CN110512027B - Quintuple RT-PCR detection method for pig viral diarrhea pathogen - Google Patents
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Abstract
The application discloses a quintuple RT-PCR detection method of porcine viral diarrhea pathogen, wherein 5 pairs of primers are respectively detection primers of porcine delta coronavirus (also known as porcine delta coronavirus), detection primers of porcine epixon virus, detection primers of porcine epidemic diarrhea virus, detection primers of porcine acute diarrhea syndrome coronavirus and detection primers of transmissible gastroenteritis virus. The nucleic acid or reverse transcription product of the sample to be detected is used as a template, 2 XTaq Plus Master Mix is adopted, and 5 pairs of primers in the application are used for PCR amplification. This patent allows a maximum of 5 viruses causing diarrhea in a swine herd to be simultaneously detected in one PCR reaction tube with a sensitivity of 1X 10 initial template concentration per microliter3One copy (1X 10)3 copies/. mu.L) and has excellent specificity and repeatability, and can be directly applied to clinical sample detection.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a quintuple RT-PCR detection technology for pig viral diarrhea pathogens.
Background
In recent years, the morbidity and mortality of viral diarrhea diseases of Chinese and even global swinery are high, and huge economic losses are brought to the live pig breeding industry. In various places of China, reports of various porcine diarrhea virus mixed infection swinery frequently appear, and the mixed infection often misleads clinical veterinarians to diagnosis and analysis of disease prevalence conditions. Therefore, it is not very gentle to strengthen the control of porcine viral diarrhea-type diseases with strong pathogenicity and infectivity, including PDCoV, PEDV, PToV, Sads-CoV, TGEV. Firstly, establishing a convenient, rapid and economic detection method for porcine viral diarrhea diseases for clinical diagnosis is very necessary.
The quintuple RT-PCR detection technology can identify five pathogens simultaneously, greatly shortens the material cost and the time cost of detection personnel in the detection process, and is particularly suitable for large-scale sample detection and mixed infection detection of samples. However, because there are many pairs of primers in the quintuple RT-PCR system, it is necessary to establish an effective quintuple RT-PCR detection method, and it is necessary to maintain high specificity among the primer pairs to avoid the occurrence of non-specific amplification products, to ensure that there is a significant difference in the size of the amplified target fragments, to conveniently distinguish different detection results by electrophoresis or other methods, to avoid the interaction among the primers used to avoid the generation of primer dimers, and to maintain relatively consistent amplification efficiency to facilitate the estimation of template amount. In addition, the more complex nucleic acid environment in the template, the different annealing temperatures required by different primer pairs, are also major factors that restrict the establishment of quintuple, even more, RT-PCR detection methods. Therefore, the establishment of a stable, efficient, economic and convenient quintuple RT-PCR detection technology for the porcine diarrhea disease has great clinical production value. The barrier is heavy, so the quintuple RT-PCR technology is valuable, but is difficult to realize.
Disclosure of Invention
The technical problem to be solved is as follows: the application mainly provides a quintuple RT-PCR detection method of the pig viral diarrhea pathogen, and solves the technical problems that the effect of the multiplex RT-PCR technology is not good when the multiplex RT-PCR technology is applied to the diagnosis of the pig viral diarrhea disease and the like.
