摘要
为了建立牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)巢式RT-PCR方法,试验通过对我国近5年流行的BVDV毒株的全基因序列进行比对分析,选择BVDV的保守5′端非编码区(5′UTR)片段作为靶点,设计巢式RT-PCR引物,对内外套引物进行筛选,优化反应条件,并分析建立的巢式RT-PCR方法的敏感性和特异性,同时利用该方法对172份临床样品进行检测并与实时荧光定量PCR方法进行比较。结果表明:巢式RT-PCR方法的敏感性为1.8×10^(1)copies/μL,敏感性是仅使用一对引物进行一轮PCR扩增的100倍;该方法对BVDV具有较好的特异性,与水疱性口炎病毒(VSV)、猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、小反刍兽疫病毒(PPRV)及蓝舌病病毒(BTV)均无交叉反应。从172份临床样品中共检出27份BVDV阳性样品,阳性率为15.70%,与实时荧光定量RT-PCR方法比较,符合率为81.82%。说明试验建立的BVDV巢式RT-PCR方法特异性、敏感性较好,可作为基层检测机构中诊断BVD的辅助检测方法。
In order to establish the nested RT-PCR method of Bovine viral diarrhea virus(BVDV),in this experiment,the whole gene sequences of the BVDV strains circulating in China in the past 5 years was compared and analyzed.Conservative 5′UTR fragments of BVDV were selected as targets and nested RT-PCR primers were designed.Inner and outer primers were screened;reaction conditions were optimized,and sensitivity and specificity of the method were analyzed.At the same time,172 clinical samples were detected by this method and compared with real-time RT-PCR method.The results showed that the sensitivity of the nested RT-PCR method was 1.80×10^(1) copies/μL.The sensitivity was the use of one pair of primers for one-step PCR,which was 100-fold increased.This method had good specificity for BVDV,and there was no cross-reaction with Vesicular stomatitis virus(VSV),Classical swine fever virus(CSFV),Foot-and-mouth disease virus(FMDV),Peste des petits ruminants virus(PPRV)and Bluetongue virus(BTV).A total of 27 positive BVDV samples were detected from 172 clinical samples,with a positive rate of 15.70%;compared with the real-time RT-PCR method,the compliance rate was 81.82%.The results showed that the BVDV nested RT-PCR method established by the test had good specificity and sensitivity,and could be used as an auxiliary detection method for the diagnosis of BVD in primary testing institutions.
作者
周思佳
韩佃刚
董俊
杨云庆
叶玲玲
李静
尹尚莲
张冲
信吉阁
ZHOU Sijia;HAN Diangang;DONG Jun;YANG Yunqing;YE Lingling;LI Jing;YIN Shanglian;ZHANG Chong;XIN Jige(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;Kunming Customs Technology Center,Kunming 650200,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2023年第7期71-77,共7页
Heilongjiang Animal Science And veterinary Medicine
基金
云南省科技厅重点研发计划项目(2018BB004)
云南省科技厅科技人才和平台计划项目(2018IC078)。