鸭坦布苏病毒一步法RT-PCR与荧光定量RT-PCR检测方法的建立  

Establishment and comparison of the one-step RT-PCR and real-time RT-PCR for the detection of duck tembusu virus

作　　者：王建昌[1] 王金凤[1] 袁万哲[2] 刘立兵[1] 

机构地区：[1]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051 [2]河北农业大学动物医学院,河北保定071001

出　　处：《中国预防兽医学报》2016年第8期629-633,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：质检总局科研项目(2015IK093)

摘　　要：为建立鸭坦布苏病毒(DTMUV)的检测方法,本研究根据GenBank中登录的DTMUV E基因保守区域设计引物和探针,建立了检测DTMUV的一步法RT-PCR和荧光定量RT-PCR方法,并比较了两种方法的敏感性和特异性。结果显示,荧光定量RT-PCR标准曲线的相关系数(R^2)为0.999,DTMUV RNA在10~2拷贝/μL^10~7拷贝/μL范围内与Ct值呈现良好的线性关系;两种方法均仅对DTMUV RNA具有特异性扩增,而对其他常见鸭病原核酸的扩增均为阴性,具有较强的特异性;一步法RT-PCR对鸭胚纯培养病毒RNA的检测下限为10~3拷贝/μL,而荧光定量RT-PCR的检测下限为10~2拷贝/μL;重复性试验的变异系数均小于1.5%。对20份模拟临床样品的检测结果表明,一步法RT-PCR的阳性率为75%(15/20),而荧光定量RT-PCR的阳性率为90%(18/20)。本研究表明所建立的DTMUV荧光定量RT-PCR特异性强、重复性好、敏感性更高,更适用于DTMUV的临床诊断和流行病学调查。To establish the sensitive detection protocol of duck tembusu virus （DTMUV）, the one-step RT-PCR and real-time RT-PCR were developed with the specific primers and probe targeting the conserved region of the DTMUV E gene. The results showed that the real-time RT-PCR possessed a linear relationship between the viral RNA concentration （10^2 to 10^7 copies/μL） and the Ct values, and the correlation coefficient （R^2） was 0.999. The detecting limit was 10^3 and 10^2 of DTMUV RNA copies/μL for one-step RT-PCR and real-time RT-PCR, respectively; the methods demonstrated the high specificity, which could only detect the DTMUV RNA, but not the nucleotides of the other common pathogens in duck. The real-time RT-PCR was highly reproducible with the coefficient of variation （CV） less than 1.5%. Moreover, 20 samples from DTMUV artificially infected ducks were tested by the one-step RT-PCR and real-time RT-PCR, the positive rate was 75% （15/20） and 90% （18/20）, respectively. The data demonstrated that the real-time RT-PCR was specific, reproducible and more sensitive, which was suitable in the clinical diagnosis and epidemiological investigation of DTMUV infection.

关 键 词：鸭坦布苏病毒 荧光定量RT-PCR 一步法RT-PCR E基因 

分 类 号：S852.65[农业科学—基础兽医学]