The technical scheme is as follows: the quintuple RT-PCR detection method of the pathogeny of the porcine viral diarrhea disease comprises the following steps:
firstly, designing specific RT-PCR detection primer pair sequences of 5 viruses:
primer pair sequences for detecting Porcine Deltacoronavirus (PDCoV):
the primer sequence of the upstream primer PD-detection (F) is 5'-TACAACCTAAGGCTAACCAAC-3',
the primer sequence of the downstream primer PD-detection (R) is 5'-ACAACTTTACCTGCCTTACATA-3';
primer pair sequences for detecting Porcine epiovirus (PToV):
the primer sequence of the upstream primer PT-detection (F) is 5'-TCCTCGTGACACTTTATCTCA-3',
the primer sequence of the downstream primer PT-detection (R) is 5'-TTCACTAACATCCTCTACCACA-3';
primer pair sequences for detecting Porcine Epidemic Diarrhe Virus (PEDV):
the primer sequence of the upstream primer PE-detection (F) is 5'-TGAAGACTTTTGACAATCCAC-3',
the primer sequence of the downstream primer PE-detection (R) is 5'-ACACAGCATTACCATCTAC-3';
primer pair sequences for detecting porcine Acute Diarrhea Syndrome Coronavirus (Swine Acute Diarrhae Syndrome Coronavir, Sads-CoV):
the primer sequence of the upstream primer SD-detection (F) is 5'-GCTCAGTACATCTTTTCAACT-3',
the primer sequence of the downstream primer SD-detection (R) is 5'-CATAGTCATACTCGCTACCTT-3';
primer pair sequences for detecting Transmissible gastroenteritis virus (TGEV):
the primer sequence of the upstream primer TG-detection (F) is 5'-TACATACCACCTGCTTATGCAA-3', and the primer sequence of the downstream primer TG-detection (R) is 5'-TACACAATTGACCAATCAACAC-3';
step two, obtaining cDNA of a sample to be detected: extracting total RNA of a diarrhea fecal sample or intestinal tissues, and performing reverse transcription to obtain a cDNA template;
and step three, detecting by using 5 pairs of RT-PCR primers: placing the primer pairs of the PDCoV, the PToV, the PEDV, the Sads-CoV and the TGEV into a PCR reaction system, and performing PCR amplification by taking the cDNA in the second step as a template;
the fourth step: the amplified products are detected by agarose gel electrophoresis, and whether PDCoV (148 bp), PToV (274 bp), PEDV (411 bp), Sads-CoV (574 bp) and TGEV (833 bp) exist in the sample can be determined according to the position of an electrophoretic band.
As a preferred technical scheme of the invention: in the second step, reverse transcription is carried out by using a reverse Aid First Strand cDNA Synthesis Kit, wherein the reaction overall system is 20 mu L, and the reaction overall system comprises the following steps: 1 mu L Random Hexamer Primer, 1 mu L Oligo (dT)18Primer, 5 mu L nuclear-free Water, 5 mu L total RNA, uniformly mixing, incubating at 65 ℃ for 5min, carrying out ice bath for 3 min, and continuously adding: 4 μ L5 × Reaction Buffer, 2 μ L10 × dNTP premix, 1 μ L RevertAId M-MuLV Reverse Transcriptase (RT) and 1 μ L RiboLock RNase Inhibitor (RI).
As a preferred technical scheme of the invention: the PCR reaction system in the third step is as follows: 10 μ L2 XTaq Plus Master Mix, 5 μ L ddH2O, PD-detection (F), PD-detection (R), PT-detection (F), PT-detection (R), PE-detection (F), PE-detection (R), SD-detection (F), SD-detection (R), TG-detection (F) and TG-detection (R), each 0.4 mu L and cDNA template 1 mu L. Wherein the concentration of each primer is 10 muM, and the total volume of the system is 20 muL.
As a preferred technical scheme of the invention: the reaction procedure during reverse transcription in the second step is as follows: 5min at 25 ℃, 60min at 42 ℃ and 5min at 70 ℃ to obtain a cDNA template after reaction.
As a preferred technical scheme of the invention: the PCR reaction procedure in the third step is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10 s, annealing at 56 ℃ for 10 s, extension at 72 ℃ for 50 s, and 35 cycles; finally, extension was carried out at 72 ℃ for 2 min.
As a preferred technical scheme of the invention: in the third step, the concentration of each primer of PDCoV, PToV, PEDV, Sads-CoV and TGEV is 10 mu M.
