Kit and detection method for detecting swine foot and mouth disease virus based on MB-RT-PCR

Abstract

The invention provides a kit and a detection method for detecting a swine foot and mouth disease virus based on MB-RT-PCR, and relates to the technical field of in-vitro nucleic acid detection.The specific detection substance comprises a forward primer, a reverse primer and a molecular beacon probe, the fluorescent RT-PCR primer and the molecular beacon probe are designed by taking conserved regions of conserved genes VP1 of seven serotypes such as O, A, C, SAT1, SAT2, SAT3 and Asia1 of the foot-and-mouth disease virus as targets, real-time monitoring reaction can be realized, and the fluorescent RT-PCR primer and the molecular beacon probe have the advantages of high specificity, high sensitivity, rapidness and high efficiency; the foot and mouth disease virus of each serotype in a sample can be rapidly and qualitatively detected; the technical problems that in the prior art, an FMDV detection method is complex in operation, tedious in process, poor in specificity and low in sensitivity, or pig foot-and-mouth disease viruses of different serotypes cannot be rapidly and accurately quantitatively detected are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro nucleic acid detection, and in particular to a kit and a detection method for detecting swine foot-and-mouth disease virus based on MB-RT-PCR. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, contagious disease common among even-toed ungulates, caused by infection with the foot-and-mouth disease virus (FMDV). The most susceptible animals are cattle, buffalo, pigs, camels, sheep, and deer. Wildlife such as gazelles, musk deer, wild boars, and bison are also susceptible. Currently, there are seven serotypes of FMDV: O, A, C, SAT1, SAT2, SAT3 (South Africa 1, 2, and 3), and Asia 1. There is little immune protection between these serotypes, and animals infected with one FMDV serotype can still become infected with another and develop disease. FMDV belongs to the Picornaviridae family and the genus Aphthovirus. It is a non-enveloped, single-stranded, positive-sense RNA virus. To date, seven serotypes, over 80 subtypes, and numerous distinct strains have been identified. In my country, serotypes O, A, and Asia 1 are predominantly prevalent. The core of the virus consists of a single-stranded, positive-sense RNA, consisting of approximately 8,000 bases, which is the basis for infection and inheritance. The surrounding protein envelope determines the virus's antigenicity, immunogenicity, and serological reactivity. The viral capsid is a symmetrical icosahedron. FMDV toxin levels are highest in the blisters within the skin and lymph fluid of infected animals. During the fever period, the toxin is highest in the blood, and FMDV is also found in milk, urine, saliva, tears, and feces. Foot-and-mouth disease is generally not fatal, but it causes numerous blisters on the mouth and hooves of infected animals, persistent high fever, and a sharp decline in livestock production. Furthermore, some variants of the foot-and-mouth disease virus are transmissible to humans. Therefore, after each outbreak, infected livestock must be slaughtered and collectively incinerated to prevent future infections. Therefore, effective detection methods for porcine foot-and-mouth disease virus are urgent.

[0003] Existing FMDV detection methods primarily include virus isolation, conventional PCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescent quantitative PT-PCR. Virus isolation requires cell culture and anti-contamination procedures, which is time-consuming and cannot be met by grassroots testing agencies. Conventional PCR is the most commonly used detection tool, but because the amplified products are often exposed to the environment during the detection process, they are prone to environmental nucleic acid contamination and false-positive results. Furthermore, it is impossible to quantify the virus and determine the extent of the disease. RT-LAMP testing requires highly conserved target sequences and primer design positions to effectively ensure the high specificity of LAMP testing. The target sequence selection and primer design steps are more complex than those of conventional PCR. Furthermore, the target amplified fragments used in LAMP testing are shorter, resulting in higher amplification efficiency. Repeated handling of the reaction tube caps after the isothermal reaction can easily cause aerosol contamination, leading to false-positive results.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a specific detection substance for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, so as to solve the technical problems in the existing technology of FMDV detection methods, such as complex operation, cumbersome process, poor specificity, low sensitivity or inability to quickly and accurately perform quantitative detection of different serotypes of swine foot-and-mouth disease virus.

