Multiplex fluorescent quantitative PCR kit for simultaneously detecting PRRSV (porcine reproductive and respiratory syndrome virus), PCV2 (porcine circovirus 2) and SIV (swine influenza virus) and detection method thereof

Abstract

The invention belongs to the field of virus detection, and particularly relates to a multiplex fluorescent quantitative PCR (polymerase chain reaction) kit for simultaneously detecting PRRSV (porcine reproductive and respiratory syndrome virus), PCV2 (porcine circovirus 2) and SIV (swine influenza virus) and a detection method thereof. PRRSV, PCV2 and SIV can be used as hosts of pigs, part of infection symptoms are similar, although an independent detection means exists in the prior art, the defects that the detection efficiency is low, the detection time is long and distinguishing is difficult exist, and in order to overcome the defects of the prior art, the invention provides a multi-fluorescent quantitative PCR kit for simultaneously detecting PRRSV, PCV2 and SIV and a detection method of the multi-fluorescent quantitative PCR kit. The kit comprises a reaction solution A, a reaction solution B, a negative control and a positive control, multiple fluorescent quantitative PCR is performed on a specific sequence of a virus through a specific primer probe group, whether a host is infected with the specific virus or not is determined by detecting a fluorescent signal, and the kit has the advantages of short detection time and high sensitivity; and the problem of difficulty in distinguishing after mixed infection clinically is solved.

Description

Technical Field

[0001] The invention belongs to the field of virus detection, and specifically relates to a multiplex fluorescent quantitative PCR kit for simultaneously detecting PRRSV, PCV2 and SIV and a detection method thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRSV), commonly known as blue ear disease, is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), which can cause reproductive disorders in sows and severe respiratory diseases in piglets, seriously affecting economic benefits. Porcine circovirus type 2 (PCV2) is a virus of the genus Porcine circovirus, which is closely related to porcine post-weaning multisystemic wasting syndrome (PMWS). Pigs infected with PCV2 usually show symptoms such as growth retardation, emaciation, jaundice and dyspnea. PCV2 has a suppressive effect on the pig's immune system, leading to an increased risk of secondary infection, which further exacerbates the severity and complexity of the disease. Swine influenza virus (SIV) can cause swine influenza, an acute respiratory infectious disease, also known as swine influenza. The disease often occurs in winter and spring, and sick pigs often show acute respiratory symptoms such as coughing, dyspnea, fever and loss of appetite. Although SIV infection is usually not fatal, its high contagiousness and impact on the health of the pig herd cannot be ignored. In addition, SIV has the potential to spread across species, posing a potential threat to public health. Therefore, timely detection and prevention of SIV is of great significance.

[0003] The above three viruses can all infect pigs as hosts, and some of the symptoms are similar. If the infected virus strain is not determined in time, the spread of the virus cannot be intervened in a targeted manner. At present, the detection methods of PRRSV, PCV2 and SIV mainly include ELISA and PCR. Although these technologies can meet the needs to a certain extent, they are only for single pathogens, with long detection cycles, cumbersome operations and high costs, and are difficult to meet the actual needs of monitoring multiple pathogens at the same time. In order to further improve detection efficiency and reduce detection costs, and to intervene and treat infected hosts in time, it is urgent to develop a more comprehensive detection method with higher detection efficiency. Based on this, the present invention is specially proposed.

[0004] Chinese invention patent 2012105059657 discloses a multiplex ligation probe amplification detection kit and primers and probes for simultaneously detecting five swine disease viruses. The primers, probes, and / or multiplex ligation probe amplification detection kit containing the primers and probes described in the present invention can simultaneously detect five important swine disease pathogens, including swine influenza virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gastroenteritis virus, and foot-and-mouth disease virus. The viruses and genes detected by the detection kit are different from those of the present invention, and the steps are cumbersome, which is not suitable for rapid detection in farms.

