some more specific embodiments, the integrated SNP detection method specifically includes the following steps: 1. Sample nucleic acid preparation Clinical samples (such as genital swabs, blood, saliva, tissue samples, etc.) are processed using conventional nucleic acid extraction methods (such as column extraction, magnetic bead extraction, boiling lysis, etc.) to obtain purified nucleic acid sample solutions (DNA or RNA, RNA needs to be reverse transcribed into cDNA); the nucleic acid sample solution concentration is ≥10 copies / μL, and the purity OD260 / OD280 is 1.8-2.0, avoiding impurities from interfering with the enzymatic reaction.

[0019] 2. Construction of integrated reaction system and isothermal reaction Construct a 50 μL integrated reaction solution with the following components and final concentrations: nucleic acid sample solution (quantitative), 1 μL lambda exonuclease, 31.9 μL RPA reaction solution, specific probe, RPA upstream primer, RPA downstream primer, and add water to 50 μL; the concentration of the specific probe in the integrated reaction solution is 0.1-0.5 μM, and the concentrations of the RPA upstream and downstream primers are 0.1-0.5 μM.

[0020] Incubate the reaction solution in an isothermal environment of 37-42℃ for 15-30 minutes. The optimal incubation temperature is 38℃ and the optimal incubation time is 20 minutes.

[0021] 3. Result Judgment The fluorescence signal intensity was monitored in real time using a fluorescence detector, with the fluorescence signal read every 30 seconds. The threshold was set as the negative control fluorescence value + 3 standard deviations: if the sample fluorescence signal intensity was ≥ the threshold and a clear fluorescence growth curve appeared, it was judged as positive (the sample contains the target SNP site, and the probe and target are perfectly matched); if the fluorescence signal intensity was < the threshold, it was judged as negative (the sample does not contain the target SNP site, and the probe and target are mismatched).

[0022] In another aspect, the present invention provides a specific probe and primers for detecting the DEFB126 wild-type recombinant plasmid, which are applicable to the above-mentioned integrated SNP detection method; The nucleotide sequence of the specific primer probe is shown in SEQ ID No: 7. The 5' end is phosphorylated, corresponding to the SNP site located at 15BP. The T base at 21BP is modified with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group. The nucleotide sequence of the upstream primer for RPA is shown in SEQ ID No:4; The nucleotide sequence of the downstream primer for RPA is shown in SEQ ID No:5.

[0023] The core principles of this invention lie in the following three points. The recombinase in the RPA reaction solution binds to the primers, rapidly scanning the target nucleic acid and unwinding the double strand. Single-strand binding proteins stabilize the single-strand structure, and DNA polymerase synthesizes a new strand using the target as a template, achieving exponential amplification of the target sequence. During amplification, the probe simultaneously binds to the newly synthesized target strand. If the target and probe form a completely complementary double-stranded DNA, the lambda exonuclease recognizes the phosphorylation modification at the 5' end of the probe and specifically cleaves it, separating the fluorescent group from the quenching group and continuously releasing a fluorescent signal. If there is an SNP mismatch between the target and probe, forming a mismatched double-stranded DNA, the lambda exonuclease cannot cleave the probe, and the fluorescent signal remains quenched (no signal). As RPA amplification continues, the target product accumulates, probe binding and cleavage reactions continue, and the fluorescent signal increases synchronously with the amplification process, achieving "integrated amplification and detection."

[0024] The present invention has the following beneficial effects: 1. Integrated amplification and detection, extremely simple operation: No need to add reagents step by step, all components are mixed at once, and the entire process is completed by a single isothermal reaction, avoiding sample contamination, reducing the difficulty of operation, and suitable for non-professionals and on-site testing.

[0025] 2. Extremely specific and precise: Through screening with "lambda exonuclease mismatch inertness + probe specific binding + RPA primer specificity", only perfectly matched targets generate fluorescent signals, while mismatches produce no signal, solving the problem of single base mismatch identification.

[0026] 3. Fast and efficient: The entire reaction is isothermal (37-42℃), and the total detection time is ≤40min, which is 1 / 3 shorter than the conventional step-by-step detection, meeting the needs of emergency and rapid testing.

