猪繁殖与呼吸综合征病毒变异株荧光定量RT-PCR检测方法的建立OA

To achieve rapid diagnosis of porcine reproductive and respiratory syndrome virus(PRRSV)variant strains,a fluorescent quantitative PCR detection method targeting different PRRSV variants was established.In this study,PRRSV-positive serum samples were collected,and viral nucleic acids were extracted via column-based methods.Re-verse transcription and PCR amplification were performed,followed by purification,sequencing,and genotyping of the PCR products to obtain nucleic acid standards.A SYBR Green I-based fluorescent quantitative PCR method was devel-oped for different genotypes,and its specificity,sensitivity,and repeatability were validated.Results demonstrated that the established method exhibited strong specificity,showing no cross—reactivity with African swine fever virus,pseudorabies virus,porcine circovirus,canine parvovirus,canine coronavirus,or canine distemper virus.A standard curve was plotted with the Ct value as the ordinate and the dilution concentration of the positive standard as the ab-scissa.The correlation coefficients(R2)of standard curves for nearly all variant strains met the required criteria(R2≥0.99),with slopes ranging from-3.10 to-3.60 and amplification efficiencies between 90％and 110％,indicating high sensitivity.The coefficients of variation for intra-and inter-assay repeatability were both below 5％.In conclusion,the established method can distinguish highly pathogenic,American,classical,NADC30-like,NADC34-like,and univer-sal PRRSV strains,providing robust technical support for rapid diagnosis of PRRSV.