猪繁殖与呼吸综合征病毒经典株与变异株RT-PCR鉴别诊断方法的建立及应用

本试验旨在建立一种快速的猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)经典株和变异株的鉴别诊断方法。根据GenBank中已发表的猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)经典株与变异株Nsp2基因序列,设计1对特异性引物,建立能鉴别诊断PRRSV经典株和变异株的RT-PCR检测方法;该方法能从经典株与高致病性株PRRSV基因组中分别扩增出549和459 bp的特异性片段;该方法对猪瘟病毒、猪乙型脑炎病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病毒、猪圆环病毒2型、猪细小病毒的扩增结果均为阴性;该方法能分别检测出1 pg经典株和0.1 pg变异株RNA含量。建立的RT-PCR鉴别检测方法具有良好的特异性、敏感性、重复性,可以准确、快速鉴别出PRRSV经典株与变异株,将为猪繁殖与呼吸综合征病毒的临床诊断、病料检测和分子流行病学调查等提供一种高效、快速、特异、灵敏的鉴别诊断方法。

Development and Application of A RT-PCR Method for Detection and Differentiation of Classical and Variant Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid differential diagnosis method of porcine reproductive and respiratory syndrome virus(PRRSV) classic and variant strains,a pair of primers corresponding to the highly conserved regions of Nsp2 gene of PRRSV genome were synthesized according to the sequences of PRRSV on GenBank,a RT-PCR method for detection and differentiation of classical and variant PRRSV was established.A fragment of 549 and 459 bp was amplified from genomic RNA of classical or high-pathogenic PRRSV respectively,no specific products were achieved after differentiating detection of porcine originated viral pathogens including HCV,JEV,PEDV,TGEV,PRV,PCV2 and PPV.The RT-PCR could detect 1 pg RNA of classical and 0.1 pg RNA of variant PRRSV respectively.The established RT-PCR method had good specificity,sensitivity,repeatability,could accurately and quickly identify the classical and variant PRRSV,it provided a kind of efficient,rapid,specific and sensitive detection method for clinical diagnosis,disease detection and molecular epidemiology of material such as porcine reproductive and respiratory syndrome virus.