[0006] A second object of the present invention is to provide the use of the above-mentioned specific detection substance in the preparation of a detection kit for swine foot-and-mouth disease virus.

[0007] The third object of the present invention is to provide a kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR.

[0008] A fourth object of the present invention is to provide a method for detecting swine foot-and-mouth disease virus for non-diagnostic purposes.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted: In a first aspect, the present invention provides a specific detection substance for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, wherein the specific detection substance comprises a forward primer, a reverse primer and a molecular beacon probe; The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO.1, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO.1 or deleting 1 to 5 bases; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO. 2, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 2 or deleting 1 to 5 bases; The nucleotide sequence of the molecular beacon probe is as shown in SEQ ID NO. 3, or a complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 3, or a nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 3 or deleting 1 to 5 bases; The swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

[0010] Furthermore, the 3' end of the molecular beacon probe is labeled with a fluorescent quencher group, and the 5' end of the molecular beacon probe is labeled with a fluorescent reporter group; Preferably, the fluorescent reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red; Preferably, the fluorescence quenching group is selected from BHQ-1, BHQ-2, BHQ-3 or TAMRA.

[0011] In a second aspect, the present invention provides the use of the above-mentioned specific detection substance in the preparation of a detection kit for swine foot-and-mouth disease virus.

[0012] Furthermore, the swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

[0013] In a third aspect, the present invention provides a kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, wherein the kit comprises the above-mentioned specific detection substance.

[0014] Furthermore, the kit also includes RT-PCR reaction solution and enzyme mixture; Preferably, the RT-PCR reaction solution comprises RT PCR buffer, PCR enhancer, magnesium chloride, deoxyribonucleotide triphosphate mixture, and buffer containing magnesium ions; Preferably, the RT-PCR buffer specifically comprises Tris-HCl with a pH of 7.5 to 8.9 and a concentration of 20 mM to 80 M, 10 mM to 5 M ammonium sulfate, 100 mM to 4 M potassium chloride, and 0.01 to 0.3% Triton X-100 by volume; Preferably, the PCR enhancer is selected from at least one of tetramethylammonium chloride, carnitine, trehalose and non-ionic detergent NP-40; Preferably, the enzyme mixture contains 2U / μL to 6U / μL of Taq DNA polymerase, 5U / μL to 12U / μL of a highly thermostable reverse transcriptase, and 1U / μL to 5U / μL of an RNase inhibitor.

[0015] Furthermore, the kit also includes a negative quality control product and a positive quality control product; Preferably, the positive quality control product is a swine foot-and-mouth disease virus positive plasmid; Preferably, the negative control substance is ddH2O.

[0016] In a fourth aspect, the present invention provides a method for detecting swine foot-and-mouth disease virus for non-diagnostic purposes, comprising using the total DNA / RNA of the sample as a template and performing RT-qPCR amplification using the above-mentioned kit. If the amplification curve is S-shaped and 37<Ct≤40, it is determined to contain swine foot-and-mouth disease virus.

[0017] Furthermore, the reaction system per 25 μL was: 10 μL template, 12.79 μL RT-PCR reaction solution, 1 μL enzyme mixture, 0.6 μL 40 μM forward primer, 0.6 μL 40 μM reverse primer, and 0.01 μL 40 μM molecular beacon probe.

[0018] Furthermore, the reaction conditions of the RT-qPCR amplification are: 50°C, 20 min; 95°C, 5 min; 95°C, 15 s, 55°C, 30 s, 40 cycles.

