【摘要】 针对猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)N基因保守序列设计引物与TaqMan探针,在建立常规PCR的基础上,设计并优化了TaqMan荧光定量PCR方法用于PRRSV核酸的检测。应用新建立的荧光定量PCR方法检测其它主要猪病病原,无任何非特异性反应;针对PRRSV阳性克隆质粒的检测灵敏度可以达到1.2×101copy/ml,比常规PCR高100倍;通过对48份临床疑似样本的检测表明有29份样本为PRRSV阳性,与巢式PCR检测结果一致,而常规PCR只能检出其中19份阳性样本,灵敏度优于常规PCR方法。结果表明,试验建立的PRRSV TaqMan荧光定量PCR方法具有良好的特异性、敏感性和重复性,可为PRRSV临床诊断提供一个更为有效的方法。更多还原

【Abstract】 A TaqMan-based real-time PCR assay was developed to rapidly detect the porcine reproductive and respiratory syndrome virus(PRRSV).Primers and probe specific to the conserved region of the PRRSV N gene were selected,and the reactive system and conditions were optimized to improve the sensitivity,specificity and repetition of the assay.The results showed that the real-time PCR assay was specific and there were no cross reactions with other common porcine viruses. The sensitivity of the assay was 1.2×101copy/ml of plasmid coding partial PRRSV N gene,which is 100 times higher than conventional PCR.It took no more than three hours from viral RNA extraction to complete the real-time PCR,and the assay was simple and had good repeats.Among tissue samples of 48 clinical diseased pigs,29 were confirmed PRRSV infected by real-time PCR,in which only 19 were confirmed by conventional PCR.This TaqMan-based real-time PCR assay is a specific,sensitive and quick tool suitable for early and quick detection of PRRSV in clinical labs.更多还原