1种牛轮状病毒Taq-Man荧光定量RT-PCR检测方法的建立  

Establishment of a Taq-Man fluorescent quantitative RT-PCR detection method for bovine rotavirus

作　　者：王秀明 陈君彦 础鲁 刘艳霞 Wang Xiuming;Chen Junyan;Chu Lu;Liu Yanxia(Liaoning Yikang Bio-Pharmaceutical Co.,Ltd.,Liaoning Liaoyang 111000;Jinyu Baoling Bio-Pharmaceutical Co.,Ltd.,Inner Mongolia Hohhot 010111;Alashan Left Banner Agricultural and Rural Comprehensive Administrative Law Enforcement Brigade,Inner Mongolia Bayannur 750300)

机构地区：[1]辽宁益康生物股份有限公司,辽宁辽阳111000 [2]金宇保灵生物药品有限公司,内蒙古呼和浩特010111 [3]阿拉善左旗农牧业综合行政执法大队,内蒙古巴彦浩特750300

出　　处：《现代畜牧兽医》2025年第3期11-15,共5页Modern Journal of Animal Husbandry and Veterinary Medicine

摘　　要：试验旨在建立一种可简便、快速地检测牛轮状病毒(BRV)的Taq-Man荧光定量RT-PCR方法,试验通过分析比对NCBI下载的BRV VP7基因序列,在VP7基因的保守区设计两对特异性引物及Taq-Man探针,经反应条件优化,建立BRV的Taq-Man荧光定量RT-PCR方法。利用建立的检测方法检测标准品,绘制标准曲线,检测方法的特异性、灵敏性和稳定性,并对临床样品及疫苗半成品进行检验。结果显示,建立的Taq-Man荧光定量RT-PCR方法的最低检测限为10 copies/μL,组内和组间重复变异系数(CV)均小于3.50%。检测临床样品和疫苗半成品,疫苗半成品均检测阳性,临床样品检测阳性率69%。研究表明,BRV Taq-Man荧光定量RTPCR检测方法操作简便、特异性强、灵敏度高,对BRV的诊断检测及疫苗生产具有重要的参考价值。The experiment aimed to establish a simple and rapid Taq-Man fluorescent quantitative RT-PCR method for the detection of bovine rotavirus(BRV).By analyzing and comparing the BRV VP7 gene sequences downloaded from NCBI,two pairs of specific primers and a Taq-Man probe were designed in the conserved region of the VP7 gene.After optimizing the reaction conditions,a TaqMan fluorescent quantitative RT-PCR method for BRV was established.The established detection method was used to test the standard products,plot the standard curve,and evaluate the specificity,sensitivity,and stability of the detection method.Clinical samples and vaccine semi-finished products were also tested.The results showed that the lowest detection limit of the established Taq-Man fluorescent quantitative RT-PCR method was 10 copies/μL,and the intra-group and inter-group repeatability coefficients of variation(CV)were both less than 3.50%.The detection of clinical samples and vaccine semi-finished products showed that all vaccine semi-finished products were positive,and the positive rate of clinical samples was 69%.The study demonstrated that the BRV Taq-Man fluorescent quantitative RT-PCR method is simple to operate,highly specific,and sensitive,and has important reference value for the diagnosis and detection of BRV and vaccine production.

关 键 词：牛轮状病毒(BRV) Taq-Man探针 荧光定量RT-PCR VP7基因 

分 类 号：S852.653[农业科学—基础兽医学]