摘要
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Taq Man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.
In order to develop a TaqMan based real-time RT-PCR method for porcine rotavirus,specific primers and probe were designed 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^(-1) with 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 virus. A standard curve generated had a wide dynamic range with a linear correlation( R2) of 0.999. Excellent reproducibility was obtained 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 Taq Man 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 tested positive by the TaqMan based real-time RT-PCR method,with coincidence rate was 100%. The Taq Man based real-time RT-PCR method established in this study may be used for earlier diagnosis for porcine rotavirus.
作者
陈如敬
章秋月
陈秋勇
修金生
吴学敏
严山
周伦江
CHEN Rujing;ZHANG Qiuyue;CHEN Qiuyong;XIU Jinsheng;WU Xuemin;YAN Shan;ZHOU Lunjiang(Institute of Animal Husbandry and Veterinary Medicine/Fujian Animal Disease Control Technology Development Center,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350013,China;Nanping Xingxing Animal Husbandry Co.,Ltd.,Nanping,Fujian 363000,China;College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)
出处
《福建农林大学学报（自然科学版）》
CSCD
北大核心
2018年第2期212-216,共5页
Journal of Fujian Agriculture and Forestry University:Natural Science Edition
基金
福建省生猪产业体系项目(2017No.129)
福建省属公益类科研专项(2018R1023-92015R1102)
福建省农业科学院科技下乡"双百"行动项目(sbmb1701)
福建省农科院重点创新团队(STIT2017-1-5)