[0019] The present invention provides a specific detection substance for detecting swine foot-and-mouth disease virus based on MB-RT-PCR. The fluorescent RT-PCR primers and molecular beacon probes are designed to target the conserved region of the VP1 gene of seven serotypes of swine foot-and-mouth disease virus, including O, A, C, SAT1, SAT2, SAT3 and Asia1, and can realize real-time monitoring reaction. It has the advantages of high specificity, high sensitivity, rapidity and high efficiency. It can quickly and qualitatively detect each serotype of swine foot-and-mouth disease virus in a sample. It solves the technical problems of the existing FMDV detection method, which is complex in operation, cumbersome in process, poor in specificity, low in sensitivity, or inability to quickly and accurately perform quantitative detection of different serotypes of swine foot-and-mouth disease virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the molecular beacon probe for swine foot-and-mouth disease virus provided in Example 1 of the present invention; Figure 2 The test results of the amplification efficiency of the five sets of primer probes designed for the conserved region of the target gene VP1 in Example 1 of the present invention are as follows; Figure 3 The amplification results of different MB-RT-PCR-based kits for detecting swine foot-and-mouth disease virus provided in Example 5 of the present invention, wherein the horizontal axis represents the number of amplification cycles and the vertical axis Rn represents the fluorescence intensity; Figure 4 The FAM channel linearity test results of the kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR provided in Example 6 of the present invention; Figure 5 The FAM standard curve of the kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR provided in Example 6 of the present invention; Figure 6 The detection limit test results of the kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR provided in Example 6 of the present invention; Figure 7 This is the specific test result of the kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0022] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0023] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0024] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] On one hand, the present invention provides a specific detection substance for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, wherein the specific detection substance comprises a forward primer, a reverse primer and a molecular beacon probe; The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO.1, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO.1 or deleting 1 to 5 bases; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO. 2, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 2 or deleting 1 to 5 bases; The nucleotide sequence of the molecular beacon probe is as shown in SEQ ID NO. 3, or a complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 3, or a nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 3 or deleting 1 to 5 bases; The swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

[0026] The fluorescent RT-PCR primers and molecular beacon probes are designed to target the conserved region of the VP1 gene of seven serotypes of swine foot-and-mouth disease virus, including O, A, C, SAT1, SAT2, SAT3 and Asia1. They can realize real-time monitoring reactions and have the advantages of high specificity, high sensitivity, rapidity and high efficiency. They can quickly and qualitatively detect each serotype of swine foot-and-mouth disease virus in a sample. They solve the technical problems of the existing FMDV detection methods, which are complex operation, cumbersome process, poor specificity, low sensitivity or inability to quickly and accurately perform quantitative detection of different serotypes of swine foot-and-mouth disease virus.

[0027] In some specific embodiments, the 3' end of the molecular beacon probe is labeled with a fluorescent quencher group, and the 5' end of the molecular beacon probe is labeled with a fluorescent reporter group; in some specific embodiments, the fluorescent reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red; in some specific embodiments, the fluorescent quencher group is selected from BHQ-1, BHQ-2, BHQ-3 or TAMRA.

[0028] According to another aspect of the invention, there is also provided the use of the above-mentioned specific detection substance in the preparation of a detection kit for swine foot-and-mouth disease virus.

[0029] In some specific embodiments, the swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

[0030] According to another aspect of the invention, a kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR is provided, comprising the above-mentioned specific detection substance. The detection kit can rapidly and qualitatively detect various serotypes of swine foot-and-mouth disease virus in a sample.

[0031] In some specific embodiments, the kit further comprises an RT-PCR reaction solution and an enzyme mixture; in some specific embodiments, the RT-PCR reaction solution comprises an RT PCR buffer, a PCR enhancer, magnesium chloride, a deoxyribonucleotide triphosphate mixture, and a buffer containing magnesium ions; in some specific embodiments, the RT-PCR buffer specifically comprises Tris-HCl with a pH of 7.5 to 8.9 and a concentration of 20 mM to 80 M, 10 mM to 5 M ammonium sulfate, 100 mM to 4 M potassium chloride, and a volume ratio of 0.01 to 0.3% Triton X-100; in some specific embodiments, the PCR enhancer is selected from at least one of tetramethylammonium chloride, carnitine, trehalose, and the non-ionic detergent NP-40; in some specific embodiments, the enzyme mixture comprises 2 U / μL to 6 U / μL of Taq DNA polymerase, 5 U / μL to 12 U / μL of a highly thermostable reverse transcriptase, and 1 U / μL to 5 U / μL of an RNase inhibitor.

