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
袁野 1董雅琴 2张慧 2刘爽 2崔进 2尼博 2魏荣 2顾丛丛 3段纲 4张锋 2李晓成 2代飞燕4
作者信息
- 1. 云南农业大学动物医学院,昆明 650201;中国动物卫生与流行病学,青岛 266032
- 2. 中国动物卫生与流行病学,青岛 266032
- 3. 桓台县畜牧渔业服务中心,淄博 256400
- 4. 云南农业大学动物医学院,昆明 650201
- 折叠
摘要
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.
Abstract
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-1 1a 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.
关键词
牛病毒性腹泻病毒/荧光RT-PCR/诊断/流行病学调查Key words
Bovine viral diarrhea virus/reverse real-time RT-PCR/diagnosis/epidemiological investigation引用本文复制引用
基金项目
国家重点研发计划(十四五)(2021YFD180030)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据