[0027] 4. Intuitive signal response: The fluorescence signal is directly related to the target matching state, eliminating the need for complex data analysis. The results can be determined with a simple fluorescence detector, and the results are reliable and easy to read.

[0028] 5. High sensitivity: RPA amplification can amplify the target sequence by 10^6-10^9 times. Combined with the specific cleavage signal amplification of lambda exonuclease, the detection limit can reach as low as 10 copies / μL, which can effectively detect low abundance target nucleic acids.

[0029] 6. Wide adaptability: Compatible with conventional nucleic acid extraction technology, it can be used for various sample types such as swabs, blood, saliva, and tissue. By changing the probe and primer, it can achieve detection of any SNP site, and its application scenarios cover multiple fields such as genetic diseases, tumors, and pharmacogenomics.

[0030] 7. Cost-controllable: No expensive thermal cycling instruments and special reagents are required. The reaction conditions are mild, making it suitable for large-scale application in primary healthcare institutions and resource-limited on-site testing scenarios. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0032] Figure 1 This is a real-time fluorescence signal monitoring diagram of wild-type plasmid-specific probe 1 in Example 1; Figure 2 This is a real-time fluorescence signal monitoring diagram of wild-type plasmid-specific probe 2 in Example 2; Figure 3 This is a real-time fluorescence signal monitoring diagram of wild-type plasmid-specific probe 3 in Example 2; Figure 4 This is a real-time fluorescence signal monitoring diagram of wild-type plasmid-specific probe 4 in Example 2; Figure 5 This is a real-time fluorescence signal monitoring graph of the sensitivity test of wild-type plasmid-specific probe 3 in Example 3. Detailed Implementation

[0033] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0034] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] An integrated SNP detection method specifically includes the following steps: 1. Sample nucleic acid preparation Clinical samples (such as genital swabs, blood, saliva, tissue samples, etc.) are processed using conventional nucleic acid extraction methods (such as column extraction, magnetic bead extraction, boiling lysis, etc.) to obtain purified nucleic acid sample solutions (DNA or RNA, RNA needs to be reverse transcribed into cDNA); the nucleic acid sample solution concentration is ≥10 copies / μL, and the purity OD260 / OD280 is 1.8-2.0, avoiding impurities from interfering with the enzymatic reaction.

[0037] 2. Construction of integrated reaction system and isothermal reaction Construct a 50 μL integrated reaction solution with the following components and final concentrations: 10 μL nucleic acid sample solution (adjustable according to specific conditions, add water to the integrated reaction solution to bring it to 50 μL), 2 μL specific probe (4 μM), 1 μL lambda exonuclease (NEB, M0262S, 0.5-2 U / reaction), 2 μL RPA upstream primer (5 μM), 2 μL RPA downstream primer (5 μM), 31.9 μL RPA reaction solution (containing recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer), and add water to bring it to 50 μL.

[0038] Incubate the reaction solution in an isothermal environment of 37-42℃ for 15-30 minutes. The optimal incubation temperature is 38℃ and the optimal incubation time is 20 minutes.

[0039] The specific probe can specifically bind to the target sequence, which contains the target SNP. The specific probe is 20-35 bp in length, corresponding to the SNP at positions 10-15. It is phosphorylated at the 5' end, and a fluorescent group is modified on the T base below the 3' end of the SNP site. A quenching group is labeled at the 3' end, forming a fluorescence resonance energy transfer (FRET) system. Fluorescence is quenched when uncut, and cleaved and released when perfectly matched. The fluorescent group is selected from FAM, HEX, and ROX. The quenching group is selected from BHQ1 and BHQ2. The corresponding SNP site is located in the middle region of the probe (positions 10-15), ensuring a significant decrease in double-strand stability in the event of a single-base mismatch.

[0040] RPA upstream and downstream primers are used to amplify the target sequence. The length of the RPA upstream and downstream primers is 30-35 bp. The RPA upstream and downstream primers avoid overlapping with the probe sequence or forming secondary structures, ensuring that the primers and probe can bind to the target sequence synchronously. The final primer concentration in the reaction solution is 0.1-0.5 μM, with an optimal concentration of 0.2 μM, ensuring efficient RPA amplification.