[0032] In some specific embodiments, the kit further comprises a negative control and a positive control; in some specific embodiments, the positive control is a swine foot-and-mouth disease virus positive plasmid; in some specific embodiments, the negative control is ddH2O.

[0033] According to another aspect of the invention, a method for detecting swine foot-and-mouth disease virus for non-diagnostic purposes is also provided, comprising using the total DNA / RNA of the sample as a template and performing RT-qPCR amplification using the above-mentioned kit. If the amplification curve is S-shaped and 37<Ct≤40, it is determined to contain swine foot-and-mouth disease virus.

[0034] In some specific embodiments, the reaction system per 25 μL is: 10 μL template, 12.79 μL RT-PCR reaction solution, 1 μL enzyme mixture, 0.6 μL 40 μM forward primer, 0.6 μL 40 μM reverse primer, and 0.01 μL 40 μM molecular beacon probe.

[0035] In some specific embodiments, the reaction conditions of the RT-qPCR amplification are: 50°C, 20 min; 95°C, 5 min; 95°C, 15 s, 55°C, 30 s, 40 cycles.

[0036] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0037] Example 1 Specific primer probe for detecting swine foot-and-mouth disease virus based on MB-RT-PCR The present invention designs specific forward primers, reverse primers and molecular beacon probes for swine foot-and-mouth disease virus targeting the conserved region of the VP1 gene of seven serotypes, including O, A, C, SAT1, SAT2, SAT3 and Asia1. The specific nucleotide sequences are shown in Table 1: Table 1 PCR primer and probe sequences

[0038] Primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0039] The beacon probe diagram is as follows Figure 1 As shown, the 3' end of the molecular beacon probe is labeled with a fluorescent quenching group, and the 5' end is labeled with different fluorescent reporter groups. The fluorescent reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red. In this embodiment, the FAM fluorescent group is selected; the fluorescent quenching group is selected from BHQ-1, BHQ-2, BHQ-3 or TAMRA. In this embodiment, BHQ-1 is selected.

[0040] The amplification efficiency of 5 sets of primer probes was tested, and the results were as follows Figure 2 As shown, it can be seen that F1R1P1 has the best amplification efficiency.

[0041] Example 2: Kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR The kit provided in this example includes the specific primer probe provided in Example 1, RT-PCR reaction solution, enzyme mixture, negative quality control product, and positive quality control product.

[0042] Among them, the positive quality control product is a synthetic swine foot-and-mouth disease virus positive plasmid solution; The negative control was ddH2O; The RT-PCR reaction solution includes RT PCR buffer, a PCR enhancer, magnesium chloride, a deoxyribonucleotide triphosphate mixture, and a buffer containing magnesium ions; the RT PCR buffer specifically includes 50 mM Tris HCl at a pH of 8.3, 20 mM ammonium sulfate, 100 mM potassium chloride, and 0.1% Triton X 100 by volume; the PCR enhancer is 2 M tetramethylammonium chloride and 50 mM carnitine; The enzyme mixture contains 5 U / μL of Taq polymerase, 10 U / μL of highly thermostable reverse transcriptase, and 2 U / μL of RNase inhibitor.

[0043] The concentration of the forward primer was 40 μM; The concentration of the reverse primer was 40 μM; The concentration of the probe was 40 μM.