Has the advantages that: compared with the prior art, the quintuple RT-PCR detection method for the pig viral diarrhea pathogen has the following technical effects by adopting the technical scheme:
1. the method can detect the porcine viral diarrhea virus in one PCR reaction tube at most, and provides a simple, convenient, efficient and low-cost method for detecting the porcine viral diarrhea pathogen;
2. the quintuple RT-PCR detection method is used for detecting positive samples of the target viruses, the detection result is consistent with the single RT-PCR detection result, and the feasibility of the method is further proved. Meanwhile, the verification proves that the detection sensitivity of the kit to each pathogen can reach the initial template concentration of 1 multiplied by 103 Individual copies (copies) with excellent specificity and reproducibility;
3. the establishment of the detection method in the application provides reliable basis for the prevention and control of the diseases, thereby greatly reducing the workload of single RT-PCR detection and greatly improving the working efficiency;
4. the detection method established in the application has high repeatability and sensitivity, can be directly used for clinical detection, and can be used for diagnosing and monitoring various porcine viral diarrhea pathogens through the rapid and efficient detection of the five diarrhea viruses.
Description of the drawings:
FIG. 1 is a diagram showing the detection result of five-fold RT-PCR detection method for detecting the positive clinical disease of five target viruses in the swine viral diarrhea pathogen.
FIG. 2 is a diagram showing the results of the five-fold RT-PCR detection method for detecting five target viruses of porcine viral diarrhea pathogens according to the present application.
FIG. 3 is a diagram showing the results of the specificity test for detecting various non-target viruses by the quintuple RT-PCR detection method for porcine viral diarrhea pathogens of the present application.
FIG. 4 is a result diagram of the validity test for detecting five target viruses by the application of the quintuple RT-PCR detection method for porcine viral diarrhea pathogens.
FIG. 5 is a result diagram of a repeat test for detecting five target viruses by the method for detecting pig viral diarrhea pathogen by quintuple RT-PCR.
FIG. 6 is a result diagram of the optimal primer concentration screening of the quintuple RT-PCR detection method for the pig viral diarrhea pathogen of the present application.
FIG. 7 is a diagram showing the result of the screening of the optimal annealing temperature of the primers in the reaction procedure of the quintuple RT-PCR detection method for the pathogen of porcine viral diarrhea of the present application.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples, but the present invention is not limited to these examples.
The sequence of the specific RT-PCR detection primer pair for 5 viruses designed by the application is synthesized by Biotechnology engineering (Shanghai) GmbH, the purification mode is HAP purification, and reverse transcription reaction is purchased from Sammer Feishale science and technology (China) GmbH by using a reverse air First Strand cDNA Synthesis Kit.
Example 1
A quintuple RT-PCR detection method of porcine viral diarrhea pathogeny,
firstly, designing a specific RT-PCR detection primer pair of 5 viruses:
primer pair sequences for detecting Porcine Deltacoronavirus (PDCoV):
the primer sequence of the upstream primer PD-detection (F) is 5'-TACAACCTAAGGCTAACCAAC-3',
the primer sequence of the downstream primer PD-detection (R) is 5'-ACAACTTTACCTGCCTTACATA-3';
primer pair sequences for detecting Porcine epiovirus (PToV):
the primer sequence of the upstream primer PT-detection (F) is 5'-TCCTCGTGACACTTTATCTCA-3',
the primer sequence of the downstream primer PT-detection (R) is 5'-TTCACTAACATCCTCTACCACA-3';
primer pair sequences for detecting Porcine Epidemic Diarrhe Virus (PEDV):