[0041] The RPA reaction solution includes recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer. This RPA reaction system can achieve exponential amplification of the target sequence at 37-42℃, and has no inhibitory interaction with lambda exonuclease or probe.

[0042] The RPA reaction solution was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., with product number WLB8201KIT.

[0043] The lambda exonuclease has an activity ≥5 U / μL, and cleaves only perfectly complementary probe-target double strands, exhibiting no cleavage activity against mismatched double strands. The final concentration of the lambda exonuclease in the reaction solution is 0.5-2 U / reaction.

[0044] 3. Result Judgment The fluorescence signal intensity was monitored in real time using a fluorescence detector, with the fluorescence signal read every 30 seconds, or at the endpoint fluorescence value after the reaction was completed. A threshold was set at the negative control fluorescence value + 3 standard deviations: if the sample fluorescence signal intensity was ≥ the threshold and a clear fluorescence growth curve appeared, it was considered positive (the sample contained the target SNP site, and the probe and target were perfectly matched); if the fluorescence signal intensity was < the threshold, it was considered negative (the sample did not contain the target SNP site, and the probe and target were mismatched).

[0045] Example 1. Feasibility verification of the integrated reaction system 1. Experimental Objective The compatibility and feasibility of the integrated reaction system of "RPA amplification-probe detection-lambda exonuclease cleavage" were verified, and it was confirmed that the synchronous reaction can achieve SNP site identification.

[0046] 2. Experimental Materials Target nucleic acids: DEFB126 wild-type recombinant plasmid (sequence shown in SEQ ID No:1) and DEFB126 mutant recombinant plasmid (sequence shown in SEQ ID No:2), with the mutation site being 152: T>C.

[0047] To target the SNP site of the DEFB126 wild-type recombinant plasmid, this embodiment designed a wild-type plasmid-specific probe 1 and RPA amplification primers according to conventional methods in the art. The nucleotide sequence of the wild-type plasmid-specific probe 1 is shown in SEQ ID No:3, with 5' phosphorylation modification, corresponding to the SNP site at 15BP, a FAM fluorescent group modified on the T base at 22BP, and a BHQ1 quencher group labeled at the 3' end; the nucleotide sequence of the RPA upstream primer is shown in SEQ ID No:4, and the nucleotide sequence of the RPA downstream primer is shown in SEQ ID No:5. The structures of each target nucleic acid, specific probe, and RPA upstream and downstream primer sequences are shown in Table 1.

[0048] Table 1 The preferred reactivity of lambda exonuclease is 1 U / reaction.

[0049] RPA reaction reagents were purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., with product number WLB8201KIT.

[0050] Instruments: Isothermal fluorescence detector, pipette.

[0051] 3. Experimental Methods Construct a 50 μL integrated reaction solution: Add 10 μL of wild-type or mutant plasmid, 2 μL of wild-type plasmid-specific probe 1 (4 μM), 1 μL of lambda exonuclease (NEB, M0262S, 0.5-2 U / reaction), 2 μL of RPA upstream primer (5 μM), 2 μL of RPA downstream primer (5 μM), and 31.9 μL of RPA reaction solution (containing recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer) to two reaction tubes respectively, and add water to make up to 50 μL.

[0052] • Reaction conditions: Incubate at 38℃ for 20 min, monitor fluorescence signal intensity in real time, and read fluorescence signal every 30 s.

[0053] 4. Experimental Results Test results as follows Figure 1 The wild-type plasmid sample showed strong fluorescence signal and obvious growth curve, proving that RPA amplification, probe binding and lambda exonuclease cleavage can be carried out simultaneously and efficiently in the integrated system; the mutant plasmid showed weak fluorescence signal after 15 min, proving that lambda exonuclease cleavage of the probe-target double strand is completely matched, while cleavage of the probe-target double strand is inefficient, and the feasibility of the integrated system meets the design requirements.

[0054] Example 2. Probe Design Optimization 1. Experimental Objective More mutation sites were artificially introduced at the 1st, 2nd, and 3rd BP positions (i.e., 16th, 17th, and 18th BP of the sequence) at the 3' end of the SNP site corresponding to wild-type plasmid-specific probe 1, respectively, to obtain wild-type plasmid-specific probe 2, wild-type plasmid-specific probe 3, and wild-type plasmid-specific probe 4. The sequence structures are shown in Table 2, which reduces the detection signal of mutants.

