Specific visual detection reagent for influenza A virus drug-resistant point mutation and detection method thereof

Abstract

The invention belongs to the field of biological detection, and particularly relates to a specific visual detection reagent for influenza A virus drug resistance point mutation and a detection method thereof. The reagents comprise a primer pair X, a connecting probe pair Y and a detection probe pair Z; the primer pair X is a primer pair which does not participate in the recognition complementation of a detection target and is used in a subsequent RPA (recombinase polymerase amplification) reaction; the connection probe pair Y is composed of a probe SP (Specific Probe) and a probe CP (Common Probe), the detection probe pair Z is composed of a detection probe WT-DP (Wildtype Determination Probe) and a detection probe MT-DP (Maximum Determination Probe), and the connection probe pair Y and the detection probe pair Z are single-stranded DNA molecules composed of 46-52 nucleotides. According to the present invention, the ligation reaction and the RPA probe method are combined, such that the SNP site detection under the normal temperature condition can be achieved, the precise thermal cycle instrument is not required, and the turnover time is short; the background signal is very low, and the sensitivity and the specificity are high. As the design process of the probe is simple and universal, the probe has a wide application prospect in the aspect of diagnosing new infectious diseases and new variants.

Description

Technical Field

[0001] This technology belongs to the field of biological detection. Specifically, it is a specific visualization detection reagent and detection method for influenza A virus resistance point mutations that utilizes sequence-specific ligase (SplintR ligase) to connect two ligation probes complementary to the target sequence to generate a complete single-stranded product through recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA). Background Art

[0002] At present, there are many SNP (single nucleotide polymorphism) detection methods, among which the most common is the molecular technology based on polymerase chain reaction (PCR). These technologies include: amplification retardation mutation PCR (ARMS-PCR) using allele-specific primers; allele-specific PCR (ASB-PCR) using blocker probes to achieve the difference in amplification efficiency between wild type and mutant type; PCR that controls the amplification of different allele DNA templates through toehold strand displacement hairpin primers; PCR that performs co-amplification at a lower denaturation temperature (COLD-PCR); and PNA-mediated PCR clamping technology. In addition, it also includes genotype PCR technology based on melting curve analysis, DNA microarray technology and whole genome sequencing (WGS) technology.

[0003] These traditional genetic analysis techniques usually require strict experimental environments, complex sample processing procedures, standardized experimental operations, and professional result analysis, and rely on high-precision experimental instruments. The rapid mutation of epidemic viruses such as influenza A and COVID-19 highlights the urgent need for versatile diagnostic testing methods that can promptly distinguish emerging variants in epidemics. However, these traditional technologies cannot be well applied to the instant detection of SNPs in pathogenic microorganisms. Therefore, it is of great significance to reduce detection time and labor costs.

[0004] In contrast, isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and the combination of nucleic acid amplification and CRISPR-Cas enzymology in technologies such as SHERLOCK and DETECTR, do not require complex thermal cycling instruments. These methods significantly reduce the limitations of the experimental environment and minimize the detection time. Among them, the more common SNPs isothermal detection techniques include competitive SNP-LAMP probes, OSD probes, allele-specific primers, etc., which identify SNPs on LAMP amplification templates through chain displacement reactions; technologies such as SHERLOCK and DETECTR can distinguish SNPs by introducing synthetic mismatches in gRNA. However, chain displacement methods usually rely on precise temperature control and long detection times, and point mutation detection based on CRISPR-Cas enzymology relies on the mutation site design of crRNA. The placement of sites for distinguishing SNPs and artificially synthesized mismatch sites is not intuitive, and some are also restricted by PAM sites. These characteristics limit their general applicability in detecting common point mutations associated with epidemic diseases. RPA is rarely used for SNP detection because the recombinase in it has a certain tolerance to mismatched nucleotides. Overall, these methods show certain limitations in the instant detection of SNPs in pathogenic microorganisms.

