AU2021105359A4

AU2021105359A4 - Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus

Info

Publication number: AU2021105359A4
Application number: AU2021105359A
Authority: AU
Inventors: Liqun Hu; Tong Li; Guoping Liu; Yu Wang; Hongtao XIE
Original assignee: Yangtze University
Current assignee: Yangtze University
Priority date: 2021-08-12
Filing date: 2021-08-12
Publication date: 2021-10-14
Anticipated expiration: 2029-08-12

Abstract

The invention provides Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus. The RT-nested PCR primers are two pairs of primers with degenerate bases obtained by coding the base sequence in the NSP2 gene of porcine porcine reproductive and respiratory syndrome virus. The detection method comprises the following steps of: Extracting RNA of a sample to be detected, carrying out PCR amplification twice by using two pairs of primers, carrying out electrophoresis detection on the amplified products in each time, and distinguishing porcine classic strains, highly pathogenic variant strains and NADC 30 strains according to the size of target gene fragments. The RT-nested PCR detection detection method provided by the invention is simple, reliable, strong in specificity, high in sensitivity and good in anti-interference performance, overcomes the problems of low sensitivity and poor specificity of ordinary RT-PCR detection, and needs to detect the classical strain of porcine reproductive and respiratory syndrome , the highly pathogenic variant strain and NADC-30 respectively, and has low cost, short detection period and good application prospect.

Description

Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus

TECHNICAL FIELD The invention relates to the technical field of molecular diagnosis, in particular to a universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus

BACKGROUND Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), also known as Porcine porcine reproductive and respiratory syndrome virus, is characterized by abortion, stillbirth, weak birth, mummy birth, dyspnea, septicemia and high mortality of piglets and finishing pigs. The disease is widespread all over the world, causing serious economic losses to the pig industry in China. Porcine Reproductive and Respiratory Syndrome Virus belongs to the order Nidovirales, Arteriviridac and Arterivirirus. Its genome is a single-stranded positive strand RNA virus, which contains 5' non-coding region and 10 open reading frames (ORF1a, ORF2a, ORF1b, ORF2b, ORF3-7, ORF5a) and encoded viral structural proteins (GP2, GP3, GP4, GP5, M, N) and 3'-terminal UTR, with a length of about 15kb. At present, according to the antigenic characteristics of PRRSV, it can be divided into two serotypes: European serotype and American serotype, and American serotype strain can be divided into three subtypes according to the deletion of NSP2 base number of nonstructural protein, which are classic strain, highly pathogenic variant strain and NADC-30 strain. In recent years, the variation of PRRSV genome has attracted people's attention, and it is also one of the important reasons that make the disease difficult to control. At present, the detection method of porcine reproductive and respiratory syndrome virus is mainly laboratory detection. Etiological detection techniques mainly include ordinary RT-PCR, fluorescent quantitative RT-PCR, etc. The main serological detection method is antibody detection. However, using a single pair of primers to amplify three subtypes by PCR has some disadvantages, such as low sensitivity, complicated operation, long detection time, etc., and it is impossible to know the genetic characteristics of PRRS in test samples in time. According to the invention, a nested RT PCR method is established on the basis of a single conventional RT-PCR, and different target fragments are amplified by two pairs of primers to distinguish pig classic strains, highly pathogenic variant strains and NADC-30 strains, so as to improve the sensitivity and specificity of detection. RT-nested PCR is a variant polymerase chain reaction, which uses two pairs of PCR primers to amplify the target fragment. After twice PCR amplification, the sensitivity of ordinary PCR can be greatly improved, and the probability of nonspecific binding of primers in the second PCR is extremely low. According to the merger of codons, one symbol is often used to replace two or more bases, and the changed symbols are degenerate bases. R and Y in the primers designed in the patent are degenerate bases, so that different bands of classical strains, highly pathogenic variant strains and NADC can be amplified. And only one can really be paired with the template. Sequence analysis showed that the amino acids of NSP2 protein of epidemic strain were highly conserved and could be used as the target antigen for clinical diagnosis.