[0044] Example 3 Optimization of conditions for detecting FMDV In this example, the PCR reaction was performed with reverse transcription at 50°C for 20 minutes, pre-denaturation at 95°C for 5 minutes, and denaturation at 95°C for 15 seconds, followed by 40 cycles. The primer volume, MB probe volume, and annealing temperature were optimized. The basic principles of optimization were to obtain clear and stable target bands, minimize costs, and minimize time.

[0045] 1. Optimization of the optimal primer and probe volume Eleven different primer-probe ratios, as shown in Table 2, were used to screen for optimal primer and probe volumes. The experimental data are shown in Table 2. Upstream and downstream primers were used with the probe at the primer:probe ratios listed in Table 2. The concentrations and volumes of the upstream and downstream primers were consistent. After optimization, the optimal primer-probe volume ratio was determined to be 0.6:0.6:0.01.

[0046] Table 2 Experimental data of PCR amplification screening for optimal primer and probe volume

[0047] 2. Optimal annealing temperature screening The screening experiment was performed with 8 different Tm values ​​as shown in Table 3. The experimental data are shown in Table 3. The optimal Tm value after optimization was 55°C.

[0048] Table 3 Experimental data of PCR amplification screening for optimal Tm value

[0049] The optimized amplification program obtained after the above experiments is as follows: 50°C, 20 min; 95°C, 5 min; (95°C, 15 s; 55°C, 30 s) × 40 cycles.

[0050] Example 4 MB-RT-PCR method for detecting FMDV The MB-RT-PCR method for detecting FMDV specifically includes: 1. Extract viral nucleic acid: Use YXN-NB-43 (complex sample nucleic acid extraction kit) developed by Guangzhou Yixin Biotechnology Co., Ltd. to extract FMDV DNA / RNA from inactivated samples.

[0051] 2. Using the DNA / RNA from step 1 as a template, perform RT-PCR amplification using the kit provided in Example 2. The total volume of the amplification system is 25 μL, including 12.79 μL of RT-PCR reaction solution, 0.6 μL of forward primer, 0.6 μL of reverse primer, 0.01 μL of molecular beacon probe, 1 μL of enzyme mix, and 10 μL of template.

[0052] Amplification program: 50°C, 20 min; 95°C, 5 min; (95°C, 15 s; 55°C, 30 s) × 40 cycles.

[0053] Quality control was performed based on positive and negative controls. If the amplification curve of the positive control channel showed an S-shaped curve and the Ct value was ≤37, and the negative control channel had no Ct value or the Ct value was >37, the result was normal.

[0054] When there is no Ct value in the sample channel or Ct>37, the sample is negative. When the amplification curve of the sample channel is an S-shaped curve and Ct≤37, the sample is positive. When the amplification curve of the sample channel is an S-shaped curve and 37<Ct≤40, the sample is an uncertain sample and the nucleic acid needs to be re-extracted for testing. If the re-test amplification curve is S-shaped and 37<Ct≤40, it is judged as positive, otherwise it is judged as negative. When there is no Ct value in the sample channel or the Ct value is>40, it is invalid.

[0055] Example 5 Comparison of amplification effects of different kits This example uses product 1 of company 1 and product 2 of company 2 for comparison with the kit provided in Example 2, selects swine foot-and-mouth disease virus nucleic acid as a sample, and amplifies according to the method provided in Example 3.

[0056] The results are as follows Figure 3 As shown in the figure, under the same plate concentration, the detection results of this kit are about 5 Ct higher than those of other companies' products, and the reproducibility is better.

[0057] Example 6 Kit Performance 1. Linearity test This example uses the kit provided in Example 2, and the linear reference product of the kit consists of a plasmid containing the target fragment (positive plasmid).