the primer sequence of the upstream primer PE-detection (F) is 5'-TGAAGACTTTTGACAATCCAC-3',
the primer sequence of the downstream primer PE-detection (R) is 5'-ACACAGCATTACCATCTAC-3';
primer pair sequences for detecting porcine Acute Diarrhea Syndrome Coronavirus (Swine Acute Diarrhae Syndrome Coronavir, Sads-CoV):
the primer sequence of the upstream primer SD-detection (F) is 5'-GCTCAGTACATCTTTTCAACT-3',
the primer sequence of the downstream primer SD-detection (R) is 5'-CATAGTCATACTCGCTACCTT-3';
primer pair sequences for detecting Transmissible gastroenteritis virus (TGEV):
the primer sequence of the upstream primer TG-detection (F) is 5'-TACATACCACCTGCTTATGCAA-3',
the primer sequence of the downstream primer TG-detection (R) is 5'-TACACAATTGACCAATCAACAC-3';
step two, obtaining cDNA of a sample to be detected: extracting total RNA of a diarrhea fecal sample or intestinal tissues, carrying out reverse transcription to obtain a cDNA template, carrying out reverse transcription by using a reverse Aid First Strand cDNA Synthesis Kit, wherein the reaction total system is 20 mu L and comprises the following steps: 1 mu L Random Hexamer Primer, 1 mu L Oligo (dT)18Primer, 5 mu L nuclear-free Water, 5 mu L total RNA, uniformly mixing, incubating at 65 ℃ for 5min, carrying out ice bath for 3 min, and continuously adding: 4 mu.L 5 × Reaction Buffer, 2 mu.L 10 × dNTP premix, 1 mu.L RevertAId M-MuLV Reverse Transcriptase (RT) and 1 mu.L RiboLock RNase Inhibitor (RI); the reaction procedure during reverse transcription is as follows: 5min at 25 ℃, 60min at 42 ℃ and 70 DEG C5min, and obtaining a cDNA template after reaction;
and step three, detecting by using 5 pairs of RT-PCR primers: putting primer pairs of PDCoV, PToV, PEDV, Sads-CoV and TGEV with the concentration of 10 mu M into a PCR reaction system, wherein the PCR reaction system is as follows: 10 μ L2 XTaq Plus Master Mix, 5 μ LddH2O, PD-detection (F), PD-detection (R), PT-detection (F), PT-detection (R), PE-detection (F), PE-detection (R), SD-detection (F), SD-detection (R), TG-detection (F) and TG-detection (R) are respectively 0.4 mu L, the cDNA template is 1 mu L, and the total volume of the system is 20 mu L; and (3) performing PCR amplification by using the cDNA in the second step as a template, wherein the PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10 s, annealing at 56 ℃ for 10 s, extension at 72 ℃ for 50 s, 35 cycles; finally, extending for 2 min at 72 ℃;
the fourth step: the amplified products are detected by agarose gel electrophoresis, and whether PDCoV (148 bp), PToV (274 bp), PEDV (411 bp), Sads-CoV (574 bp) and TGEV (833 bp) exist in the sample can be determined according to the position of an electrophoretic band.
As shown in fig. 1, five clinical specimens positive for the target viruses were tested, including 31 PDCoV clinical positive samples, 31 PEDV clinical positive samples, 15 PToV clinical positive samples, 3 Sads-CoV clinical positive samples, and 7 TGEV clinical positive samples. 8 mixed infection samples of PDCoV and PEDV, 4 mixed infection samples of PDCoV and PToV, and 4 mixed infection samples of PEDV and PToV. The detection result completely accords with the actual infection condition of the sample, which indicates that the kit can be applied to clinical detection.
FIG. 3 shows the result of the specificity test for detecting various non-target viruses by the quintuple RT-PCR detection method for porcine viral diarrhea pathogens of the present application. The DNA or cDNA templates of the non-target viruses in the sample wells 1-12 are respectively Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), Porcine Parvovirus (PPV), porcine circovirus type 2 (PCV 2), porcine circovirus type 3 (PCV 3), Japanese Encephalitis Virus (JEV), pseudorabies virus (PRV), Porcine Sapelovirus (PSV), Porcine Kobuvirus (PKV), Porcine Astrovirus (PASTV), Porcine Teschovirus (PTV) and porcine Sapovirus (PSaV). No non-specific amplified band was observed.