[0005] Chinese invention patent application 201110221749.5 discloses a CSFV, PRRSV, PCV2 and SIV H9 subtype multiple SYBR Green I real-time fluorescence PCR primer and a detection method thereof, which respectively designs specific primers for amplifying CSFV, PRRSV, PCV2 and SIVH9 subtypes, and simultaneously detects CSFV, PRRSV, PCV2 and SIV H9 subtypes through a quadruple SYBR Green I real-time quantitative PCR detection method, but the genes amplified by its primer pairs are different from those of the present invention, and its positive judgment is based on the range of TM value changes, which has a large error rate. Summary of the invention

[0006] PRRSV, PCV2 and SIV can all use pigs as hosts, and some of their infection symptoms are similar. Although there are separate detection methods in the prior art, they have the defects of low detection efficiency, long detection time and difficulty in distinguishing, thereby delaying the intervention and treatment process of the infected host. In order to overcome the shortcomings of the above-mentioned technology, the present invention provides a multiple fluorescent quantitative PCR kit for simultaneously detecting PRRSV, PCV2 and SIV and its inspection method. The kit includes reaction solution A, reaction solution B, negative control and positive control. It uses a specific primer probe group to perform multiple fluorescent quantitative PCR on a specific sequence of the virus, and determines whether the host is infected with a specific virus by detecting its fluorescent signal. The kit and detection method can simultaneously detect whether there are the above three viruses, and have the advantages of short detection time and high sensitivity, solving the problem of difficulty in distinguishing after mixed infection in clinical practice, and have important clinical significance.

[0007] The present invention adopts the following technical solutions to achieve the above technical effects:

[0008] The present invention provides a primer probe group for multiplex fluorescent quantitative PCR for simultaneously detecting PRRSV, PCV2 and SIV. The primer probe group comprises an upstream primer, a downstream primer and a probe primer, wherein the upstream primer for detecting PRRSV is shown as SEQ ID No.1, the downstream primer for detecting PRRSV is shown as SEQ ID No.2, and the probe primer 1 is shown as SEQ ID No.3; the upstream primer for detecting PCV2 is shown as SEQ ID No.4, the downstream primer for detecting PCV2 is shown as SEQ ID No.5, and the probe primer 2 is shown as SEQ ID No.6; the upstream primer for detecting SIV is shown as SEQ ID No.7, the downstream primer for detecting SIV is shown as SEQ ID No.8, and the probe primer 3 is shown as SEQ ID No.9.

[0009] Preferably, the 5′ ends of the probe primer 1, the probe primer 2 and the probe primer 3 are connected to different fluorescent groups, and the 3′ ends are connected to corresponding quenching groups.

[0010] Preferably, the fluorescent group connected to the 5′ end of the probe primer 1 is FAM, and the quencher group connected to the 3′ end is BHQ1; the fluorescent group connected to the 5′ end of the probe primer 2 is ROX, and the quencher group connected to the 3′ end is BHQ2; the fluorescent group connected to the 5′ end of the probe primer 3 is HEX, and the quencher group connected to the 3′ end is TAMRA.

[0011] Preferably, the primer probe set is used to amplify the ORF6 gene segment of PRRSV, the ORF2 gene segment of PCV2 and the NA gene segment of SIV.

[0012] The present invention also provides a multiplex fluorescent quantitative PCR kit for simultaneously detecting PRRSV, PCV2, and SIV, the kit comprising:

[0013] (1) reaction solution A;

[0014] (2) reaction solution B;

[0015] (3) Positive control;

[0016] (4) Negative control;

[0017] The reaction solution A is 2×PCR Mix, which consists of Taq enzyme 0.2U / μL, dNTP 0.4mM, Tris-HCl (pH 8.3) 20mM, MgCl 2 4mM, KCl 100mM, BSA 2mg / mL; reaction solution B is Primer Mix, which contains the above-mentioned primer probe sets for PRRSV, PCV2, and SIV.

