猪轮状病毒TaqMan实时荧光定量RT-PCR方法的建立

摘要

In order to develop a TaqMan based real-time RT-PCR method for porcine rotavirus,specific primers and probe were de-signed by Oligo7 software targeting the sequences of VP6 gene for porcine rotavirus. The established real-time RT-PCR method could detect at 192 copies.μL-1with high sensitivity. No positive signal was detected from common porcine infectious diseases such as porcine epidemic diarrhea virus,transmissible gastroenteritis virus of swine,classical swine fever virus and Japanese encephalitis vi-rus. A standard curve generated had a wide dynamic range with a linear correlation(R2) of 0.999. Excellent reproducibility was ob-tained for detecting constructed positive plasmids with intra-assay of 0.25%-1.26% and inter-assay of 0.31%-1.49%,respectively. Seventy-nine clinical samples were tested by the development TaqMan based real-time RT-PCR method and conventional RT-PCR method with the positive ratio were 5.06%(4/79) and 7.59%(6/79),respectively. RT-PCR method positive samples were all test-ed positive by the TaqMan based real-time RT-PCR method,with coincidence rate was 100%.The TaqMan based real-time RT-PCR method established in this study may be used for earlier diagnosis for porcine rotavirus.%根据GenBank中登录的猪轮状病毒(PoRV)VP6基因序列特征,设计特异性的引物和探针,条件优化后,建立检测PoRV TaqMan实时荧光定量RT-PCR方法.结果表明:建立的TaqMan实时荧光定量RT-PCR方法敏感性高,最低可检测192拷贝.μL-1;特异性强,对猪常见病原(如猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒和猪流行性乙型脑炎病毒等)检测均为阴性;重复性好,扩增相关系数为0.999,其组内和组间变异系数分别为0.25%~1.26%和0.31%~1.49%.对临床收集的79份猪腹泻病料进行常规RT-PCR检测,检测出阳性样品4份,阳性率为5.06%(4/79);对这79份病料进行TaqMan实时荧光定量 RT-PCR方法检测,检测出阳性样品6份,阳性率为7.59%(6/79);同时,4份经RT-PCR检测为阳性的样品经TaqMan实时荧光定量RT-PCR方法检测均为阳性,符合率达100%.本研究建立的方法为PoRV早期感染诊断提供检测手段.