As shown in FIG. 5, the result of the repetitive test for detecting five target viruses by the quintuple RT-PCR detection method for porcine viral diarrhea pathogens of the application is shown, and the initial template concentration is 1 × 104copies/. mu.L of a single target-positive plasmid standard and an initial template concentration of 1X 104Duplicate experiments were performed using 5 premixes of positive plasmid standards, copies/μ L, as cDNA templates, with 3 replicates per batch in each case at three different times. It can be seen that 9 parallel test groups all can obtain consistent detection results.
As shown in FIG. 7, the test result of the optimal primer annealing temperature screening in the reaction program of the quintuple RT-PCR detection method for the pig viral diarrhea pathogen is shown. The annealing temperatures in the sample wells 1-12 were set to 50.0 deg.C, 50.3 deg.C, 51.0 deg.C, 52.2 deg.C, 53.7 deg.C, 55.3 deg.C, 56.7 deg.C, 58.3 deg.C, 59.8 deg.C, 61.0 deg.C, 61.7 deg.C, and 62.0 deg.C, respectively. The annealing temperature is recommended to be 56 ℃ in the present detection method.
Example 2
The quintuple RT-PCR detection method of the pathogeny of the porcine viral diarrhea disease comprises the following steps:
firstly, designing a specific RT-PCR detection primer pair of 5 viruses:
primer pair sequences for detecting Porcine Deltacoronavirus (PDCoV):
the primer sequence of the upstream primer PD-detection (F) is 5'-TACAACCTAAGGCTAACCAAC-3',
the primer sequence of the downstream primer PD-detection (R) is 5'-ACAACTTTACCTGCCTTACATA-3';
primer pair sequences for detecting Porcine epiovirus (PToV):
the primer sequence of the upstream primer PT-detection (F) is 5'-TCCTCGTGACACTTTATCTCA-3',
the primer sequence of the downstream primer PT-detection (R) is 5'-TTCACTAACATCCTCTACCACA-3';
primer pair sequences for detecting Porcine Epidemic Diarrhe Virus (PEDV):
the primer sequence of the upstream primer PE-detection (F) is 5'-TGAAGACTTTTGACAATCCAC-3',
the primer sequence of the downstream primer PE-detection (R) is 5'-ACACAGCATTACCATCTAC-3';
primer pair sequences for detecting porcine Acute Diarrhea Syndrome Coronavirus (Swine Acute Diarrhae Syndrome Coronavir, Sads-CoV):
the primer sequence of the upstream primer SD-detection (F) is 5'-GCTCAGTACATCTTTTCAACT-3',
the primer sequence of the downstream primer SD-detection (R) is 5'-CATAGTCATACTCGCTACCTT-3';
primer pair sequences for detecting Transmissible gastroenteritis virus (TGEV):
the primer sequence of the upstream primer TG-detection (F) is 5'-TACATACCACCTGCTTATGCAA-3',
the primer sequence of the downstream primer TG-detection (R) is 5'-TACACAATTGACCAATCAACAC-3';
step two, obtaining cDNA of a sample to be detected: extracting total RNA of a diarrhea fecal sample or intestinal tissues, carrying out reverse transcription to obtain a cDNA template, carrying out reverse transcription by using a reverse Aid First Strand cDNA Synthesis Kit, wherein the reaction total system is 20 mu L and comprises the following steps: 1 mu L Random Hexamer Primer, 1 mu L Oligo (dT)18Primer, 5 mu L nuclear-free Water, 5 mu L total RNA, uniformly mixing, incubating at 65 ℃ for 5min, carrying out ice bath for 3 min, and continuously adding: 4 mu.L 5 × Reaction Buffer, 2 mu.L 10 × dNTP premix, 1 mu.L RevertAId M-MuLV Reverse Transcriptase (RT) and 1 mu.L RiboLock RNase Inhibitor (RI); the reaction procedure during reverse transcription is as follows: 5min at 25 ℃, 60min at 42 ℃ and 5min at 70 ℃ to obtain a cDNA template after reaction;