[0018] Preferably, in the above-mentioned multiplex fluorescent quantitative PCR kit for simultaneous detection of PRRSV, PCV2, and SIV, the positive control is a mixture of plasmids constructed according to the ORF6 gene fragment of PRRSV, the ORF2 gene fragment of PCV2, and the NA gene fragment of SIV in a ratio of 1:1:1, and the negative control is sterilized diethyl pyrocarbonate treated water (DEPC-H 2 O).

[0019] The present invention provides a multiplex fluorescence quantitative PCR detection method for detecting whether a sample to be tested is infected with PRRSV, PCV2 and SIV using the above kit, which comprises the following steps:

[0020] 1) Extract the whole genome (including DNA and RNA) from the sample to be tested, and use reverse transcriptase to obtain the reverse transcription product (DNA / cDNA);

[0021] 2) adding the reverse transcription product of the sample to be tested, the positive control, and the negative control as templates into a multiplex fluorescence quantitative PCR reaction system for fluorescence quantitative PCR amplification, wherein the PCR reaction system comprises reaction solution A and reaction solution B in the multiplex fluorescence quantitative PCR kit;

[0022] 3) Start the PCR reaction program and collect the fluorescent signal during the amplification process, and determine whether PRRSV, PCV2 and SIV exist in the sample based on the presence and type of the fluorescent signal.

[0023] Preferably, in the multiplex fluorescent quantitative PCR detection method for simultaneously detecting PRRSV, PCV2 and SIV using the kit as described above, the reaction system of the kit is: 10 μL of reaction solution A, 8 μL of reaction solution B, 2 μL of template, and the total reaction volume is 20 μL.

[0024] Preferably, the step 3) specifically includes the following steps: the PCR program is: performing fluorescent quantitative PCR reaction according to 40 cycles of pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 10 seconds, annealing / extension at 60°C for 30 seconds; collecting the fluorescent signal during the PCR amplification process at the end of each cycle, and making a positive judgment based on the Ct value and amplification curve of a channel gene in the sample to be tested.

[0025] Preferably, the positive judgment standard described in step 3) is: if there is an amplification curve and the Ct value is ≤35, it means that the virus corresponding to the multiple fluorescent quantitative PCR primers and the probe fluorescent group is present in the sample to be tested; if there is no amplification curve, it means that there is no PRRSV, PCV2, or SIV in the sample to be tested.

[0026] The beneficial effects of the present invention are as follows: the present invention uses real-time fluorescence quantitative PCR technology and specific primers for three viruses, PRRSV, PCV2 and SIV, to develop a multiplex real-time fluorescence quantitative PCR rapid diagnosis kit for three viruses. The detection method based on primer pair / probe combination and kit provided by the present invention has high sensitivity, strong specificity and real-time quantitative detection, which lays a foundation for early diagnosis of PRRSV, PCV2 and SIV and providing a reasonable drug administration scheme.

[0027] 1) The present invention sets primer probe groups specifically for the ORF6 gene fragment of PRRSV, the ORF2 gene fragment of PCV2 and the NA gene fragment of SIV, which is more accurate than amplifying the whole genome sequence of the virus to be tested or other genome fragments of the virus to be tested. The present invention has made creative efforts in selecting the gene fragments of the above viruses, so that the specificity of the detection method of the present invention is higher than other detection methods in the prior art. Example 5 of the present invention extracts the genomes of three common viruses that infect pigs, including pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and porcine parvovirus (PPV), to carry out overall specificity experiments. At the same time, a 1:1:1 mixture of plasmids constructed with the ORF6 gene fragment of PRRSV (PQ062578.1), the ORF2 gene fragment of PCV2 (AY651850) and the NA gene fragment of SIV (MT372515) was used as a positive control, and DEPC was used as a negative control, and fluorescent quantitative PCR amplification and detection were performed. The results showed that there was no specific amplification curve for pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and porcine parvovirus (PPV), indicating that the primer probe set selected by the present invention has a strong specificity with the gene sequence of the virus detected by the present invention.