[0055] Table 2 2. Experimental Materials Target nucleic acids: DEFB126 wild-type recombinant plasmid (sequence shown in SEQ ID No:1), DEFB126 mutant recombinant plasmid (sequence shown in SEQ ID No:2), with the mutation site being 152: T>C; To address the SNP sites of the DEFB126 wild-type recombinant plasmid, this embodiment further introduces mutation sites based on wild-type plasmid-specific probe 1 from Example 1. A mutation site is artificially introduced at the 1st BP position (i.e., 16 BP of the sequence) at the 3' end of the SNP site corresponding to wild-type plasmid-specific probe 1, resulting in wild-type plasmid-specific probe 2; mutation sites are artificially introduced at the 1st and 2nd BP positions (i.e., 16th and 17th BP of the sequence) at the 3' end of the SNP site corresponding to wild-type plasmid-specific probe 1, resulting in wild-type plasmid-specific probe 3; and mutation sites are artificially introduced at the 1st, 2nd, and 3rd BP positions (i.e., 16th, 17th, and 18th BP of the sequence) at the 3' end of the SNP site corresponding to wild-type plasmid-specific probe 1, resulting in wild-type plasmid-specific probe 4.

[0056] The preferred reactivity of lambda exonuclease is 1 U / reaction.

[0057] The RPA reaction reagent was purchased from Anpu Future (Changzhou) Biotechnology Co., Ltd., product number WLB8201KIT.

[0058] Instruments: Isothermal fluorescence detector, pipette.

[0059] 3. Experimental Methods Constructing a 50 μL integrated reaction system: Add 10 μL of wild-type or mutant plasmid to each reaction tube, along with 2 μL of wild-type plasmid-specific probe 2-4 (4 μM) (SEQ ID No: 6 or SEQ ID No: 7 or SEQ ID No: 8), 1 μL of lambda exonuclease (NEB, M0262S, 0.5-2 U / reaction), 2 μL of RPA upstream primer (5 μM), 2 μL of RPA downstream primer (5 μM), and 31.9 μL of RPA reaction solution (containing recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer). Add water to bring the volume to 50 μL.

[0060] Reaction conditions: Incubate at 38℃ for 20 min, monitor fluorescence signal intensity in real time, and read fluorescence signal every 30 s.

[0061] 4. Results Analysis When a mutation site is artificially created at the first base at the 3' end of the SNP site corresponding to the specific probe ( Figure 2 Wild-type plasmid samples showed strong fluorescence signals and obvious growth curves, while mutant plasmids showed no fluorescence signals. When mutation sites were artificially created at the first and second bases at the 3' end of the SNP site corresponding to the specific probe (…),… Figure 3Wild-type plasmid samples showed fluorescence signals and a clear growth curve, with the highest fluorescence intensity at the endpoint, while mutant samples showed the lowest fluorescence intensity, with a specificity ratio of 11.44, representing the optimal concentration. When mutation sites were artificially created at the 1st, 2nd, and 3rd bases at the 3' end of the SNP site corresponding to the specific probe (…),… Figure 4 The fluorescence signal in wild-type plasmid samples appeared significantly later. Introducing too many mutation sites artificially reduced probe matching efficiency and cleavage efficiency, resulting in a delayed fluorescence signal. Introducing too few mutation sites artificially may cause non-specific cleavage, leading to increased fluorescence intensity and decreased specificity in mutant samples.

[0062] Example 3. Sensitivity verification of the detection method 1. Experimental Objective The lowest detection limit of the method of the present invention was determined, and its ability to detect low-abundance target nucleic acids was verified.

[0063] 2. Experimental Materials Target nucleic acids: DEFB126 wild-type recombinant plasmid (sequence shown in SEQ ID No:1), serially diluted to 10^4, 10^3, 10^2, 10^1, 10^0 copy / μL; DEFB126 mutant recombinant plasmid (sequence shown in SEQ ID No:2), with the mutation site 152: T>C.

