猪繁殖与呼吸综合征病毒通用型实时荧光定量RT-PCR检测方法的建立及初步应用

根据GenBank登录的猪繁殖与呼吸综合征病毒（PRRSV）北美型毒株和欧洲型毒株N蛋白基因保守序列,设计1对特异性引物和1条探针,以ABI公司PRRSV RNA为标准品,优化了PRRSV实时荧光定量RT-PCR检测方法,25μL反应体系引物prrsv-f/prrsv-r（20μmol/L）各0.5μL,探针prrsv-p（10μmol/L）0.5μL为反应最佳工作浓度,标准曲线Ct值和模板启始浓度的log值之间具有良好的线性相关性。特异性、敏感性和重复性检测结果显示,该方法能特异的检测PRRSV北美型毒株和欧洲型毒株,与其他主要猪病病原检测无交叉反应;针对PRRSV RNA标准品检测,灵敏度可以达到10拷贝;重复性检测Ct值变异系数为0.74%～1.52%。用建立的实时荧光定量RT-PCR检测方法,对40份临床样品检测,与商品化试剂盒检测结果完全一致。

Establishment of one-step real-time Taqman RT-PCR assay for porcine reproductive and respiratory syndrome virus

A one-step real-time Taqman RT-PCR assay for porcine reproductive and respiratory syndrome virus(PRRSV) was developed to detect the two type of PRRSV,American and European strain,by targeting the ORF7 gene of the virus.This new assay was compared to commercial methods developed by applied biosystems company.Quantitation was performed against a standard samples of the PRRSV for which a minimum of 10 copies per reaction were detected with a corresponding Ct value of 0.74%-1.52% and it shows no cross reaction with other swine pathogens.By detecting 40 clinical samples of the virus,the results are the same as the commercial kits.