新疆地区4种猪病多重PCR检测方法的建立与初步应用  

Establishment and Preliminary Application of the Multiplex PCR Method for Detection of 4 Kinds of Swine Diseases

作　　者：张勋[1] § 吴鹏[1] 李世震 伏寅峰 许追[1] 梁田[1] 杨素芳[1] 陈新凯[1] 齐亚银[1] 盛金良[1] 

机构地区：[1]石河子大学动物科技学院基础兽医学重点实验室,石河子832003 [2]石河子市第八师142团兽医站,石河子932029 [3]石河子市第八师136团,石河子834018

出　　处：《中国畜牧兽医》2015年第10期2612-2618,共7页China Animal Husbandry & Veterinary Medicine

基　　金：绵羊新抗微生物多肽基因的筛选、酵母表达及抗性分析(30960276);新疆不同品种绵羊抗菌肽NK-Lysin多态性与抗肿瘤活性关系研究(31360589)

摘　　要：本试验旨在建立一种可同时检测猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒2型(porcine circovirus type 2,PCV2)、猪伪狂犬病病毒(porcine pseudorabies virus,PRV)、猪肺炎支原体(Mycoplasma hyopneumoniae,Mh)的多重PCR检测方法。参考相关文献合成PRRSV、PCV2、PRV、Mh的特异性引物,通过对反应条件优化、特异性、敏感性测定,建立了可同时检测以上4种猪多发传染病的PCR诊断方法。扩增的片段长度分别为424bp(PRRSV)、490bp(PCV2)、298bp(PRV)和360bp(Mh),对其他常见猪病病原无特异性扩增,采用Multiplex PCR Master Mix对PRRSV、PCV2、PRV、Mh的核酸最低检出量为3个拷贝。采用多重PCR方法对60份临床样本反复检测,与单重PCR相比,4种病原符合率均为100%。结果表明,建立的多重PCR诊断方法可用于猪群中上述4种病原的单一或混合感染的快速鉴别诊断和流行病学调查。The aim of this study was to establish a multiplex PCR method for simultaneously detecting porcine reproductive and respiratory syndrome virus（PRRSV）,porcine circovirus type 2（PCV2）,porcine pseudorabies virus（PRV）and Mycoplasma hyopneumoniae（Mh）.Four pairs of primers were synthesized according to the reference.The multiplex PCR method was developed by optimizing the reaction condition,specificity and sensitivity detection.Four different amplicons with size of 424,490,298 and 360bp for PRRSV,PCV2,PRV and Mh,respectively,were yielded.The sensitivity of multiplex PCR indicated that the detection limit was 3copies by using Multi-plex PCR Master Mix,and other common pathogens were not amplified.A total of 60 specimens from piglets were tested by multiplex PCR method.The positive accordance rate between simple and multiplex PCR was 100%.This study indicated that multiplex PCR might be a useful tool for rapid and sensitive etiological diagnosis and provided an effective technical support for pathogenic molecular epidemiology investigation.

关 键 词：猪繁殖与呼吸综合征病毒 猪圆环病毒2型 猪伪狂犬病病毒 猪肺炎支原体 多重PCR 检测 

分 类 号：S858.28[农业科学—临床兽医学]