[1]宋艳华,魏后军,范志宇,等.兔出血症病毒经典毒株和变异毒株的RT-PCR鉴定[J].江苏农业学报,2016,(05):1117-1121.[doi:10.3969/j.issn.1000-4440.2016.05.026]
　SONG Yan-hua,WEI Hou-jun,FAN Zhi-yu,et al.Development of a RT-PCR technique for rapid identification of classical and variant rabbit hemorrhagic disease virus[J].,2016,(05):1117-1121.[doi:10.3969/j.issn.1000-4440.2016.05.026]
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兔出血症病毒经典毒株和变异毒株的RT-PCR鉴定()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年05期
页码:
1117-1121
栏目:
畜牧兽医·水产养殖
出版日期:
2016-11-22

文章信息/Info

Title:
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1; 陈萌萌1; 李明勇2; 薛家宾1; 徐为中1; 王芳1
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
Author(s):
SONG Yan-hua1; WEI Hou-jun1; FAN Zhi-yu1; ZUO Yuan-yuan1; HU Bo1; QIU Ru-long1; CHEN Meng-meng1; LI Ming-yong2; XUE Jia-bin; XU Wei-zhong; WANG Fang
1. 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-products, Nanjing 210014,China；2. Qingdao Kangda-Eurolap Rabbit Selection Co.,LTD., Qingdao 266400,China
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
Keywords:
classical RHDV; RHDV2; RT-PCR identification
分类号:
S858.2912.65
DOI:
10.3969/j.issn.1000-4440.2016.05.026
文献标志码:
A
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GenBank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pMD19-T-VP60-2和pMD19-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pMD19-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。
Abstract:
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 sensitivity and specificity. The RT-PCR was also used to detect the samples experimentally infected with RHDV and RHDV-suspected 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 results were negative for Bordetella bronchiseptidca, Pasteurellamultocide, Clostridium perfringens, Escherichia coli and pMD19-T- EBHSV. The diagnostic rate was 100% for the samples experimentally 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 detecting the epidemic of RHDV, especially RHDV2.

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备注/Memo

备注/Memo:
收稿日期：2016-08-08 基金项目：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） 作者简介：宋艳华（1985-），女，博士，山东聊城人，副研究员，主要从事畜禽传染病防控研究。(Tel) 025-84390337；(E-mail) songyanhua8507@126.com 通讯作者：王芳，(Tel)025-84390337；(E-mail)rwangfang@126.com
更新日期/Last Update: 2016-11-22