Eestablished The Dual FQ-PCR Diagnostic Method And Analysised Nsp2and ORF5Gene Mutation

Since2006,HP-PRRS and LP-PRRS have been mixed infected and its Nsp2andORF5have been varied,which increases the difficulty in controlling these two kinds ofdisease to cause severe economic losses in the pig industry. The study established a kindof duplex FQ-PCR detection method to identify HP/LP-PRRSV fastly and reliably, at thesame time,we cloned.and analysised Nsp2and ORF5gene of PRRSV to understandpopular dynamic and main genetic variation of PRRSV of2012in henan province.Ahighly sensitive and specific duplex real-time TaqMan MGB-fluorescent-quantitativePCR(FQ-PCR)assay has been developed to identify and quantitate the Highly and lowlypathogenic porcine reproductive and respiratory syndrome virus（HP/LP-PRRSV）infection using PRRSV nonstructural2(Nsp2)gene-derived primers and TaqManfluorescent probe. The sensitivity, specificity and repetition assay of duplex FQ-PCRwere tested, and25clinic suspicious PRRSV infected samples were detected by theFQ-PCR assay in contrast to the routine Duplex PCR method. Choose the detectionpositive samples in this method，using RT-PCR method to amplificate and clone Nsp2and ORF5genes, And then to determine the sequence and the analysis of geneticevolution.The results indicated that the quantitative standard carves for HP/LP-PRRSV ofFQ-PCR assay possess good linearities0.998and0.997respectively, which wassuccessfully established. The duplex FQ-PCR assay was able to detect as little as1.0×101copy/μL of recombinant plasmid DNA.The specificity assay exhibited thatpositive signals could be obtained from the HP/LP PRRSV positive control, but not fromthe genomic DNA of the other5kinds of pathogenic microorganism acting as the control.The repetition test indicated that the FQ-PCR was reproducible by repeatedly amplifying3times of different concentrations of HP/LP-PRRSV recombinant plasmids. Ninty-two percent positive results from25clinic suspicious PRRSV infected samples were obtained,which showed the better sensitivity than the detection results of the same25suspectedsamples by duplex PCR.We cloned Nsp2and ORF5genes of PRRSV and analysisedgenetic variation and evolution from eight representative clinical samples.The sequenceanalysis results showed that the homology of Nsp2nucleotide is93.5%～93.5%among8positive samples, and is82.8%～83.7%with VR-2332nucleotide, and is94.5～97.3%With JXA1strain, which is HP-PRRSV representative.Missing30amino acids comparedwith the LP-PRRSV. The8strains and JXA1is on a big branchand the LV strain alonelocated a branch, suggested that Nsp2nucleotide homology rate is higher, all belong tothe americas strains； Which showed that strains are all HP-PRRSV in this study. Therate of ORF5gene nucleotide homology among8strains are93.9%～98.2%;and93.5～96.6%with JXA1;Of ORF5gene, only HN-GY strainsand CH-1are in the same branch,the remaining7strains and JXA1strains are in the same branch, and with HP-PRRSVhaving a close genetic relationship. Nsp2amino acid mutation gene is small and ORF5amino acid exist multiple locus mutation, so we should strengthen the study of themolecular variation to provide reference basis for vaccine selection.