[0058] The positive plasmid was diluted 10-fold using TE buffer to obtain the following gradient solutions (E10: 1.0×10 10 copies / μL, E9: 1.0×10 9 copies / μL, E8: 1.0×10 8 copies / μL, E7: 1.0×10 7 copies / μL, E6: 1.0×10 6 copies / μL, E5: 1.0×10 5 copies / μL, E4: 1.0×10 4 copies / μL, E3: 1.0×10 3 copies / μL, E2: 1.0×10 2 copies / μL, E1: 1.0×101 copies / μL, 0.5E1: 1.0×10 1 / 2 copies / μL, 0.25E1: 1.0×10 1 / 4 copies / μL) as a linear reference, the results are as follows Figure 4 The standard curve is shown as Figure 5 shown.

[0059] The results showed that the kit was 10 copies / μL to 1.0×10 2 There is a good linear relationship between the two. The linear equation fitted by the kit is: Y = -3.504x + 37.386, and the correlation coefficient R 2 =0.9997, where Y represents the Ct value and x represents the logarithmic value of the positive reference.

[0060] 2. Minimum detection limit test The linear reference material E8 was used: 1.0×10 8 copies / μL, E7: 1.0×10 7 copies / μL, E6: 1.0×10 6 copies / μL, E5: 1.0×10 5 copies / μL, E4: 1.0×10 4 copies / μL, E3: 1.0×10 3 copies / μL, E2: 1.0×10 2 copies / μL, E1: 1.0×10 1 copies / μL, 0.5E1: 1.0×10 1 / 2 copies / μL, 0.25E1: 1.0×10 1 / 4 The positive plasmid with the same concentration of 10 copies / μL was used as the detection limit reference, and 10 replicates were performed for each concentration. Figure 6 As shown in the figure, the detection limit of reference products E2 and E1 all showed amplification curves, which were positive data. The 0.5E1 and 0.25E1 ​​reference products were not detected and did not meet the requirements, indicating that the detection limit of the kit was 1.0×10 1 copies / μL, that is, 5 copies / reaction.

[0061] 3. Precision test To verify the intra-batch precision of the kit, the linear reference product E7 was used: 1.0×10 7 copies / μL, E6: 1.0×10 6copies / μL, E5: 1.0×10 5 Three high and low concentrations of positive plasmids with 10 copies / μL were used as precision references, and ten replicates were made for each. The results are shown in Table 4. The coefficient of variation of the three concentration data was less than 5%.

[0062] Table 4

[0063] 4. Specificity test This experiment selected common pathogens with the same infection site as porcine foot-and-mouth disease virus as specific reference substances. The FMDV positive samples used were inactivated positive samples, and FMDV E4 and FMDV E5 were selected from the linear reference substances. The specific reference substances were classical porcine blue ear disease virus, highly pathogenic porcine blue ear disease virus, African swine fever virus, porcine circovirus type 2, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus. The above specific reference substances were all derived from live vaccines and extracted after inactivation. Among them, porcine circovirus type 2 was derived from Yuanjian (inactivated porcine circovirus type 2 vaccine (SH strain)), purchased from Pleco Bioengineering Co., Ltd., veterinary drug code 160021069; porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus were derived from porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus (G5 type) triple live vaccine (attenuated Huadu pig + attenuated CV777 strain + NX strain), purchased from Huamu Biotechnology Co., Ltd. African swine fever virus (ASFV) was obtained from Haiwen'an (a cell line used for the passage of live classical swine fever vaccine) and purchased from Sichuan Hailinger Biopharmaceutical Co., Ltd., with a veterinary drug code number of 220521084. Classical porcine reproductive and respiratory syndrome (PRRS) virus was obtained from Lanlikang porcine reproductive and respiratory syndrome chimeric virus live vaccine (PC strain) and purchased from Sinopharm Animal Health Co., Ltd., with a veterinary drug code number of 170261139. Highly pathogenic porcine reproductive and respiratory syndrome (PRRS) virus was obtained from Lanerwei porcine reproductive and respiratory syndrome (JXA1-R strain) live vaccine and purchased from Zhaoqing Dahuanong Animal Health Products Co., Ltd. DNA / RNA was extracted from these inactivated samples using the YXN-NB-43 (complex sample nucleic acid extraction kit) developed by Guangzhou Yixin Biotechnology Co., Ltd. as a template.

