牛病毒性腹泻病毒TaqMan探针荧光RT-PCR方法的建立和临床应用

Development and Clinical Application of TaqMan Reverse Real Time PCR for Detection of Bovine Viral Diarrhea Virus

为建立一种牛病毒性腹泻病毒(BVDV)的快速检测方法,本研究根据国内BVDV流行毒株5'-UTR序列保守区域设计1对特异性引物和TaqMan探针,并优化了反应条件,最终建立了一种检测BVDV基因1型(BVDV-1)的TaqMan探针荧光RT-PCR检测方法,并开展7省份BVDV-1型检测和流行情况分析。结果显示:所建立方法能够特异性检测出BVDV-1,与基因2型BVDV、猪瘟病毒、边界病毒、牛冠状病毒、牛传染性鼻气管炎病毒、牛轮状病毒等病原无交叉反应;灵敏度高,最低检测下限为4.3 copies/μL;批内和批间变异系数均小于2%,重复性良好。7省份规模化牛养殖场BVDV-1型平均阳性率为17.40%(161/925),序列分析表明我国主要流行的为BVDV-1a,BVDV-1c亚型。本研究成功建立了一种良好的诊断BVDV的方法,监测地区优势流行毒株为BVDV-1a与1c亚型,为临床中BVDV早期诊断和流行病学调查监测提供了技术和数据支持。

A TaqMan-based real-time RT-PCR assay was developed in the present study for rapid detection of bovine viral diarrhea virus(BVDV)using a pair of primers and TaqMan probe targeting the BVDV-1 conserved 5'-UTR gene sequence of domestic epidemic strains.Then this detection method was used to conduct surveillance and epidemic analysis of BVDV-1 in 7 provinces.The results showed that the developed real-time RT-PCR assay was specific for BVDV-1 and had no cross-reaction with BVDV-2,classical swine fever virus,border disease virus,bovine coronavirus,infectious bovine rhinotracheitis virus and bovine rotavirus.The detection limit of the assay was as low as 4.3 copies/μL.In addition,this assay has good reproducibility with less than 2%of the coefficient of variation within batches and between batches.Surveillance and sequence analysis of BVDV performed on large-scale cattle farms in 7 provinces showed that 17.40%samples(161/925)were positive for BVDV-1.Further phylogenetic analysis showed that BVDV-11a and 1c were prevalent in cattle populations.The development of a fast and sensitive diagnostic method of BVDV and findings of the dominant epidemic strain of BVDV-1a and 1c in China provided technical and data support for the early diagnosis and surveillance of BVDV.