[0028] 2) The present invention sets different probe primers for different viruses, and sets different fluorescence detection signals on the probe primers. Specifically, the fluorescent group connected to the 5′ end of the probe primer 1 for detecting PRRSV is FAM, and the quenching group connected to the 3′ end is BHQ1; the fluorescent group connected to the 5′ end of the probe primer 2 for detecting PCV2 is ROX, and the quenching group connected to the 3′ end is BHQ2; the fluorescent group connected to the 5′ end of the probe primer 3 for detecting SIV is HEX, and the quenching group connected to the 3′ end is TAMRA. When performing multiple fluorescence quantitative PCR, it can be accurately judged whether one or more of PRRSV, PCV2, and SIV are infected according to the presence or absence of fluorescence detection signals and the types of detected fluorescence signals. The judgment is simple and accurate, which is superior to other judgment criteria, such as using the TM value variation range as the judgment basis, and is more suitable for on-site application in farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1(A) PRRSV positive amplification curve and negative control; (B) PCV2 positive amplification curve and negative control; (C) SIV positive amplification curve and negative control;

[0030] Figure 2 This is the triple fluorescence quantitative PCR amplification curve of PRRSV, PCV2, SIV and blank control;

[0031] Figure 3 (A)PRRSV, (B)PCV2, (C)SIV fluorescence quantitative PCR sensitivity test;

[0032] Figure 4 (A) PRRSV, (B) PCV2, (C) SIV fluorescence quantitative PCR standard curve; DETAILED DESCRIPTION

[0033] To further illustrate the present invention, the technical scheme of the present invention is further described below by specific embodiments, but is not intended to limit the scope of the present invention. The materials used in the following examples are not limited to the above-mentioned examples, and can be replaced by other similar materials. If the specific conditions of the instruments are not indicated, conventional conditions or conditions recommended by the manufacturer are used; unless otherwise specified, the reagents and methods used in the present invention are conventional reagents and conventional methods in the art.

[0034] Example 1 Primer probe set for multiplex fluorescence quantitative PCR for simultaneous detection of PRRSV, PCV2, and SIV of the present invention

[0035] The primer probe set of the multiplex fluorescence quantitative PCR for simultaneously detecting PRRSV, PCV2 and SIV of the present invention is mainly used for amplifying the ORF6 gene fragment of PRRSV (PQ062578.1), the ORF2 gene fragment of PCV2 (AY651850) and the NA gene fragment of SIV (MT372515).

[0036] The 5' end of the probe is connected to different fluorescent groups, namely FAM, ROX or HEX; the 3' end is connected to the corresponding quenching group, namely BHQ1, BHQ2 or TAMRA. The primer and probe sequences included in the primer probe set in this embodiment are as follows:

[0037] (1) PRRSV-specific primers and probes

[0038] SEQ ID No. 1 Upstream primer ORF6_purF_F 5′-GGACCGGGAAGAAAGAAA-3′;

[0039] SEQ ID No. 2 downstream primer ORF6_purF_R 5′-TCTCCCGTGTCTTTTCTC-3′;

[0040] SEQ ID No.3 Fluorescent probe ORF6_purF_P 5′-FAM-CCACCAGCGACGAAGAGACG-BHQ1-3′.

[0041] (2) PCV2-specific primers and probes

[0042] SEQ ID No. 4 upstream primer ORF2_purF_F 5′-GGGAAGAAAGTCATTAATATTG-3′;

[0043] SEQ ID No. 5 downstream primer ORF2_purF_R 5′-GCACCTTCGGATATACTA-3′;

[0044] SEQ ID No.6 Fluorescent probe ORF2_purF_P 5′-ROX-ATCTCATCATGTCCACCGCC-BHQ2-3′.