and step three, detecting by using 5 pairs of RT-PCR primers: putting primer pairs of PDCoV, PToV, PEDV, Sads-CoV and TGEV with the concentration of 10 mu M into a PCR reaction system, wherein the PCR reaction system is as follows: 10 mu L2 XTaq Plus Master Mix, 5 muddH of L2O, PD-detection (F), PD-detection (R), PT-detection (F), PT-detection (R), PE-detection (F), PE-detection (R), SD-detection (F), SD-detection (R), TG-detection (F) and TG-detection (R) are respectively 0.4 mu L, the cDNA template is 1 mu L, and the total volume of the system is 20 mu L; and (3) performing PCR amplification by using the cDNA in the second step as a template, wherein the PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10 s, annealing at 56 ℃ for 10 s, extension at 72 ℃ for 50 s, 35 cycles; finally, extending for 2 min at 72 ℃;
the fourth step: the amplified products are detected by agarose gel electrophoresis, and whether PDCoV (148 bp), PToV (274 bp), PEDV (411 bp), Sads-CoV (574 bp) and TGEV (833 bp) exist in the sample can be determined according to the position of an electrophoretic band.
As shown in figure 2, the result of the accuracy test for detecting five target viruses by the quintuple RT-PCR detection method for the porcine viral diarrhea pathogens of the application shows that the detection accuracy of PDCoV, PToV, PEDV, Sads-CoV and TGEV reaches the initial template concentration of 1 × 103The sensitivity of copies and the simultaneous detection of 5 virus mixed solutions can still reach the initial template concentration of 1 × 103 copies。
As shown in FIG. 4, the result of the effectiveness test for detecting five target viruses by the application of the quintuple RT-PCR detection method for the pig viral diarrhea pathogen is shown. The templates in sample wells 1-29 are PDCoV, PToV, PEDV, Sads-CoV, TGEV and mixtures of different mixed modes thereof. It can be seen that 5 viruses can be effectively detected under the condition of 29 combinations.
As shown in FIG. 6, the result of the primer final concentration screening test in the reaction system of the quintuple RT-PCR detection method for the pig viral diarrhea pathogen is shown. In sample wells 1-9, the final concentrations of 5 pairs of specific amplification primers were set at 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 75 nM, 50 nM, respectively, and ddH was used2O as a negative control. The concentration of the primer in the present detection method is recommended to be 200 nM.
The above examples are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the examples, and any other changes and modifications without departing from the principle of the present invention should be considered as the protection scope of the present invention.