[0005] In summary, existing traditional SNP detection technologies such as PCR-based methods, DNA microarray technology, and whole genome sequencing (WGS) technology require strict experimental environments, standardized experimental operations, and professional experimental result analysis, and rely on sophisticated experimental instruments; and these reactions all require pre-processing of samples, further increasing the time required for detection. Commonly used isothermal SNP detection technology has many limitations in the probe design process and cannot be widely used in common SNP detection. Summary of the invention

[0006] The present invention aims to provide a specific visualization detection reagent and a detection method for influenza A virus drug-resistant point mutations based on SNPs isothermal detection technology of ligation reaction, which is simple to operate and sensitive to reaction.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A specific visual detection reagent for influenza A virus drug resistance point mutation, the reagent comprising a primer pair X, a ligation probe pair Y and a detection probe pair Z;

[0009] The primer pair X is complementary to the target and does not participate in the detection, and serves as a primer pair in the subsequent RPA reaction;

[0010] The ligation probe pair Y is composed of probes SP (Specific Probe) and CP (Common Probe)

[0011] The detection probe pair Z is composed of detection probes WT-DP (Wildtype Detection Probe) and MT-DP (Mutation Detection Probe), both of which are single-stranded DNA molecules consisting of 46-52 nucleotides;

[0012] Wherein, the probe SP in the ligation probe pair Y contains the same sequence as the detection probe pair Z.

[0013] The ligation probe pair Y is composed of a probe SP (Specific Probe) (WT-SP) and a CP (Common Probe), wherein the ligation probe SP is DNA fragment A, DNA fragment B and DNA fragment C in order from 5'→3' direction; the ligation probe CP is DNA fragment D and DNA fragment E in order from 5'→3' direction; wherein the sequence of DNA fragment A in the ligation probe SP is consistent with the sequence of F in the primer pair X; the sequence of DNA fragment B is consistent with the sequences of DNA fragments F and H in the WT-DP of the detection probe pair Z; DNA fragment C is a specific DNA fragment (10-12 nt) of influenza A virus; and the 3' terminal nucleotide of DNA fragment C is a complementary nucleotide to the SNP site for detecting influenza A virus;

[0014] The DNA fragment D in the ligation probe CP is a specific DNA fragment (10-12 nt) of influenza A virus, the 5' terminal nucleotide of the DNA fragment D is modified with a phosphate group to form a phosphodiester bond in sequence, the sequence of the DNA fragment F is consistent with the complementary sequence of the R sequence in the primer pair X, and the 3' end of the DNA fragment F contains a blocking group C3 that prevents non-specific extension.

[0015] The specific DNA fragment of the DNA fragment C is a sequence present at the 5' end of the SNP site, including the SNP site itself;

[0016] The specific DNA fragment of the DNA fragment D is a sequence present at the 3' end of the SNP site, and does not include the SNP site itself.

[0017] The detection probe pair Z is composed of detection probes WT-DP (Wildtype Detection Probe) and MT-DP (Mutation Detection Probe), both of which are single-stranded DNA molecules consisting of 46-52 nucleotides, wherein each single-stranded DNA molecule may include a DNA fragment F, a cutting site G of an exonuclease exo III, and a DNA fragment H in sequence from 5' to 3'.

[0018] The DNA fragments F and H are independent single-stranded DNA molecules composed of 30-35 nucleotides respectively, and the cutting site G of the exonuclease exo III is specifically tetrahydrofuran; wherein the DNA fragments F and H of WT-DP and MT-DP are different independent sequences that are not complementary, and the left and right ends of the cutting site of the exonuclease exo III of WT-DP are modified by a FAM fluorophore and a BHQ-1 quencher respectively; the left and right ends of the cutting site of the exonuclease exo III of MT-DP are modified by a ROX fluorophore and a BHQ-3 quencher respectively; and the 3' ends of WT-DP and MT-DP are both modified with a blocking group C3.

[0019] The influenza A virus drug resistance point mutation is one or more of A / G, U / G, A / C, U / C, and G / C; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and G-SP at the A / G mutation site correspond to T and C respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe U-SP and G-SP at the U / G mutation site correspond to A and C respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and C-SP at the A / C mutation site correspond to T and G respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe U-SP and C-SP at the U / C mutation site correspond to A and G respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and G-SP at the G / C mutation site correspond to C and G respectively.

[0020] The specific DNA fragment recorded above is derived from the influenza A virus sequence in the NCBI database, GenBank: GQ894817.1.

[0021] The visual detection reagent

[0022]

[0023]

[0024] An application of the specific visualization detection reagent for influenza A virus drug resistance point mutation, and the application of the visualization detection reagent in portable rapid visualization fluorescence detection of influenza A virus point mutation.

[0025] An application of the specific visualization detection reagent for influenza A virus drug resistance point mutation, and the application of the visualization detection reagent in detecting influenza A virus drug resistance point mutation.