[0045] (3) SIV-specific primers and probes

[0046] SEQ ID No. 5 Upstream primer ORF2_purF_F 5′-CATGCTCTCATTTGGAATG-3′;

[0047] SEQ ID No.6 downstream primer ORF2_purF_R 5′-GCTCATTAAGGTTCTATAAGG-3′;

[0048] SEQ ID No.7 Fluorescent probe ORF2_purF_P 5′-ROX-CCTTGACGGTTCCATTAGAATGCC-BHQ 2-3′.

[0049] Example 2 Establishment of a multiplex fluorescence quantitative PCR detection kit for simultaneous detection of PRRSV, PCV2, and SIV

[0050] A multiplex fluorescence quantitative PCR detection kit for simultaneously detecting PRRSV, PCV2, and SIV, comprising reaction solution A, reaction solution B, positive control, negative control, instructions, and a box body. Reaction solution A is 2×PCR Mix, and the components include: Taq enzyme 0.2U / μL, dNTP 0.4mM, Tris-HCl (pH 8.3) 20mM, MgCl 24mM, KCl 100mM, BSA 2mg / mL; reaction solution B is a mixture of primers and probes including PRRSV, PCV2, and SIV. The positive control is a mixture of plasmids constructed according to the ORF6 gene fragment of P RRSV (PQ062578.1), the ORF2 gene fragment of PCV2 (AY651850) and the NA gene fragment of SIV (MT372515) in a ratio of 1:1:1, the plasmid concentration is 1.0ng / μL, and the corresponding Ct value is 15-25. The negative control is DEPC water.

[0051] Example 3 Construction of the Multiplex Fluorescence Quantitative PCR Reaction System of the Present Invention

[0052] 1) Extract the DNA of the sample to be tested (using a bacterial genomic DNA extraction kit or other recognized extraction methods with the same effectiveness) and store at -20°C for future use.

[0053] 2) After optimizing the conditions of the multiplex fluorescence quantitative PCR reaction system, the final total reaction system of 20 μL is as follows: 10 μL of reaction solution A, 8 μL of reaction solution B, and 2 μL of sample DNA template. The reaction conditions are 95°C pre-denaturation for 10 s; 95°C denaturation for 5 s, 60°C annealing / extension for 30 s, for a total of 40 cycles.

[0054] 3) Result judgment: if a gene channel of the sample to be tested has an amplification curve and the Ct value is ≤35, then the sample to be tested is judged to contain the virus; b) If a gene channel of the sample to be tested does not have an amplification curve and the Ct value is ≥38, then the sample to be tested does not contain the virus; c) If a gene channel of the sample to be tested has an amplification curve and 35<Ct<38, then it is judged to be an uncertain sample and the sample needs to be retested.

[0055] Example 4 Specificity verification of the multiplex fluorescence quantitative PCR detection system of the present invention

[0056] In order to verify the single specificity of the multiplex fluorescence quantitative PCR detection system constructed by the present invention, PRRSV, PC V2, SIV genomic DNA / RNA were extracted and reverse transcribed as PCR reaction templates, and a blank control group and a negative control group were set up at the same time. The blank control was DEPC water, and the negative control was sterilized diethyl pyrocarbonate treated water. The PCR reaction system and reaction conditions were the same as those in Example 3. The multiplex fluorescence quantitative PCR specificity verification results are shown in FIG. Figure 1 As shown, (A) PRRSV, (B) PCV2 and (C) SIV only have amplification curves in the corresponding fluorescence channels, while the blank control and the negative control have no amplification curves in the above three channels. This result shows that the multiplex fluorescence quantitative PCR detection system of the present invention has high single specificity.