[0064] Reagents: Wild-type plasmid-specific probe 3 SEQ ID No: 7, RPA upstream primer SEQ ID No: 4, RPA downstream primer SEQ ID No: 5, lambda exonuclease (1U / reaction), RPA reaction solution; Instrument: ABI 7500 real-time quantitative PCR instrument.

[0065] 3. Experimental Methods Construct a 50 μL integrated reaction system: Add 10 μL of wild-type plasmid, 2 μL of wild-type plasmid-specific probe 3 (4 μM), 1 μL of lambda exonuclease (NEB, M0262S, 0.5-2 U / reaction), 2 μL of RPA upstream primer (5 μM), 2 μL of RPA downstream primer (5 μM), and 31.9 μL of RPA reaction solution (containing recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer) to the reaction tube, and add water to make up to 50 μL.

[0066] • Reaction conditions: Incubate at 38℃ for 20 min, and monitor the fluorescence signal intensity in real time.

[0067] 4. Results Analysis When the plasmid concentration is greater than 10^0 copies / μL ( Figure 4 All samples tested positive, indicating that the detection limit of the method of this invention is 10 copies / μL, which can meet the detection requirements of low abundance clinical samples.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made to the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for SNP detection based on lambda exonuclease-mediated RPA amplification-detection integration, characterized in that, Includes the following steps: Nucleic acid sample solution is added to the integrated reaction system to obtain integrated reaction solution. The integrated reaction solution is incubated in an isothermal environment of 37-42℃ for 15-30 min. The intensity of the associated fluorescence signal of the target SNP is monitored in real time during the reaction. If the intensity of the associated fluorescence signal of the target SNP is greater than the threshold and an obvious fluorescence growth curve appears, it is judged as positive; otherwise, it is negative. The integrated reaction system includes: a specific probe, RPA amplification primers, lambda exonuclease, and RPA reaction solution; The specific probe binds specifically to the target containing the target SNP, and a fluorescent group is modified on a T base below the 3' end of the corresponding SNP site. The RPA amplification primers include upstream and downstream primers, used to amplify the target containing the target SNP; The RPA reaction solution includes recombinase, single-stranded binding protein, DNA polymerase, dNTPs, and reaction buffer.

2. The SNP detection method according to claim 1, characterized in that, The specific probe is 20-35 bp in length, with the corresponding SNP site located at position 10-15, phosphorylated at the 5' end, and labeled with a quenching group at the 3' end.

3. The SNP detection method according to claim 1, characterized in that, The fluorescent group is selected from one of FAM, HEX, and ROX.

4. The SNP detection method according to claim 2, characterized in that, The quenching group is selected from one of BHQ1 and BHQ2.

5. The SNP detection method according to claim 1, characterized in that, The upstream or downstream primer is 30-35 bp in length.

6. The SNP detection method according to claim 1, characterized in that, The lambda exonuclease has an activity ≥5 U / μL and cleaves only perfectly complementary probe-target double strands, with no cleavage activity against mismatched double strands.

7. The SNP detection method according to claim 1, characterized in that, Each 50 μL integrated reaction solution includes nucleic acid sample solution, 1 μL lambda exonuclease, 31.9 μL RPA reaction solution, specific probe, RPA upstream primer, RPA downstream primer, and water added to 50 μL; the concentration of the specific probe in the integrated reaction solution is 0.1-0.5 μM, and the concentrations of the RPA upstream primer and RPA downstream primer are 0.1-0.5 μM.

8. The SNP detection method according to claim 1, characterized in that, The target nucleic acid sample solution has a concentration ≥10 copies / μL and a purity OD260 / OD280 of 1.8-2.

0.

9. The SNP detection method according to claim 1, characterized in that, The incubation temperature was 38°C and the time was 20 minutes.

10. A specific probe and primers for detecting the DEFB126 wild-type recombinant plasmid, characterized in that, The detection method is as described in claim 1; The nucleotide sequence of the specific primer probe is shown in SEQ ID No:

7. The 5' end is phosphorylated, corresponding to the SNP site located at 15BP. The T base at 21BP is modified with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quencher group. The nucleotide sequence of the upstream primer for RPA is shown in SEQ ID No:4; The nucleotide sequence of the downstream primer for RPA is shown in SEQ ID No:5.

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