[0026] The design principles of the ligation probe of the present invention are as follows:

[0027] Design probes according to the SNP sites present in the sample to be tested, and select 10-12nt sequences at both ends of the SNP site as the recognition region of the ligation probe (where the Tm value is around 30-32°C); when the sequence near the target SNP site is AT-enriched, the length of the ligation probe recognition region can be appropriately extended to ensure a suitable Tm value; similarly, when CG is enriched, the length of the ligation probe recognition region should be appropriately shortened. The terminal nucleotide of one of the probes is complementary to the SNP site to perform specific recognition of the SNP. The penultimate site is artificially mismatched, where the 5' end nucleotide of the A / G mutation site is T, then the corresponding ligation probe A-SP and the penultimate site nucleotide of the 3' end of the DNA fragment C of G-SP are designed to be G (artificially set as a TG mismatch site); similarly, the 5' end nucleotide of the U / G mutation site is A, then the corresponding ligation probe U-SP and the penultimate site nucleotide of the 3' end of the DNA fragment C of G-SP are designed to be C (artificially set as an AC mismatch site); the 5' end nucleotide of the A / C mutation site is A, then the corresponding ligation probe A-S The penultimate nucleotide at the 3' end of DNA fragment C of P and C-SP is designed to be C (artificially set as AC mismatch site); the 5' end nucleotide of the U / C mutation site is T, then the corresponding penultimate nucleotide at the 3' end of DNA fragment C of the connecting probe U-SP and C-SP is designed to be G (artificially set as TG mismatch site); the 5' end nucleotide of the G / C mutation site is T, then the corresponding penultimate nucleotide at the 3' end of DNA fragment C of the connecting probe G-SP and C-SP is designed to be G (artificially set as TG mismatch site). -

[0028] Detection principle:

[0029] The present invention can accurately identify the SNP in the template by connecting the penultimate artificial mismatch site and the last SNP recognition site of the probe. The design of the connection probe is simple and unrestricted. Under the action of sequence-specific ligase, when a template containing a matching site exists, a complete connection product is formed to facilitate subsequent RPA amplification; when there is no template or the mutation site does not match, no connection product is formed. The present invention combines the connection reaction with the RPA technology, and its core is a specific connection probe containing a recognition site. The connection probe can not only identify the SNP in the template, but also the generated connection product can be used as a template for amplification and signal amplification by RPA technology. In the RPA reaction containing detection probes labeled with different fluorophores, the method of the present invention can realize multiple detection of SNPs. The present invention can distinguish point mutations in DNA and RNA with high specificity, and has outstanding advantages in isothermal SNP detection.

[0030] Advantages of the present invention

[0031] The present invention utilizes a designed specific ligation probe to identify the target, so that hybridization between the target and the ligation probe can be achieved at room temperature of 25°C. The uniqueness of the ligation probe design is that the SNP recognition region is designed at the end of one of the ligation probes, and an artificial mismatch nucleotide is set at the penultimate site. In this way, accurate hybridization between the probe and the matching SNP target RNA is achieved, and the SNP site is accurately identified in the ligation reaction. The existing RPA detection technology cannot detect SNPs through ordinary detection probes due to the tolerance of recombinase polymerase to mismatch sites; the present invention can not only recognize the target with high specificity through the ligation probe, but also cleverly convert the ordinary RPA detection method that uses target amplification into an RPA detection method with single nucleotide resolution for amplification of ligation products. Through this design, we combine the rapidity and sensitivity of the RPA detection technology itself and expand the application of RPA technology in the field of SNP detection. In addition to the region that recognizes the target, the ligation probe also designs a universal primer region and a detection probe recognition region. Through the reaction of detection probes labeled with different fluorophores with different ligation products in the RPA system, multiple detections of SNPs can be achieved;

[0032] The universal primers and detection probes used in the present invention can be repeatedly used for different SNP detections, and only different connection probes need to be designed according to the target, thereby further reducing the detection cost.

[0033] The present invention combines the ligation reaction with the RPA probe method, which can detect SNP sites at room temperature, does not require a sophisticated thermal cycler, and has a short turnaround time; the background signal is very low, and the sensitivity and specificity are high; the detection procedure is simple, and can be directly judged by the naked eye without complicated result analysis. Given that the probe design process is simple and universal, the present invention has broad application prospects in the diagnosis of emerging infectious diseases and newly emerging variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the specific visual detection principle of influenza A virus drug-resistant point mutations provided by the present invention.