SUMMARY In view of the shortcomings of the prior art, the invention aims to provide a rapid and simple nested RT-PCR detection method for porcine porcine reproductive and respiratory syndrome , which not only can quickly distinguish different virus subtypes infected with porcine porcine reproductive and respiratory syndrome , but also has the characteristics of strong specificity, high sensitivity and the like. The invention is realized by the following technical scheme: Design of nested RT-PCR primers for porcine reproductive and respiratory syndrome virus: Two pairs of nested detection primers with degenerate bases were obtained by coding the base sequence in NSP2 gene of porcine reproductive and respiratory syndrome virus. The two pairs of detection primers were outer primers PRRSV-O-F, PRRSV-O-R and inner primers PRRSV-1-F, PRRSV-1-R. The sequences are as follows: PRRSV-O-F: AATCTTGARGAATGCTTGGC(SEQ ID NO.1)

PRRSV-O-R: GCTGAGTAYTTTGGGCGTG(SEQ ID NO.2) PRRSV-1-F: TTCTTTGATTGGRATGTTGTGC(SEQ ID NO.3) PRRSV-1-R: CAAGGAGCTGCTTGAYGACAC(SEQ ID NO.4) In which degenerate base R represents A or G, and degenerate base Y represents C or T. The establishment of a triple nested RT-PCR detection method for porcine reproductive and respiratory syndrome virus comprises the following steps: S1. Extracting RNA of a sample to be detected. S2. The first amplification: the RNA extracted from S1 is used as the template, and the outer primers are used for the first amplification. the PCR reaction system is: 2x1Step Buffer 1 2 .5pl, PrimeScript 1Step Enzyme Mix 1pl, 20pmol/L PRRSV-O-F and 20pmol/L PRRSV-0-R each 1pl, and RNA template 0.5pl I. PCR reaction procedure is as follows: reverse transcription at 500 C for 30min, pre-denaturation at 940 C for 2min, denaturation at 940 C for 30s, annealing at 530 C for 30s, extension at 720 C for 1min, 35 cycles, extension at 720 C for 8min, electrophoresis detection of PCR products with 1% agarose gel, and second amplification if there is no target band. S3. Second-round amplification: using S2 amplification product diluted 50 times as template, using inner primers to carry out second-round amplification, the PCR reaction system is: Mix 1 2 .5pl, PRRSV-1-F 20pmol/L and PRRSV-1-R 20pmol/L each 1pl, template 1pl, RNase Free dH20 to 25 pl. The procedure of PCR reaction is as follows: pre-denaturation at 94 0C for 2min, denaturation at 980C for 30s, annealing at 550 C for 30s, extension at 72 0C for 45s, 35 cycles, extension at 720 C for 8min. The PCR products were detected by electrophoresis with 1% agarose gel, and judged according to the detection results. Further, S1, the RNA to be detected is extracted from colostrum, semen, serum, tissue (including placenta) or lochia of pigs. Further, the target band in S2 is 1124bp, 1034bp or 731bp. Further, the target band is 1124bp, and it is judged that the sample contains porcine classical porcine reproductive and respiratory syndrome virus. The target band is 1034bp, and it is determined that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus. The target band was 731bp, and it was determined that the sample contained porcine NADC-30 porcine reproductive and respiratory syndrome virus. Further, the results of S3 are specifically judged as follows: the target band is 1029bp, and it is judged that the sample contains porcine classical porcine reproductive and respiratory syndrome virus. The target band is 939bp, and it is determined that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus. The target band was 636bp, and it was determined that the sample contained porcine NADC-30 porcine reproductive and respiratory syndrome virus. Compared with the prior art, the invention has the beneficial effects that: (1) the invention provides two pairs of primers with degenerate bases, which can detect that the infected porcine reproductive and respiratory syndrome virus in the tested sample is a classic strain or a highly pathogenic variant strain or NADC-30 at one time, thus solving the defects of time consumption, consumables, low sensitivity and the like caused by the separate detection of three strains of porcine reproductive and respiratory syndrome virus. (2) The nested RT-PCR detection primers provided by the invention are four specific primers designed by coding the base sequence of the highly conserved NSP2 protein gene, and have strong specificity. (3) The RT-PCR detection method provided by the invention has wide application range through multi-step amplification detection, and the sensitivity of the lowest detection limit is 2-3 orders of magnitude higher than that of the common PCR detection method. (4) The detection method can amplify specific bands from the mixed template, with good interference and strong reliability, but also suitable for the detection of colostrum or semen, lochia and other samples of pigs, which has strong practicability and is beneficial to popularization and popularization.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a gel electrophoresis diagram of nested RT-PCR external primer specificity detection, where m is DL2000DNAMarker. 1 is recombinant positive plasmid. 2 is the classical porcine reproductive and respiratory syndrome virus plasmid. 3 is a highly pathogenic porcine reproductive and respiratory syndrome virus plasmid. 4 is NADC-30 virus plasmid. 5-8 were porcine epidemic diarrhea virus (PEDV), porcine coronavirus