[0064] The results are as follows Figure 7 As shown in the figure, none of the seven specific reference products had a typical "S"-shaped amplification curve, indicating that the kit had good specificity.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A specific detection substance for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, characterized in that: The specific detection substances include a forward primer, a reverse primer and a molecular beacon probe; The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO.1, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO.1 or deleting 1 to 5 bases; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO. 2, or the complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 2, or the nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 2 or deleting 1 to 5 bases; The nucleotide sequence of the molecular beacon probe is as shown in SEQ ID NO. 3, or a complementary chain sequence of the nucleotide sequence shown in SEQ ID NO. 3, or a nucleotide sequence obtained by extending 1 to 5 bases from the 5' end and / or the 3' end of the nucleotide sequence shown in SEQ ID NO. 3 or deleting 1 to 5 bases; The swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

2. The specific detection substance according to claim 1, characterized in that The 3' end of the molecular beacon probe is labeled with a fluorescent quenching group, and the 5' end of the molecular beacon probe is labeled with a fluorescent reporter group; Preferably, the fluorescent reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red; Preferably, the fluorescence quenching group is selected from BHQ-1, BHQ-2, BHQ-3 or TAMRA.

3. Use of the specific detection substance according to claim 1 or 2 in the preparation of a detection kit for swine foot-and-mouth disease virus.

4. The use according to claim 3, characterized in that The swine foot-and-mouth disease virus includes at least one of type O, type A, type C, SAT1, SAT2, SAT3 and Asia1.

5. A kit for detecting swine foot-and-mouth disease virus based on MB-RT-PCR, characterized in that: The kit comprises the specific detection substance according to claim 1 or 2.

6. The kit according to claim 5, characterized in that The kit also includes RT-PCR reaction solution and enzyme mixture; Preferably, the RT-PCR reaction solution comprises RT PCR buffer, PCR enhancer, magnesium chloride, deoxyribonucleotide triphosphate mixture, and buffer containing magnesium ions; Preferably, the RT-PCR buffer specifically comprises Tris-HCl with a pH of 7.5 to 8.9 and a concentration of 20 mM to 80 M, 10 mM to 5 M ammonium sulfate, 100 mM to 4 M potassium chloride, and 0.01 to 0.3% Triton X-100 by volume; Preferably, the PCR enhancer is selected from at least one of tetramethylammonium chloride, carnitine, trehalose and non-ionic detergent NP-40; Preferably, the enzyme mixture contains 2U / μL to 6U / μL of Taq DNA polymerase, 5U / μL to 12U / μL of a highly thermostable reverse transcriptase, and 1U / μL to 5U / μL of an RNase inhibitor.

7. The kit according to claim 5, characterized in that The kit also includes a negative quality control product and a positive quality control product; Preferably, the positive quality control product is a swine foot-and-mouth disease virus positive plasmid; Preferably, the negative control substance is ddH2O.

8. A method for detecting swine foot-and-mouth disease virus for non-diagnostic purposes, characterized in that: The method comprises using the total DNA / RNA of the sample as a template and performing RT-qPCR amplification using the kit according to claim 5. If the amplification curve is S-shaped and 37<Ct≤40, it is determined that the sample contains swine foot-and-mouth disease virus.

9. The method according to claim 8, characterized in that The reaction system for each 25 μL is: 10 μL template, 12.79 μL RT-PCR reaction solution, 1 μL enzyme mixture, 0.6 μL 40 μM forward primer, 0.6 μL 40 μM reverse primer, and 0.01 μL 40 μM molecular beacon probe.

10. The method according to claim 8, characterized in that The reaction conditions for the RT-qPCR amplification were: 50°C, 20 min; 95°C, 5 min; 95°C, 15 s, 55°C, 30 s, 40 cycles.

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