[0035] Figure 2Visual detection of influenza A virus SNP provided by the embodiment of the present invention; wherein A is the single (A / G) visual detection of different SNP sites of influenza A virus; B is the single (U / G) visual detection of different SNP sites of influenza A virus; C is the single (A / C) visual detection of different SNP sites of influenza A virus; D is the single (U / C) visual detection of different SNP sites of influenza A virus; E is the single (G / C) visual detection of different SNP sites of influenza A virus; F is the dual detection of A / G SNP. According to the difference between the sequence of the detection probe and the labeled fluorophore, WT and MT detection can be performed simultaneously; G is the ARMS-PCR detection result of the virus simulating clinical influenza A samples; H is the visual detection result of the simulated clinical influenza A samples. Specific implementation methods

[0037] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0038] Unless otherwise specified, all methods are conventional methods.

[0039] The nucleic acid of the sample to be tested is used as a template, and the following set of reagents is used for ligation reaction and RPA, and then the following evaluation is performed: if the set of reagents can detect a fluorescent signal, the sample to be tested contains or is suspected of containing a candidate influenza A virus resistance point mutation; if the set of reagents cannot detect a fluorescent signal, the sample to be tested does not contain or is suspected of not containing a candidate influenza A virus resistance point mutation.

[0040] The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all repeated three times, and the results were averaged. The portable fluorescence amplification detector is a product of Anpu Future (Changzhou) Company. The DNA / RNA nucleic acid extraction kit is a product of Tiangen (Beijing) Company. Hieff UNICON Advanced qPCR SYBR Master Mix is ​​a product of Yishen Biotechnology (Shanghai) Co., Ltd. The sequence of the influenza A virus strain (2009pandemic) was obtained from the NCBI database Influenza A virus (A / Illinois / 07 / 2009 (H1N1)) segment 6neuraminidase (-Nucleotide-NCBI (nih.gov). Among them, the PxpAx2; pMD2.G; pLVX-IRES-Puro plasmid sequence information comes from Changsha Aibiwei Biotechnology Co., Ltd. (pLVX-IRES-Puro_Vector and Strain Library_Product Center_HonorGene-HonorGene-Gene Research Resource Provider).

[0041] Example 1

[0042] 1. Design the connection probe according to the sequence of influenza A virus (2009 pandemic); primer pair X and detection probe pair Z; synthesize the plvx-IA plasmid (the target sequence length is 200 bp) according to the specific DNA fragment containing SNP in influenza A virus and the pLVX-IRES-Puro_ vector;

[0043] The primer pairs, ligation probe pairs and detection probes (sequence list below) were synthesized by BoShang Biotechnology (Shanghai) Co., Ltd., and the influenza A plvx-IA plasmid was obtained by BoShang Biotechnology (Shanghai) Co., Ltd. based on the above information.

[0044] 2. Prepare influenza A virus lentivirus containing the target sequence.

[0045] a. Inoculate well-growing HEK 293T cells, and the growth density reaches about 4 million per 10 cm culture dish the next day.

[0046] b. Add 20 μg of plasmid (PxpAx2: pMD2.G: Plvx-IA = 7.5 μg: 2.5 μg: 10 μg) to a total volume of 500 μL DMEM serum-free, antibody-free culture medium, referred to as solution A, and mix gently several times.

[0047] c. Add 60 μL beyopei to 500 μL of DMEM serum-free medium without double antibody TM (1 mg / ml) 60 μL, recorded as solution B, and gently mixed several times.

[0048] d. Add solution B dropwise into solution A, mix gently several times with a pipette, and let stand at room temperature for 20 minutes.

[0049] e. Slowly add the static transfection mixture to the HEK 293T cells in good condition.

[0050] f. 18 h after transfection, replace the HEK 293T cell supernatant containing the transfection reagent with 10 mL of DMEM complete medium.

[0051] g. Collect the viral supernatant 48h and 72h after transfection, filter and concentrate using an injection concentration kit for subsequent experiments.

[0052] h. Use the TIANamp Viral RNA Kit to extract RNA from lentivirus.

[0053] 3. Use the RNA extracted by the kit as the sample to be tested.

