塔城病毒Ⅰ型TaqMan探针法荧光定量PCR检测方法的建立  

Development of A TaqMan Probe Based Real-Time Quantitative PCR Method for Detection of Tacheng Virus Type I

作　　者：徐昭艳 姜峰 陈瑶[2] 金鑫[1] 陈泽良 韩小虎[1] XU Zhaoyan;JIANG Feng;CHEN Yao;JIN Xin;CHEN Zeliang;HAN Xiaohu(School of Animal Science and Medicine,Shenyang Agricultural University,Shenyang 110161,China;Liaoning Provincial Center for Animal Disease Prevention and Control,Shenyang 110161,China)

机构地区：[1]沈阳农业大学动物科学与医学学院,沈阳110161 [2]辽宁省动物疫病预防控制中心,沈阳110161

出　　处：《中国动物传染病学报》2024年第5期83-92,共10页Chinese Journal of Animal Infectious Diseases

基　　金：科技部对发展中国家科技援助项目(KY201901014)。

摘　　要：建立快速特异的蜱传塔城病毒Ⅰ型(TcTV-Ⅰ)荧光定量PCR检测方法,用于TcTV-Ⅰ的初步筛查。选用TcTV-Ⅰ糖蛋白G基因设计特异性引物以及TaqMan探针,建立实时荧光定量PCR检测方法。建立的实时荧光定量PCR检测方法具有良好的特异性,与TcTV-Ⅱ、新布尼来病毒(Severe fever with thrombocytopenia virus,SFTSV)、森林脑炎病毒(Tick-borne encephalitis virus,TBEV)、大别山病毒(Dabieshan orthohanta virus,DBSV)、阿龙山病毒(Alongshan virus,ALSV)等常见蜱传病毒均无交叉反应;标准曲线的循环阈值(Ct值)与模板拷贝数之间呈良好的线性关系,线性相关系数R^(2)=0.9999;具有良好的敏感性,最低检测限为1.10×10^(1)copies/μL,灵敏度是常规PCR的10^(3)倍;具有较好的稳定性,组内和组间的变异系数均低于2%。采用本方法对辽宁、内蒙古及新疆地区的425份蜱样本就行检测,只有新疆地区的19份蜱样本呈阳性。结果表明本研究建立的实时荧光定量PCR方法具有较高的特异性、敏感性和稳定性,可用于TcTV-Ⅰ的快速检测、流行病学调查和疫情监测等。A rapid and specifi c tick-borne Tacheng virus type I(TcTV-I)real-time PCR detection method was developed in the present study for the preliminary screening of TcTV-I.The TcTV-I glycoprotein G gene was used to design specifi c primers and TaqMan probes for development of a real-time fl uorescent quantitative PCR detection method.The real-time fl uorescence quantitative PCR detection method had good specificity as it had no cross-reaction with common tick-borne viruses such as TcTV-Ⅱ,SFTSV,TBEV,DBSV and ALSV.The cycle threshold(Ct value)of the standard curve was related to the template copy number.There was a good linear relationship with the linear correlation coeffi cient R^(2)=0.9999.This method also had good sensitivity as its minimum detection limit of 1.10×10^(1)copies/μL was 10^(3)times higher than that of conventional PCR.Good repeatability was also achieved as the coefficients of variation between intra-and inter-groups were all less than 2%.Subsequently,425 tick samples from Liaoning,Inner Mongolia and Xinjiang were tested using this method and 19 tick samples from Xinjiang were positive.The results showed that the real-time quantitative PCR method developed in this study had high specifi city,sensitivity and repeatability and might be used for rapid detection,epidemiological investigation and epidemic monitoring of TcTV-I.

关 键 词：蜱 实时荧光定量PCR 塔城病毒 TAQMAN探针 

分 类 号：S852.65[农业科学—基础兽医学]