Study On The Rapid Multiplex RT-PCR Diagnosic Technology For Major Porcine RNA Viral Diseases

【Objective】To develop new rapid diagnostic techniques for Classical swine fever virus(CSFV), Porcine reproductive and respiratory syndrome(PRRSV) and Japanese encephalitis virus(JEV), Single or multiplex PCR, RT-PCR and Taqman(?) real-time fluorescence quantitative RT-PCR assay were established, which would facilitate lab detection of these pathogens.【Methods】(1) Conserved regions were selected by align genome sequences of CSFV, PRRSV and JEV from GenBank, and NS5B gene of CSFV, Nsp2 gene of PRRSV and E gene of JEV were determined to be the target gene for PCR amplification. Three pairs of primers were designed with Primer Premier for multiplex RT-PCR, and the size of amplicon was 482bp for CSFV, 576bp for PRRSV and 375bp for JEV. Single and multiplex PCR assay were established after optimization of reaction mixture and cycling conditions, and the diagnose coincidence rate between traditional PCR and the established PCR was analyzed. (2) According to the principle of TaqMan(?) real-time fluorescence quantitative RT-PCR assay, one pair of primers and one probe for each of CSFV, PRRSV and JEV were designed with Primer Premier 5.0,Primer Express 3.0 and Beacon Designer 4, and the reporter genes dye were JOE, TAMARA and FAM respectively.【Results】(1) The optimized PCR reaction mixture contains 0.4μmol/L each primer for CSFV, PRRSV and JEV, 200μmol/L each deoxynucleotide triphosphate (dNTP), 2.0mmol/L MgCl₂, 2.5U of rTaq DNA Polymerase(5U/μL) for 25μL reaction mixture, and the optimal Tm were 58℃;Using ten fold dilutions of purified recombinant plasmid as template, the sensitivity of the PCR assay was 10² Copies/μL. No non-specific fragment was amplified when test with PCV2, PRV, PPV, PEDV, TGEV genome as template, other than the specific fragments for CSFV, PRRSV and JEV. The coincidence rate for CSFV, PRRSV and JEV was 98%, 100% and 100% when compared with traditional PCR. The developed PCR assay can be finished within one hour, saving time by at least 38% when compared with traditional PCR. (2) TaqMan(?) probe based real-time fluorescence quantitative RT-PCR assay for identification of CSFV, PRRSV and JEV were developed, and sensitivity of the assay was tested to be 10Copies/μL for detection of RNA made by in vitro transcription. Each function achieved a high squared correlation coefficient (R2 greater than 0.99), indicating a high degree of accuracy in forming the standard curves. The sensitivity could be up to 10 TCID50 when applied the assay on cell cultured PRRSV. The mean intra-and inter-assay coefficients of variation (CV %) was less than 4% for all the three viruses, which demonstrates high reproducibility. (3) One Step RQ-RT-PCR allows the identification process could be finished within 70min, and ensures less contamination, and avoids of false positive results. The development of One Step RQ-RT-PCR technique makes it possible that any of the three pathogens could be detected in the same reaction mixture simultaneously and quantitatively.【Conclusion】(1) Single and multiplex two step-PCR assay for rapid detection of CSFV, PRRSV and JEV were developed, and the sensitivity of the assay could be up to 10² Copies/μL. (2) Single and multiplex TaqMan(?) probe based RQ-RT-PCR were developed for rapid detection of CSFV, PRRSV and JEV, and sensitivity was tested to be 10Copies/μL. As for PRRSV, using FAM as reporter dye, the sensitivity of the single RQ-RT-PCR is 10 TCID50. The developed PCR assay demonstrated good stability and high sensitivity, which facilitates the identification of these three viruses mentioned above in laboratory.