荧光定量RT-PCR法对犬瘟热病毒PS株感染犬血清和粪便中病毒的检测

为研究犬人工感染犬瘟热病毒(CDV)后病毒在血清和粪便中的含量及变化,本实验根据CDV核衣壳蛋白(NP)基因序列设计一对特异引物,扩增NP基因.并以NP基因重组质粒作为阳性标准品,建立检测CDV的SYBR GreenⅠ荧光定量RT-PCR方法.结果表明,该方法102拷贝～109拷贝范围内具有良好的线性关系,相关系数为R2=0.998,扩增效率为E=98.3％,敏感度高,最低检测限为6.1拷贝/μL,重复性检测变异系数低于2.62％.该方法与常规RT-PCR方法比较,其敏感性提高102倍.两只人工感染CDV PS强毒株的犬,分别于接种后17d、19d发病死亡,采用荧光定量RT-PCR方法对人工感染犬的血液和拭子液中的病毒核酸栽量进行定量检测以及对收集的22份临床样本拭子液进行检测,均检测到CDV.本研究结果表明,该方法可以用于CDV核酸定量检测,为该病毒的致病机理及病毒传播途径的研究提供了一种快速和敏感的检测手段.

Detection of canine distemper virus in blood and feces of the infected dogs by real-time PCR

A SYBR Green Ⅰ real-time RT-PCR method was established for detection of canine distemper virus(CDV) loads in blood and swab of using primers targeting the neucleoprotein sequence.The assay had a good linear relation in the range from 102 copies/μL to 109 copies/μL with 0.998 coefficient and 98.3% amplified efficiency.The detection limit was 6.1 copies by the real-time PCR which was 102 times more sensitive than the conventional RT-PCR.The virus loads in blood and swab of artificial-infected dogs and 22 clinical fecal swab samples were detected by the real-time PCR.These results showed that all the samples were positive and it demonstrated the real-time PCR assay could be an effective method for quantitative detection of CDV.