[0057] Example 5: Verification of the overall specificity of the multiplex fluorescence quantitative PCR detection system of the present invention

[0058] The genomes of three common viruses that infect pigs, including pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and porcine parvovirus (PPV), were extracted according to the instructions of the extraction kit. A 1:1:1 mixture of plasmids constructed with the ORF6 gene fragment of PRRSV (PQ062578.1), the ORF2 gene fragment of PCV2 (AY651850) and the NA gene fragment of SIV (MT372515) was used as a positive control, and DEPC was used as a negative control. Fluorescence quantitative PCR amplification and detection were performed to determine the specificity of the established multiplex fluorescence quantitative PCR detection. The results showed that the established multiplex fluorescence quantitative PCR detection method had good specificity ( Figure 2 ).

[0059] Example 6: Sensitivity verification of the multiplex fluorescence quantitative PCR detection system of the present invention

[0060] like Figure 3 As shown, the positive plasmids (A: PRRSV, 10~10 6 copies / μL; B: PCV2, 10~10 5 copies / μL; C: SIV, 10~10 6 The sensitivity test of three pairs of primers and probes was carried out using 10 copies / μL) as template. The results showed that the minimum detection value of PRRSV, PCV2 and SIV was 10 copies / μL.

[0061] The standard curve constructed based on the average Ct value is as follows Figure 4 As shown, the correlation coefficient R 2 All of them were greater than 0.99, showing a good linear relationship. The results showed that the minimum detection values ​​of PRRSV, PCV2, and SIV were all 10 copies / μl.

[0062] Example 7: Comparison between the multiplex fluorescence quantitative PCR of the present invention and the conventional PCR+sequencing identification detection method

[0063] To verify its accuracy in clinical application, suspected infected pigs from three farms in Taizhou City, Jiangsu Province, Fengxian District, Shanghai, and Jingzhou City, Hubei Province were selected as test objects. The sample sizes of the three farms are shown in Table 1. The clinical samples were tested using the multiplex fluorescence quantitative PCR detection method established in the present invention (using the multiplex fluorescence quantitative PCR kit of the present invention). To ensure the accuracy of the results, all samples were reviewed by conventional PCR+sequencing identification methods at the same time. The positive test results and infected virus conditions measured by the two methods were compared to determine the compliance of the two detection methods.

[0064] Table 1 Validity verification of the actual detection results of the multiplex fluorescence quantitative PCR of the present invention

[0065]

[0066] The experimental results show that the virus detection results determined by the multiplex fluorescence quantitative PCR detection method are exactly the same as those determined by conventional PCR + sequencing identification, and the consistency between the two is 100%. However, the detection method of the present invention has the technical advantages of intuitive detection results and short detection time, and is suitable for use in rapid detection in farms.

[0067] The above examples show and describe the basic principles and features of the present invention. The results show that the kit of the present invention has the characteristics of good accuracy and high sensitivity, and is suitable for the rapid detection of PRRSV, PCV2 and SIV in farms.

Claims

1. A primer probe set for multiplex fluorescence quantitative PCR for simultaneous detection of PRRSV, PCV2, and SIV, characterized in that: The primer probe set includes an upstream primer, a downstream primer and a probe primer, wherein the upstream primer for detecting PRRSV is shown as SEQ ID No.1, the downstream primer for detecting PRRSV is shown as SEQ ID No.2, and the probe primer 1 is shown as SEQ ID No.3; The upstream primer for detecting PCV2 is shown as SEQ ID No. 4, the downstream primer for detecting PCV2 is shown as SEQ ID No. 5, and the probe primer 2 is shown as SEQ ID No. 6; The upstream primer for detecting SIV is shown as SEQ ID No.7, the downstream primer for detecting SIV is shown as SEQ ID No.8, and the probe primer 3 is shown as SEQ ID No.

9.

2. The primer probe set for simultaneous detection of PRRSV, PCV2, and SIV by multiplex fluorescence quantitative PCR according to claim 1, characterized in that: The 5' ends of the probe primer 1, the probe primer 2 and the probe primer 3 are connected with different fluorescent groups, and the 3' ends are connected with corresponding quenching groups.

