Establishment Of SYBR Green-Ⅰ Real Time PCR And Multiplex SYBR Green-Ⅰ Real Time PCR For Detection Of Porcine Reproductive And Respiratory Syndrome Virus, Porcine Circovirus Type 2 And Pseudorabies Virus

Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV-2) and Porcine pseudorabies virus (PRV) are the important infection disease etiologic agents, which have caused excessive lost in global swine industry. Because of the disadvantage, the traditional etiology and serodiagnoasis are not efficient in clinical. It is necessary to establish a rapid diagnosis for the three etiologic agents.Real time PCR that has been developed in 1990s of 20 century is a new technique for the detection of nucleic acid. SYBR Green-I that is fluorescent double-stranded DNA (dsDNA)-specific intercalating dyes has been used in real-time PCR extensively. The principle of the method is to add fluorescent dye in PCR; the process of PCR can be monitored with the change of fluorescence intensity. The nucleic acid copies of sample can be gained by standard curve; it also is used for qualitative analysis of sample by solubility curve. Real-time PCR based on SYBR Green-I is more simple and less cost compared with other real-time PCR. Now, the technique has been generally used in many fields. In our research, the SYBR Green-I real time PCR method was established to detect simultaneously PRRSV, PCV-2 and PRV.In the research, three pairs primers were designed based on the sequences of ORF7 gene of PRRSV, ORF1 gene of PCV-2 and gE gene of PRV in GenBank, the length of amplicons is 81bp for PRRSV, it is 130bp for PCV-2 and PRV, and then the amplicons were cloned and sequenced. The standard preparations were prepared by diluting the recombinant plasmid, then the SYBR Green-I real time PCR of the three viruses were established. The coefficient correlation (R~2) of the standard curve is 0.998 for PRRSV, 0.996 for PCV-2 and 0.983 for PRV, the efficiency is 1.01,0.92 and 0.98 respectively, The results showed that real time PCR was specific and reproductive, the sensitivity of PRRSV is -25copies/μL, and it is 50 copies/μL for PCV-2 and PRV.In the part two, three pairs primers were add in one PCR tube, the multiplex SYBR Green-I real time PCR was established successfully by the TM value disparity of the three amplicons. The solubility curve showed that the TM value of PRRSV is 82.8-83.8℃,PCV-2 is 84.96-85.68℃,PRV is 92.1-93.28℃,it can be discriminated. The specificity of PCR showed that PRRSV, PCV-2 and PRV are specific and PPV, CSFV have no amplification. The sensitivity is 250 copies/μL for PRRSV and PCV-2, 500copies/μL for PRV. The reproducibility of intra-assay is that the CV% of TM is 0.12% for PRRSV, 0% for PCV-2, 0.1% for PRV, and the CV% of TM is 0.10% for PRRSV, 0.07% for PCV-2, 0.05% for PRV in inter-assay.In the research, the SYBR Green-I real time PCR and multiplex SYBR Green-I real time PCR for detected the PRRSV, PCV-2, PRV were established successfully, and then the diagnostic kit will be developed.