牛冠状病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及初步应用  

Development and preliminary application of a SYBR GreenⅠReal-time PCR assay for detection of bovine coronavirus

作　　者：沈付娆 杨建乐[1,3] 赵贵民[1] 朱彤[1] 程凯慧[1] 王洪梅[1] 何洪彬[1] 

机构地区：[1]山东省农业科学院奶牛研究中心,山东济南250100 [2]山东师范大学生命科学学院,山东济南250000 [3]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《中国兽医杂志》2016年第6期22-24,27,共4页Chinese Journal of Veterinary Medicine

基　　金：现代农业(奶牛)产业技术体系科学家岗位(Cars-37);泰山学者海外特聘专家(tshw20100417);国家自然科学基金(31272586;31302095);山东省农业重大应用技术创新课题;兽医生物技术国家重点实验室开放课题基金(SKLVBF201510);山东省农业科学院科技创新重点项目(2014CXZ08)子课题(2014CXZ-08-3);山东省农业科学院重大成果培育项目(2015CGPY02)

摘　　要：针对牛冠状病毒(BCoV)N基因保守序列设计合成了1对特异性引物,以体外转录法制备的cDNA标准品为模板,建立了检测BCoV的SYBR GreenⅠ实时荧光定量PCR方法。在1个反应体系中模板最低检测限为7.8 copies/μL,比普通RT-PCR更加灵敏;与牛主要消化道和呼吸道病毒病病原均无交叉反应;批内、批间重复变异系数均低于1.5%;应用定量PCR检测了39份疑似临床病料,阳性检出率为51.28%(20/39),普通RT-PCR为41.03%(16/39)。结果表明,该方法特异性强、稳定性好、准确率高,本方法将为BCoV的临床诊断和流行病学调查提供技术保障。In order to detect bovine coronavirus（BCoV）,we developed a SYBR GreenⅠreal-time PCR assay. According to the sequence of bovine coronavirus N gene,a pair of primers was designed. The cDNA was prepared by in vitro transcription and used as standard positive template.Then,the SYBR GreenⅠreal-time fluorescence quantitative RT-PCR assay was established to detect BCoV.The developed SYBR GreenⅠreal-time fluorescence quantitative RT-PCR was much more sensitive than the conventional RT-PCR and could detect the template as low as 7.8 copies/μL. There were no cross reactions with the other digestive and respiratory pathogens from cattle. The Inter-and intra-assays were conducted using the standard plasmid and the coefficients of variation were less than 1.5%.The 39 suspected samples were detected both by SYBR GreenⅠreal-time RT-PCR,and gel-based RT-PCR,and the positive detection rate were 51.28% and 41.03%,respectively. The SYBR GreenⅠreal-time PCR assay was much more specific,sensitive,rapid and accurate and could be used as an effective tool for clinical diagnosis,epidemiological investigation and quantification of bovine coronavirus.

关 键 词：牛冠状病毒 SYBR GreenⅠ实时荧光定量PCR 病毒核酸检测 

分 类 号：S852.653[农业科学—基础兽医学]