同时检测五种引起猪繁殖障碍的病毒的多重PCR的建立  

Simultaneous detection of five porcine reproductive disorder pathogens by multiplex PCR

作　　者：胡凌[1] 王印[1,2] 杨泽晓[1] 姚学萍[1] 林星宇[1] 李桂黎 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国兽医科学》2015年第10期1047-1052,共6页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2013BAD12B04)

摘　　要：为建立一种能同时检测5种引起猪繁殖障碍的病毒的多重PCR方法,分别针对各病毒的保守序列设计了4对特异性引物,其中3对分别用于扩增猪瘟病毒(CSFV)E2基因、非洲猪瘟病毒(ASFV)VP72基因、伪狂犬病病毒(PRV)gB基因的目的片段;针对NSP2基因设计的1对引物用于区分猪繁殖与呼吸综合征病毒(PRRSV)和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)。通过对反应条件的优化,建立了快速检测CSFV、ASFV、PRV、PRRSV和HP-PRRSV的多重PCR方法。敏感性试验结果显示,该多重PCR对这5种病原核酸的最低检测量分别为2.10×103(HP-PRRSV),1.30×103(PRRSV),1.09×104(CSFV),1.50×103(ASFV)和8.97×102(PRV)copies/μL。对49份临床样品的检测结果显示,它们的混合感染率为51%(25/49)。该方法对阴性样本的扩增结果均为阴性,并且无交叉反应性,表明该方法特异性良好,具有快速、灵敏且成本低等优点,能够对猪繁殖障碍病毒病的单个或混合感染的临床样品进行快速鉴别诊断,并对其流行病学调查具有深远意义。To establish a multiplex PCR for simultaneous detection of five porcine reproductive disorder pathogens,four pairs of specific primers were designed according to the conservative sequence of each virus.There primers of them was used to amplify：E2gene of classical swine fever virus（CSFV）,VP72 gene of African swine fever virus（ASFV）and gB gene of pseudorabies virus（PRV）,respectively.One pair of primers for NSP2 was designed to distinguish two different types of porcine reproductive and respiratory syndrome virus（PRRSV）,and highly pathogenic PRRSV（HP-PRRSV）.After optimizing reaction conditions,a multiplex PCR for simultaneous detection of CSFV,ASFV,PRV,PRRSV and HP-PRRSV was established.The sensitivity of the multiplex PCR was 2.10×10^3,1.30×10^3,1.09×10^4,1.50×10^3 and8.97×10^2 copies/μL for HP-PRRSV,PRRSV,CSFV,ASFV and PRV,respectively.Co-infection among 49 clinical samples from Sichuan Province was 51%（25/49）.There were no cross-reactions among all five pathogens and all the uninfected controls were scored negative.This method is a rapid,sensitive,specific and cost-effective diagnostic tool for single and mixed infections.And it has great significance for diagnosis and epidemiological investigation of reproductive disorder pathogens in swine.

关 键 词：多重PCR 非洲猪瘟病毒 猪瘟病毒 高致病性猪繁殖与呼吸综合征病毒 猪繁殖与呼吸综合征病毒 伪狂犬病病毒 

分 类 号：S852.659.1[农业科学—基础兽医学]