(PDCoV), Escherichia coli and Staphylococcus aureus. 9 is the negative control (RNase free water). Fig. 2 is a gel electrophoresis diagram of nested RT-PCR inner primer specificity detection, m is DL2000DNA Marker. 1 is recombinant positive plasmid. 2 is the classical porcine reproductive and respiratory syndrome virus plasmid. 3 is a highly pathogenic porcine reproductive and respiratory syndrome virus plasmid. 4 is NADC-30 virus plasmid; 5-8 were porcine epidemic diarrhea virus (PEDV), porcine coronavirus (PDCoV), Escherichia coli and Staphylococcus aureus. 9 is the negative control (RNase free water). Fig. 3 is a gel electrophoresis diagram of nested RT-PCR outside primer sensitivity detection, m is DL2000DNA Marker. The template copy numbers of PRRSV classic strains in 1-8 were 3.8x107, 3.8x106, 3.8x105, 3.8x104, 3.8x103, 3.8x102, 3.8x101 and 3.8 respectively. The template copy numbers of PRRSV variant strains in 1-8 were 1.3x107, 1.3x106, 1.3x105,1.3x104, 1.3x103, 1.3x102, 1.3x101 and 1.3 respectively. 1-8 PRRSV 1.6x107, 1.6x106, 1.6x105, 1.6x104, 1.6x103, 1.6x102, 1.6x10 1 and 1.6 respectively. Fig. 4 is a gel electrophoresis diagram of nested RT-PCR inner primer sensitivity detection. 1-9 respectively use the first PCR product as the corresponding template; The template copy numbers of PRRSV classic strains in 1-8 were 3.8x107, 3.8x106, 3.8x105, 3.8x104, 3.8x103, 3.8x102, 3.8x101 and 3.8 respectively. The template copy numbers of PRRSV variant strains in 1-8 were1.3x107,1.3x106, 1.3x105,1.3x104,1.3x103, 1.3x102, 1.3x101 and 1.3 respectively. 1-8 PRRSV 1.6x107, 1.6x106, 1.6x105, 1.6x104, 1.6x103, 1.6x102, 1.6x101 and 1.6 respectively. Fig. 5 is a gel electrophoresis diagram of interference detection of nested RT-PCR primers. M is DL2000DNA Marker, 1 is the mixed genome amplified by outer primers, and 2-5 are RNase-free water. Fig. 6 is a gel electrophoresis diagram of interference detection of nested RT-PCR primers. M is DL2000DNA Marker, 1 is internal primer amplification mixed genome, and 2-5 is RNase-free water.