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Five-fold RT-PCR detection method for porcine viral diarrhea pathogen
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Claims (6)
1. A quintuple RT-PCR detection method for pig viral diarrhea pathogens, which is used for non-disease diagnosis and is characterized by comprising the following steps:
firstly, designing specific RT-PCR detection primer pair sequences of 5 viruses:
the primer pair sequence for detecting the porcine delta coronavirus PDCoV is as follows: the primer sequence of the upstream primer PD-Detection F is 5'-TACAACCTAAGGCTAACCAAC-3',
the primer sequence of the downstream primer PD-DetectionR is 5'-ACAACTTTACCTGCCTTACATA-3';
primer pair sequences for detecting porcine epiregulon virus PToV:
the primer sequence of the upstream primer PT-Detection F is 5'-TCCTCGTGACACTTTATCTCA-3',
the primer sequence of the downstream primer PT-Detection R is 5'-TTCACTAACATCCTCTACCACA-3';
primer pair sequences for detecting porcine epidemic diarrhea virus PEDV:
the primer sequence of the upstream primer PE-detectinfF is 5'-TGAAGACTTTTGACAATCCAC-3',
the primer sequence of the downstream primer PE-Detection R is 5'-ACACAGCATTACCATCTAC-3';
the primer pair sequence for detecting the porcine acute diarrhea syndrome coronavirus Sads-CoV is as follows:
the primer sequence of the upstream primer SD-Detection F is 5'-GCTCAGTACATCTTTTCAACT-3',
the primer sequence of the downstream primer SD-Detection R is 5'-CATAGTCATACTCGCTACCTT-3';
primer pair sequences for detecting transmissible gastroenteritis virus TGEV:
the primer sequence of the upstream primer TG-Detection F is 5'-TACATACCACCTGCTTATGCAA-3', and the primer sequence of the downstream primer TG-Detection R is 5'-TACACAATTGACCAATCAACAC-3';
step two, obtaining cDNA of a sample to be detected: extracting total RNA of a diarrhea fecal sample or intestinal tissues, and performing reverse transcription to obtain a cDNA template;
and step three, detecting by using 5 pairs of RT-PCR primers: placing the primer pairs of the PDCoV, the PToV, the PEDV, the Sads-CoV and the TGEV into a PCR reaction system, and performing PCR amplification by taking the cDNA in the second step as a template;
the fourth step: detecting the amplification product by agarose gel electrophoresis, and determining whether PDCoV, PToV, PEDV, Sads-CoV and TGEV exist in the sample according to the position of an electrophoretic band; the presence of PDCoV in the sample is determined when a band of 148bp is present, the presence of PToV in the sample is determined when a band of 274bp is present, the presence of PEDV in the sample is determined when a band of 411bp is present, the presence of Sads-CoV in the sample is determined when a band of 574bp is present, and the presence of TGEV in the sample is determined when a band of 833bp is present.
2. The quintuple RT-PCR detection method of porcine viral diarrhea pathogens of claim 1, wherein: in the second step, reverse transcription is carried out by using a reverse Aid First Strand cDNA Synthesis Kit, wherein the reaction overall system is 20 mu L, and the reaction overall system comprises the following steps: 1 mu L Random Hexamer Primer, 1 mu L OligodT18Primer, 5 mu L nuclear-free Water, 5 mu L total RNA, uniformly mixing, incubating at 65 ℃ for 5min, carrying out ice bath for 3 min, and continuously adding: 4 μ L5 × Reaction Buffer, 2 μ L10 mM dNTP premix, 1 μ L RevertAId M-MuLV Reverse transcriptase and 1 μ L RiboLock RNase Inhibitor.
3. The quintuple RT-PCR detection method of porcine viral diarrhea pathogens of claim 1, wherein: the PCR reaction system in the third step is as follows: 10 μ L2 XTaq Plus Master Mix, 5 μ L ddH2O, 0.4 mu L of each of PD-Detection F, PD-Detection R, PT-Detection F, PT-Detection R, PE-Detection F, PE-Detection R, SD-Detection F, SD-Detection R, TG-Detection F and TG-Detection R, 1 mu L of cDNA template and 20 mu L of total system volume.
4. The quintuple RT-PCR detection method of porcine viral diarrhea pathogens of claim 1, wherein: the reaction procedure during reverse transcription in the second step is as follows: 5min at 25 ℃, 60min at 42 ℃ and 5min at 70 ℃ to obtain a cDNA template after reaction.
5. The quintuple RT-PCR detection method of porcine viral diarrhea pathogens of claim 1, wherein: the PCR reaction procedure in the third step is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10 s, annealing at 56 ℃ for 10 s, extension at 72 ℃ for 50 s, 35 cycles; finally, extension was carried out at 72 ℃ for 2 min.
6. The quintuple RT-PCR detection method of porcine viral diarrhea pathogens of claim 1, wherein: in the third step, the concentrations of the primer pairs of PDCoV, PToV, PEDV, Sads-CoV and TGEV are all 10 mu M.
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