[0054] Mix 1 μl of the sample to be tested with the ligation probe corresponding to the SNP site to be detected, such as 1 μl CP (100 nM), 0.2 μl SP (100 nM) of the corresponding site, and 1 μl ligase buffer (10×), and add RNase-free H2O to 9 μl. Denature the reaction mixture at 95°C for 5 min and then anneal to room temperature.

[0055] 4. Add SplintR ligase at a final concentration of 0.5 U / μl, incubate at 25°C for 30 min in a PCR instrument for ligation reaction, then inactivate at 65°C for 10 min to terminate the ligation reaction.

[0056] 5. Use the fluorescent RPA rapid amplification kit for detection. Add 2-3 μl of the ligation reaction mixture to the RPA reaction buffer containing 2 μl of primer pair (10 μM) and 1 μl of WT-DP (10 μM), and amplify isothermally at 39°C for 90 min. The gene type is determined by the presence or absence of fluorescent signal (see Figure 2 ).

[0057] Depend on Figure 2 It can be seen that the single-plex visualization detection of different SNP sites of influenza A virus in AE (including the detection of A / GU / GA / CU / CG / C SNPs), in which Figure A (left) uses the connection probe A-SP combined with CP and RPA technology to visualize the detection of A / G SNPs. It can be seen from the real-time fluorescence curve and the fluorescence graph at the end of the reaction tube that the A site in the virus to be detected shows a strong fluorescence signal, while the H 2There is no fluorescence at the O and G sites. The specific ligation probe A-SP and CP can well distinguish the A in A / GSNP, and after combining with the RPA technology, the results that can be clearly distinguished by the naked eye can be obtained within 90 minutes; Figure A (right) uses the ligation probe G-SP combined with CP and RPA technology to visualize the G site in A / G SNP, and the final test results can be obtained without more professional result processing and analysis. Figure B (left) uses the ligation probe U-SP combined with CP and RPA technology to visualize the U / G SNP, in which the U site shows obvious fluorescence; Figure B (right) uses the ligation probe G-SP combined with CP and RPA technology to visualize the U / G SNP, in which the G site shows obvious fluorescence. Figure C (left) uses the ligation probe A-SP combined with CP and RPA technology to visualize the A / C SNP, in which the A site shows obvious fluorescence; Figure C (right) uses the ligation probe C-SP combined with CP and RPA technology to visualize the A / C SNP, in which the C site shows obvious fluorescence. Figure D (left) shows the visualization of U / C SNPs using the ligation probe U-SP in combination with CP and RPA technology, where the U site shows obvious fluorescence; Figure D (right) shows the visualization of U / C SNPs using the ligation probe C-SP in combination with CP and RPA technology, where the C site shows obvious fluorescence. Figure E (left) shows the visualization of G / C SNPs using the ligation probe G-SP in combination with CP and RPA technology, where the G site shows obvious fluorescence; Figure E (right) shows the visualization of G / C SNPs using the ligation probe C-SP in combination with CP and RPA technology, where the C site shows obvious fluorescence.

[0058] Example 2

[0059] According to the above-mentioned Example 1, double mutation detection was performed.

[0060] The RNA extracted by the kit was used as the sample to be tested.

[0061] Mix 1 μl of the sample to be tested with the ligation probe corresponding to the SNP site to be detected, such as 1 μl CP (100 nM), 0.2 μl SP (100 nM) corresponding to the site, 0.2 μl SP (100 nM) corresponding to the mutation site; and 1 μl ligase buffer (10×), and add RNase-free H2O to 9 μl. Denature the reaction mixture at 95°C for 5 minutes and then anneal to room temperature.

[0062] SplintR ligase was added at a final concentration of 0.5 U / μl, and the reaction was incubated at 25°C for 30 min in a PCR instrument for ligation reaction, followed by inactivation at 65°C for 10 min to terminate the ligation reaction.

[0063] The fluorescence RPA rapid amplification kit was used for detection.

[0064] Add 2-3 μl of the ligation reaction mixture to the RPA reaction buffer containing 2 μl of primer pair (10 μM), 1 μl of WT-DP (10 μM), and 1 μl of MT-DP (10 μM). Amplify isothermally at 39°C for 90 min. The gene type is determined by the presence or absence of fluorescent signal (see Figure 2 ).