DESCRIPTION OF THE INVENTION The following will further describe the present invention with examples, but it is not intended to limit the scope of the present invention, but only for illustration. Embodiment 1: Establishment of Triple Nested RT-PCR Method for Detection of Porcine porcine reproductive and respiratory syndrome virus. 1. Design of primers Sequence analysis of porcine reproductive and respiratory syndrome virus showed that the amino acids of NSP2 protein of epidemic strain were highly conserved and could be used as target antigen for clinical diagnosis. A pair of outer primers and a pair of inner primers are designed to encode the base sequence of NSP2 protein, and the inner primers are inside the outer primer amplification sequence. Two pairs of primers contain degenerate bases, which can amplify different bands of classical strain, highly pathogenic mutant strain and NADC-30, and achieve the purpose of detecting and distinguishing these three subtypes at one time. Among them, the sequences of two pairs of primers are: PRRSV-O-F: AATCTTGARGAATGCTTGGC; PRRSV-O-R: GCTGAGTAYTTTGGGCGTG; PRRSV-I-F: TTCTTTGATTGGRATGTTGTGC; PRRSV-I-R: CAAGGAGCTGCTTGAYGACAC; In which degenerate base R represents A or G, and degenerate base Y represents C or T. 2. Establish triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to the primer designed. S1. Sampling and RNA extraction A: Sample collection: collect sample serum, centrifuge at 3000r/min for 20 minutes, extract 200pL of RNA with DNA/RNA extraction kit, and measure the concentration for later use. B: Tissue sample treatment: take the tissue placenta of the pig sample to be detected, cut it into pieces, extract RNA with RNA extraction kit, and measure the concentration for later use.

S2. Performing the first PCR amplification by using the outer detection primers PRRSV-O-F and PRRSV-O-R. PCR reaction system: 2xlStep Buffer 1 2 .5pl, PrimeScript 1Step Enzyme Mix 1pl, 20pmol/L PRRSV-O-F and 20pmol/L PRRSV-O-R each 1pl, RNA template 0.5pl, RNase Free dH20 to 25 pl. PCR reaction procedure is as follows: reverse transcription at 500 C for 30min, pre-denaturation at 940 C for 2min, denaturation at 94 0C for 30s, annealing at 530C for 30s, extension at 720 C for 1min, 35 cycles, extension at 720 C for 8min, electrophoresis detection of PCR products with 1% agarose gel, and second amplification if there is no target band. S3. Using the inner detection primers PRRSV-1-F and PRRSV--R to dilute the template of the first amplification product by 50 times for the second amplification. The optimum amplification conditions of inner primers are as follows: Mix 1 2 .5pl, PRRSV- F 20pmol/L and PRRSV--R 20pmol/L each 1pl, template 1pl, RNase Free dH 2 0 to 25 p L; The procedure of PCR reaction is as follows: pre-denaturation at 940 C for 2min, denaturation at 98 0C for 30s, annealing at 55 0C for 30s, extension at 720 C for 45s, 35 cycles, extension at 720 C for 8min, and the PCR products were detected by electrophoresis with 1% agarose gel. After the first round of PCR amplification, the target band detected by electrophoresis was reported as positive, and the detected sample was highly toxic. Specifically, if the target band is 1124bp, it is determined that the sample contains porcine classical porcine reproductive and respiratory syndrome virus If the target band is 1034bp, it is judged that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus If the target band is 731bp, it is judged that the sample contains porcine NADC-30 porcine reproductive and respiratory syndrome virus. If there are no three target bands, the second round of amplification is carried out. After the second PCR amplification, the target band detected by electrophoresis was also judged to be positive, indicating that the sample was relatively low in toxic amount. Specifically, if the target band is 1029bp, it is determined that the sample contains porcine classical porcine reproductive and respiratory syndrome virus. If the target band is 939bp, it is judged that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus; If the target band is 636bp, it is judged that the sample contains porcine NADC-30 porcine reproductive and respiratory syndrome virus. After two rounds of PCR, if there is no target band, it will be reported as negative. Embodiment 2: RT-nested PCR detection kit for porcine reproductive and respiratory syndrome virus A detection kit for identifying porcine reproductive and respiratory syndrome virus and distinguishing three subtypes such as classical strain, highly pathogenic variant strain or NADC-30 comprises two pairs of detection primers, amplification reagents, positive control and negative control. Two pairs of primers are outer primers PRRSV-O F and PRRSV-O-R and inner primers PRRSV-1-F and PRRSV-1-R, the negative control is RNase-free water, and the positive control is recombinant positive plasmid pMD18-T NSP2. The construction method of the positive plasmid is as follows: Step 1: Cloning of NSP2 gene Genomic RNA of porcine reproductive and respiratory virus (PRRSV) was extracted, and the partial conserved sequence of NSP gene was amplified by nested RT-PCR primers PRRSV-O-F and PRRSV-O-R. PCR reaction system (25i-4I): 2 X 1Step Buffer 12.5 9 1, PrimeScript 1Step Enzyme Mix 1 9 1, 20 u mol/L PRRSV-O-F and 20 u1 mol/L PRRSV-O-R each 1 il, RNA template 0.5911, RNase Free dH20 to 2591. The PCR reaction procedure is: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 2min, denaturation at 94C for 30s, annealing at 53C for 30s, extension at 72°C for 1min, 35 cycles, extension at 72C for 8min. The PCR products were identified by electrophoresis in 1% agarose gel. As shown in Figure 1, a total of 1124bp and 1034bp were obtained. Step 2: Construction of pMD18-T-NSP2 positive plasmid The above PCR products were recovered by the gel recovery kit of TaKaRa company, and then connected with pMD18-T cloning vector of TaKaRa company. the connection system was pMD18-T vector 1 ul, PCR recovery products 3 il, solution I5 9 I and ddh201 91. Escherichia coli DH5 a competent cells were transformed after being connected at 16°C for 30min, and coated on Amp resistant LB medium. After overnight culture at 37C, the correct recombinant was screened by resistance. Embodiment 3: Feasibility analysis of nested RT-PCR primers

