兔出血症病毒经典毒株和变异毒株的RT-PCR鉴定  

Development of a RT-PCR technique for rapid identification of classical and variant rabbit hemorrhagic disease virus

作　　者：宋艳华[1] 魏后军[1] 范志宇[1] 左园园[1] 胡波[1] 仇汝龙 陈萌萌[1] 李明勇 薛家宾[1] 徐为中[1] 王芳[1] SONG Yan-hua WEI Hou-jun FAN Zhi-yu ZUO Yuan-yuan HU Bo QIU Ru-long CHEN Meng-meng LI Ming-yong XUE Jia-bin XU Wei-zhong WANG Fang(Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biologicals Engineering and Technology,Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-prodnets, Nanjing 210014, China Qingdao Kangda-Eurolap Rabbit Selection Co., LTD., Qingdao 266400, China)

机构地区：[1]江苏省农业科学院兽医研究所/农业部动物疫病诊断与免疫重点开放实验室/国家兽用生物制品工程技术研究中心,江苏南京210014 [2]山东青岛康大欧洲兔业育种有限公司,山东青岛266400

出　　处：《江苏农业学报》2016年第5期1117-1121,共5页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省自然科学基金项目(BK20140740);现代农业产业技术体系建设专项基金项目(CARS-44);国家自然科学基金项目(31502073)

摘　　要：本研究旨在建立鉴别兔出血症病毒经典毒株(RHDV)和变异毒株(RHDV2)的RT-PCR检测方法。根据Gen Bank中经典RHDV和RHDV2的VP60基因序列,设计2对分别结合两种基因的特异性引物。利用2对引物,以人工合成RHDV2的VP60基因构建的p MD19-T-VP60-2和p MD19-T-VP60为模板,进行RT-PCR体系退火温度的优化,优化后的退火温度为58.2℃。对优化后的RT-PCR体系进行RHDV和RHDV2的敏感性试验、特异性试验,并用于检测RHDV人工感染样品和疑似临床样品,结果显示,该方法具有良好的特异性和敏感性,经典RHDV和RHDV2的检测限度分别为95拷贝和76拷贝的靶基因片段,且对支气管败血波氏杆菌、多杀性巴氏杆菌、产气荚膜梭菌、大肠杆菌等病原以及重组质粒p MD19-T-EBHSV的检测结果均为阴性。RHDV人工感染的组织样品检出率为100%,15份临床样品中3份为经典RHDV阳性,其他均为经典RHDV及RHDV2阴性。该方法的建立能够实现快速、特异及敏感地检测RHDV和RHDV2,为监测RHDV变异株的流行情况提供技术支撑。This study aims to develop a RT-PCR system to distinguish the classical and variant rabbit hemorrhagic disease virus （ RHDV） . According to VP60 sequences of the classical and variant RHDV available in GenBank, two pairs of primers were designed. pMD19-T-VP60-2 containing VP60 gene of variant RHDV2 and pMD19-T-VP60 containing VP60 gene of classical RHDV were used as templates to optimize the annealing temperature of RT-PCR reactions and test sensitiv-ity and specificity. The RT-PCR was also used to detect the samples experimentally infected with RHDV and RHDV-suspec-ted clinical samples. The RT-PCR system with annealing temperature at 58-2℃ showed good sensitivity and specificity to RHDV and RHDV2. The detection limits were 96 copies for classical RHDV and 75 copies for RHDV2,respectively. The re-sults were negative for Bordetella bronchiseptidca, Pasteurellam-ultocide, Clostridium perfringens, Escherichia coli and pMD19-T-EBHSV. The diagnostic rate was 100% for the samples experi-mentally infected with RHDV. Fifteen RHDV-suspected clinical samples were detected, three of which were positive for classical RHDV infection, and others were all negative for both classical RHDV and RHDV2.The RT-PCR method is rapid, specific and sensitive for distinguishing RHDV and RHDV2 in a reaction system, and will offer a technical support for rapid detec-ting the epidemic of RHDV, especially RHDV2.

关 键 词：兔出血症病毒经典毒株(RHDV) 兔出血症病毒变异毒株(RHDV2) RT-PCR鉴定 

分 类 号：S858.291[农业科学—临床兽医学]