3. The primer probe set for the multiplex fluorescent quantitative PCR for simultaneous detection of PRRSV, PCV2, and SIV according to claim 1, characterized in that: The fluorescent group connected to the 5' end of the probe primer 1 is FAM, and the quenching group connected to the 3' end is BHQ1; the fluorescent group connected to the 5' end of the probe primer 2 is ROX, and the quenching group connected to the 3' end is BHQ2; the fluorescent group connected to the 5' end of the probe primer 3 is HEX, and the quenching group connected to the 3' end is TAMRA.

4. The primer probe set for simultaneous detection of PRRSV, PCV2, and SIV by multiplex fluorescence quantitative PCR according to claim 1, characterized in that: The primer probe set is used to amplify the ORF6 gene segment of PRRSV, the ORF2 gene segment of PCV2 and the NA gene segment of SIV.

5. A multiplex fluorescence quantitative PCR kit for simultaneous detection of PRRSV, PCV2, and SIV, characterized in that: The kit comprises: (1) reaction solution A; (2) reaction solution B; (3) Positive control; (4) Negative control; Reaction solution A is 2×PCR Mix, which consists of Taq enzyme 0.2U / μL, dNTP 0.4mM, Tris-HCl 20mM, MgCl2 4mM, KCl 100mM, BSA 2mg / mL; reaction solution B is Primer Mix, which contains the primer probe set described in claim 1.

6. The multiplex fluorescent quantitative PCR kit for simultaneous detection of PRRSV, PCV2, and SIV according to claim 5, characterized in that: The positive control is a mixture of plasmids constructed according to the ORF6 gene fragment of PRRSV, the ORF2 gene fragment of PCV2 and the NA gene fragment of SIV in a ratio of 1:1:1, and the negative control is sterilized water treated with diethyl pyrocarbonate.

7. A method for simultaneously detecting whether a sample to be tested is infected with PRRSV, PCV2 and SIV using the kit according to claim 5, comprising the following steps: 1) Extracting the whole genome of the sample to be tested and obtaining the reverse transcription product using reverse transcriptase; 2) adding the reverse transcription product of the sample to be tested, the positive control, and the negative control as templates into a multiplex fluorescence quantitative PCR reaction system for fluorescence quantitative PCR amplification, wherein the PCR reaction system comprises reaction solution A and reaction solution B in the multiplex fluorescence quantitative PCR kit; 3) Start the PCR reaction program and collect the fluorescent signal during the amplification process, and determine whether PRRSV, PCV2 and SIV exist in the sample based on the presence and type of the fluorescent signal.

8. The multiplex fluorescence quantitative PCR detection method for simultaneously detecting PRRSV, PCV2 and SIV according to claim 7, characterized in that: The reaction system of the kit is: add 10 μL of reaction solution A, 8 μL of reaction solution B, and 2 μL of template, and the total reaction volume is 20 μL.

9. The method for simultaneously detecting whether a sample to be tested is infected with PRRSV, PCV2 and SIV according to claim 7, characterized in that: The step 3) specifically includes the following steps: the PCR program is: perform fluorescence quantitative PCR reaction according to 40 cycles of pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 10 seconds, annealing / extension at 60°C for 30 seconds; collect the fluorescence signal during the PCR amplification process at the end of each cycle, and make a positive judgment based on the Ct value and amplification curve of a channel gene in the sample to be tested.

10. The multiplex fluorescence quantitative PCR detection method for simultaneously detecting PRRSV, PCV2 and SIV according to claim 9, characterized in that: The positive judgment standard is: if there is an amplification curve and the Ct value is ≤35, it means that the virus corresponding to the multiple fluorescent quantitative PCR primers and probe fluorescent groups is present in the sample to be tested; if there is no amplification curve, it means that there is no PRRSV, PCV2, or SIV in the sample to be tested.

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