中国兽医科学2024，Vol.54Issue(4)：454-464,11.DOI:10.16656/j.issn.1673-4696.2024.0052
牛诺如病毒和牛嵴病毒及牛环曲病毒三重RT-PCR检测方法的建立
Establishment of a triplex RT-PCR assay for detection of bovine norovirus,bovine kobuvirus and bovine torovirus
摘要
Abstract
In order to explore the prevalence of bovine norovirus(BNoV),bovine kobuvirus(BKV)and bovine torovirus(BToV)in China,three pairs of specific primers were designed according to the conserved regions of the RdRp genes of BNoV,the 3D genes of BKV and the N genes of BToV registered in GenBank.The triple RT-PCR method was successfully established for simultaneous and rapid detection of BNoV,BKV and BToV.This method has strong specificity,and only specific bands were amplified for BNoV,BKV and BToV,and no bands were amplified for other 5 common pathogens causing calf diarrhea.The sensitivity was high,and the minimum detection limits for pMD18-T-BNoV,pMD18-T-BKV and pMD18-T-BToV plasmid standards were 2.73 × 104 copies/μL,2.87 × 104 copies/μL and 3.18 × 103 copies/μL,respectively.The three test results of same batch samples consistently showed that the repeatability was good.A total of 153 clinical samples were detected by this method.The results showed that the positive detection rates of BNoV,BKV and BToV were 11.11％,25.49％and 38.56％,respectively.The positive mixed infection rate for all three viruses reached 7.18％.Compared with the single RT-PCR detection method established by other scholars,the coincidence rates for the three viruses mentioned above were 94.77％,93.46％and 91.50％,respectively.In this study,the triplex RT-PCR method provides technical support for rapid detection and epidemiological investigation of the above mentioned domestic emerging viruses in calves.关键词
牛诺如病毒/牛嵴病毒/牛环曲病毒/三重RT-PCRKey words
bovine norovirus/bovine kobuvirus/bovine torovirus/triplex RT-PCR分类
农业科技引用本文复制引用
孙睿雪,李玉铎,吴国军,陈天杰,车瑞香,茹毅,韩欢胜,赵建军..牛诺如病毒和牛嵴病毒及牛环曲病毒三重RT-PCR检测方法的建立[J].中国兽医科学,2024,54(4):454-464,11.基金项目
黑龙江省重点研发计划项目(GA21B004) （GA21B004）
家畜疫病病原生物学国家重点实验室开放基金课题(SKLVEB2021KFKT017) （SKLVEB2021KFKT017）