[0065] Depend on Figure 2 It can be seen from the dual detection of A / G SNP in F that, according to the difference between the sequence of the detection probe and the labeled fluorophore, WT (A site) and MT (G site) can be detected simultaneously. Among them, the WT-DP probe is modified by the FAM fluorophore and the MT-DP is modified by the ROX fluorophore. Depending on the type of A / G SNP added to the sample to be detected, a fluorescence endpoint graph of different colors can be obtained. When only the WT (A site) sample to be detected is added, the endpoint fluorescence is green, when only the MT (G site) sample to be detected is added, the endpoint fluorescence is red, and when the WT (A site) sample to be detected and the MT (G site) sample to be detected are added at the same time, the endpoint fluorescence is yellow (the visual effect of the superposition of green fluorescence and red fluorescence); only by the different colors of the endpoint fluorescence, the dual detection of A / G SNP can be well performed.

[0066] Example 3

[0067] The lentivirus obtained in Example 1 was added to the throat swab samples of healthy people to simulate the clinical influenza A virus samples. The RNA in the virus-simulated clinical influenza A samples was extracted using the TIANamp virus RNA kit as the sample to be tested.

[0068] 1. ARMS-PCR detection of virus-simulated clinical influenza A samples

[0069] Mix 1 μl of the sample to be tested with the allele-specific primers corresponding to the SNP site to be tested, such as 0.6 μl R292-F (10 μM), 0.2 μl plvx-R (10 μM), and 10 μl 2×Hieff UNICON Advanced qPCR SYBR MasterMix, and add RNase-free H2O to 20 μl. Denature the reaction mixture at 95°C for 5 min, and then perform 45 cycles of amplification according to the thermal cycle of 95°C for 10 s and 62°C for 30 s.

[0070] 2. Visual detection of virus simulation clinical influenza A samples

[0071] Mix 1 μl of the sample to be tested with the ligation probe corresponding to the SNP site to be detected, such as 1 μl CP (100 nM), 0.2 μl R292-SP (100 nM), 0.2 μl 292K-SP (100 nM); and 1 μl ligase buffer (10×), and add RNase-free H2O to 9 μl. Denature the reaction mixture at 95°C for 5 min and then anneal to room temperature.

[0072] Add SplintR ligase at a final concentration of 0.5 U / μl, incubate at 25°C for 30 min in a PCR instrument for ligation reaction, then inactivate at 65°C for 10 min to terminate the ligation reaction.

[0073] The fluorescence RPA rapid amplification kit was used for detection.

[0074] Add 2-3 μl of the ligation reaction mixture to the RPA reaction buffer containing 2 μl of the primer pair (10 μM) and 1 μl of WT-DP (10 μM), and amplify isothermally at 39°C for 90 min. The gene type is determined by the presence or absence of the fluorescent signal (see Figure 2 ).

[0075] Depend on Figure 2 GH in the figure is the ARMS-PCR test result of virus simulation clinical influenza A sample and the visualization test result performed by the present invention. It can be seen that the collected influenza A lentivirus is added to the throat swab sample of a healthy person (from this laboratory) to simulate the clinical influenza A sample. ARMS-PCR detection and visualization detection performed by the present invention are performed after RNA is extracted by RNA virus extraction kit. Figure G shows the result of using ARMS-PCR to detect the R292K SNP site. Professional processing and calculation of ΔCt are required to distinguish the WT sample or MT sample of the R292K SNP site. Figure G (left) Ct (R292-292K) should be <0 under the WT allele-specific PCR primer amplification reaction. It can be seen that the sample corresponding to the red curve is WT, that is, the R292 sample; Figure G (right) Ct (R292-292K) should be >0 under the MT allele-specific PCR primer amplification reaction. It can be seen that the sample corresponding to the green curve is MT, that is, the 292K sample. Figure H shows the results of SNP detection using the method of the present invention. The SNP type of the sample to be detected can be well distinguished according to the presence or absence of endpoint fluorescence. In the WT Sensor composed of R292-SP (i.e., WT(A)-SP) and CP, the corresponding green columnar sample has fluorescence, and the sample is R292, that is, the WT sample; in the MT Sensor composed of 292K-SP (i.e., MT(G)-SP) and CP, the corresponding red columnar sample has fluorescence, and the sample is 292K, that is, the MT sample.

[0076] Instance sequence information:

[0077]

[0078]

[0079]

[0080] The present application utilizes a designed ligation probe that can well distinguish SNPs in a ligation reaction. The ligation probe is simple in design and is not restricted. It can be widely used in various SNP detections of DNA and RNA, and combined with RPA technology, it can perform visual and instant detection of SNPs.

