鸭坦布苏病毒TaqMan荧光定量RT-PCR检测方法的建立

根据鸭坦布苏病毒(DTMUV)BYD-1株基因序列设计特异性引物和TaqMan探针,建立了快速检测DTMUV的实时荧光定量RT-PCR方法。特异性结果显示,DTMUV显示阳性,而3株非鸭坦布苏病毒均显示阴性,说明该方法具有高度特异性。其表达式为Ct=-3.32×lg Copy number+41.151,R2=0.998,R循环效率Eff%=100.072,DNA拷贝数在101～108范围内检测曲线有良好的线性关系,对质粒标准品最低检测限为1.0×101拷贝/μL,变异系数低于5%。利用该方法分别对攻毒感染具有典型鸭新型黄病毒病临床症状和病理变化的蛋鸭的脾脏和肝脏组织进行检测,阳性率均为100%,而用本实验室建立的普通RT-PCR方法检测的阳性检测率为85%(17/20)和75%(15/20)。建立的方法具有敏感性高、特异性好、稳定性强的特点,可用DTMUV早期感染的快速诊断和流行病学调查。

Development of Taq Man Fluorescent Quantitative RT-PCR for Detecting Duck Tembusu Virus

A pair of primers and one TaqMan probe were designed in conservative region of the duck tembusu virus strain BYD,and a rapid fluorescent quantitative RT-PCR assay was successfully established for detection of duck tembusu virus.In result,specificity test showed that duck tembusu virus submitted positive reactions,while 3other non-duck tembusu virus submitted negative reactions.The equation of the assay was Ct=-3.32g copy number +41.151,and the correlation coefficient was 0.998and PCR efficiency reached up to 100.072%.There was an excellent linear relation during the DNA concentration from 10 1 to 1×10 8 copies.The sensitivity of the assay was 1.0×10 1 copies / μ L and variation coefficient was less than 5%.When comparing duck tembusu virus conventional RT-PCR with fluorescent quantitative RT-PCR to detection the samples of spleen and liver from the duckling with typical clinical symptoms and pathological lesions infected with duck tembusu virus,the positive rates by duck tembusu virus conventional RT-PCR established by ourselves were 85%and 75%respectively;but 100%positive for all samples by fluorescent quantitative RT-PCR.The results showed that fluorescent quantitative RT-PCR was sensitive,specific and stable;The fluorescent quantitative RT-PCR can be used for the rapid detection of early infection with duck tembusu virus and epidemiological investigation.