1. Specificity test Genomes of porcine porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine coronavirus (PDCoV), Escherichia coli and staphylococcus aureus were extracted as templates, and two pairs of primers PRRSV-O-F, PRRSV-O-R and PRRSV--F, PRRSV-I-R used in nested RT-PCR were specifically detected results. As shown in Figures 1 and 2, both pairs of primers have good specificity and high amplification efficiency. 2. Sensitivity test The positive plasmid obtained in Embodiment 2 was diluted to single-digit copy number by 10 times, and each diluted plasmid was selected as template for the first PCR amplification with primers PRRSV-O-F and PRRSV-O-R, and the PCR products were detected by 1% agarose gel. The results are shown in Fig. 3. the results of the first PCR amplification electrophoresis show that the outer primers can detect 3.8 X 10 2 copies of PRRSV classic strain, 1.3X 10 2 copies of PRRSV variant strain and 1.6 X 10 2 copies of PRRSV NADC30 strain, with good amplification specificity. The amplified products were used as templates of corresponding gradients for the second PCR amplification with primers PRRSV-I-F and PRRSV-1-R, and the amplified products were detected with 1% agarose gel. Results as shown in Fig. 4, after nested RT-PCR amplification twice, the lowest number of copies of genes of 10 1 orders of magnitude can be detected. 3. Interference test The genome used in specificity test was mixed with porcine reproductive and respiratory syndrome virus genome in equal quantity, and PCR amplification was carried out with primers PRRSV-O-F, PRRSV-O-R, PRRSV-I-F and PRRSV--R, respectively, to test the anti-interference of primers and whether the target band could be amplified from the mixed genome template. Results as shown in Fig. 5 and Fig. 6, both pairs of primers can amplify specific bands from the mixed template, which shows that the primers have good anti-interference ability. 4. Coincidence rate test In order to verify the sensitivity of nested primers to the detection of porcine reproductive and respiratory syndrome virus in different regions, genomes of CH-1R, R98, JXA1-R, GDr180, NADC-30 and other strains were extracted, and the copy number was calculated after concentration determination, which was diluted to single-digit copy number by gradient. After two rounds of nested RT-PCR amplification, 101 orders of magnitude gene copy numbers could be detected in various genomes, which proved that the method of the present invention was feasible. Embodiment 4: Clinical trial of detection method 1. Clinical sample collection: in November, 2018, 20 blood samples of piglets were collected from a large-scale pig farm in Hubei province, centrifuged at 3000r/min for 30 minutes, sample serum was collected, and RNA was extracted according to DNA/RNA extraction kit of TaKaRa company. 2. PCR amplification and electrophoresis detection: Carry out two rounds of PCR amplification according to the method provided in Embodiment 1. The amplified products were detected by electrophoresis, and 13 highly pathogenic porcine reproductive and respiratory virus mutants and 4 classic porcine reproductive and respiratory syndrome s were detected in 20 serum samples. Sequencing results showed that all the 17 positive products were porcine porcine reproductive and respiratory syndrome genes, which proved that the detection results of this method were reliable, could be popularized and had a good application prospect. For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus is characterized in that the primers obtain two pairs of primers with degenerate bases according to the base sequence in NSP2 gene of porcine reproductive and respiratory syndrome virus, which are outer primers PRRSV-O-F and PRRSV-O-R and inner primers PRRSV-1-F, PRRSV--R, specifically are described as following: PRRSV-0-F: AATCTTGARGAATGCTTGGC(SEQ ID NO.1) PRRSV-O-R: GCTGAGTAYTTTGGGCGTG(SEQ ID NO.2) PRRSV--F: TTCTTTGATTGGRATGTTGTGC(SEQ ID NO.3) PRRSV--R: CAAGGAGCTGCTTGAYGACAC(SEQ ID NO.4) In which degenerate base R represents A or G and degenerate base y represents c or T.
2. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus is characterized in comprising steps as following: S1. Extracting RNA of a sample to be detected S2. Taking the RNA extracted from S1 as a template, carrying out the first round of PCR amplification by using PRRSV-O-F and PRRSV-O-R, carrying out agarose gel electrophoresis analysis on PCR amplification products, and carrying out the second amplification if no target band exists S3. Using S2 amplification products as templates, using PRRSV--F and PRRSV- R to carry out the second round of PCR amplification, analyzing the PCR amplification products by agarose gel electrophoresis, and distinguishing the types of strains according to the target bands.
3. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 2, is characterized in that the PCR reaction system in 3.S2 is: 2xStep Buffer 1 2 .5pl, PrimeScript 1Step Enzyme Mix 1pl, 20pmol/L PRRSV-O-F and 20pmol/L PRRSV-O-R each 1pl, RNA template 0.5pl, RNase Free dH20 supplemented to 25 pl. PCR reaction procedure is: reverse transcription at 500 C for 30min, pre-denaturation at 940 C for 2min, denaturation at 940C for 30s, annealing at 530 C for 30s, extension at 720 C for 1min, 35 cycles and extension at 720 C for 8min.
4. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 2, is characterized in that the PCR reaction system described in S3 is: Mix 1 2 .5pl, PRRSV-1-F 20pmol/L and PRRSV--R 20pmol/L each 1pl, template 1pl, RNase Free dH20 supplemented to 25 p I. PCR reaction procedure is: pre-denaturation at 940 C for 2min, denaturation at 980 C for s, annealing at 55 0C for 30s, extension at 720C for 45s, 35 cycles and extension at 72 0C for 8min.
5. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 4, is characterized in that the template is diluted 50 times by PCR products in S2.
6. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 2, is characterized in that the RNA to be detected in S1 is extracted from colostrum, semen, serum, tissues, lochia and other samples of pigs.
7. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 2, is characterized in that the target band in 7.S2 is 1124bp, 1034bp or 731bp.
8. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 7, is characterized in that the target band is 1124bp, and it is judged that the sample contains porcine classical porcine reproductive and respiratory syndrome virus. The target band is 1034bp, and it is determined that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus. The target band was 731bp, and it was determined that the sample contained porcine NADC-30 porcine reproductive and respiratory syndrome virus.
9. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 2, is characterized in that the discrimination method in S3 is as following: the target band is 1029bp, and it is judged that the sample contains porcine classical porcine reproductive and respiratory syndrome virus. The target band is 939bp, and it is determined that the sample contains porcine highly pathogenic porcine reproductive and respiratory syndrome virus. The target band was 636bp, and it was determined that the sample contained porcine NADC porcine reproductive and respiratory syndrome virus.
10. Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus, according to claim 1, is characterized in that the application of primers in the preparation of detection kits for the identification of porcine reproductive and respiratory syndrome virus and the differentiation of classical strains, highly pathogenic variants or NADC-30 strains.