Claims

1. A specific visual detection reagent for influenza A virus drug resistance point mutation, characterized in that: The reagents are primer pair X, ligation probe pair Y and detection probe pair Z; The primer pair X is complementary to the target and does not participate in the detection, and serves as a primer pair in the subsequent RPA reaction; The ligation probe pair Y is composed of probes SP (Specific Probe) and CP (Common Probe) The detection probe pair Z is composed of detection probes WT-DP (Wildtype Detection Probe) and MT-DP (Mutation Detection Probe), both of which are single-stranded DNA molecules consisting of 46-52 nucleotides; Wherein, the probe SP in the ligation probe pair Y contains the same sequence as the detection probe pair Z.

2. The specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 1, characterized in that: The ligation probe pair Y is composed of a probe SP (Specific Probe) (WT-SP) and a CP (Common Probe), wherein the ligation probe SP is DNA fragment A, DNA fragment B and DNA fragment C in order from 5'→3' direction; the ligation probe CP is DNA fragment D and DNA fragment E in order from 5'→3' direction; wherein the sequence of DNA fragment A in the ligation probe SP is consistent with the sequence of F in the primer pair X; the sequence of DNA fragment B is consistent with the sequences of DNA fragments F and H in the WT-DP of the detection probe pair Z; DNA fragment C is a specific DNA fragment (10-12 nt) of influenza A virus; and the 3' terminal nucleotide of DNA fragment C is a complementary nucleotide to the SNP site for detecting influenza A virus; The DNA fragment D in the ligation probe CP is a specific DNA fragment (10-12 nt) of influenza A virus, the 5' terminal nucleotide of the DNA fragment D is modified with a phosphate group to form a phosphodiester bond in sequence, the sequence of the DNA fragment F is consistent with the complementary sequence of the R sequence in the primer pair X, and the 3' end of the DNA fragment F contains a blocking group C3 that prevents non-specific extension.

3. The specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 2, characterized in that: The specific DNA fragment of the DNA fragment C is a sequence present at the 5' end of the SNP site, including the SNP site itself; The specific DNA fragment of the DNA fragment D is a sequence present at the 3' end of the SNP site, and does not include the SNP site itself.

4. The specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 1, characterized in that: The detection probe pair Z is composed of detection probes WT-DP (Wildtype Detection Probe) and MT-DP (Mutation Detection Probe), both of which are single-stranded DNA molecules consisting of 46-52 nucleotides, wherein each single-stranded DNA molecule may include a DNA fragment F, a cutting site G of an exonuclease exo III, and a DNA fragment H in sequence from 5' to 3'.

5. The specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 4, characterized in that: The DNA fragments F and H are independent single-stranded DNA molecules composed of 30-35 nucleotides, respectively, and the cutting site G of the exonuclease exo III is specifically tetrahydrofuran; wherein the DNA fragments F and H of WT-DP and MT-DP are different independent sequences that are not complementary, and the left and right ends of the cutting site of the exonuclease exo III of WT-DP are modified by a FAM fluorophore and a BHQ-1 quencher, respectively; The left and right ends of the cleavage site of the exonuclease exo III of MT-DP were modified with ROX fluorophore and BHQ-3 quencher, respectively; the 3' ends of WT-DP and MT-DP were modified with the blocking group C3.

6. The specific visual detection reagent for influenza A virus drug resistance point mutation according to any one of claims 1 to 5, characterized in that: The influenza A virus drug resistance point mutation is one or more of A / G, U / G, A / C, U / C, and G / C; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and G-SP at the A / G mutation site correspond to T and C respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe U-SP and G-SP at the U / G mutation site correspond to A and C respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and C-SP at the A / C mutation site correspond to T and G respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe U-SP and C-SP at the U / C mutation site correspond to A and G respectively; wherein the 3'-terminal nucleotides of the DNA fragment C of the connecting probe A-SP and G-SP at the G / C mutation site correspond to C and G respectively.

7. The specific visual detection reagent for influenza A virus drug resistance point mutation according to claim 6, characterized in that: The visual detection reagent 8. Use of the specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 1, characterized in that: The visualization detection reagent is used in portable rapid visualization fluorescence detection of influenza A virus point mutations.

9. Use of the specific visualization detection reagent for influenza A virus drug resistance point mutation according to claim 1, characterized in that: The visualization detection reagent is used in detecting influenza A virus drug-resistant point mutations.

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