四种猪呼吸道疾病综合征病原多重PCR检测方法的建立及应用  

Establishment and application of a multiplex PCR for simultaneous detection of porcine reproductive and respiratory syndrome virus,porcine circovirus type 2,Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae

作　　者：陈玉燕[1,2] 杨俊[2] 聂福平[2,3] 王昱[2,3] 张姜琳 秦敏[2,4] 肖进文[2] 王国民[2] 李应国[2] 蔡家利[1] 

机构地区：[1]重庆理工大学,重庆400054 [2]重庆出入境检验检疫局/重庆市进出口食品安全工程中心,重庆400020 [3]重庆大学生物工程学院,重庆400030 [4]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《中国兽医科学》2015年第4期339-344,共6页Chinese Veterinary Science

基　　金：国家质检公益项目(201310093);国家质检总局科技项目(2013IK049);重庆市科技攻关计划项目(cstc2012ggyyjs80014)

摘　　要：为建立同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、胸膜肺炎放线杆菌(APP)和猪肺炎支原体(M.hyo)的多重PCR方法,分别针对PRRSV的ORF1a基因、PCV2的ORF2基因、APP的OMLA基因和M.hyo的P46基因的保守区域设计4对特异性引物,优化反应体系及条件,建立了可同时扩增PRRSV(225bp)、PCV2(337bp)、APP(107bp)、M.hyo(176bp)相应基因的四重PCR方法。结果显示,该方法能单管同时特异地鉴别检测PRRSV、PCV2、APP、M.hyo 4种病原体,对猪瘟病毒、副猪嗜血杆菌、猪呼吸道冠状病毒、猪流感病毒、猪细环病毒及猪细小病毒等无特异性扩增,说明该PCR具有良好的特异性;方法的灵敏度分别为195、272、241、321pg/μL。应用该方法对重庆市的61份样品进行检测,其中PRRSV、PCV2、APP、M.hyo的阳性率分别为22.9%(14/61)、18.0%(11/61)、8.2%(5/61)和18.0%(11/61)。该方法的建立为临床上4个病原的快速鉴别诊断提供了有力的技术手段。To establish a quadruple PCR for the simultaneous detection of porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV2),Actinobacillus pleuropneumoniae(APP)and Mycoplasma hyopneumoniae(M.hyo),four pairs of specific primers were designed in the conserved regions of ORF1 agene of PRRSV,ORF2 gene of PCV2,OMLA gene of APP and P46 gene of M.hyo as target sequences.And then the reaction system and conditions of the PCR were optimized to establish the quadruple PCR for the detection of PRRSV,PCV2,APP,and M.hyo.In result,the method can identify PRRSV,PCV2,APP,and M.hyo simultaneously and specifically,but non-specifically amplify classical swine fever virus,Haemophilus parasuis,porcine respiratory coronavirus,swine influenza virus,Torque teno sus virus,and porcine parvovirus.And its detection sensitivity for four pathogens are 195pg/μL(PRRSV),272pg/μL(PCV2),241pg/μL(APP),and 321pg/μL(M.hyo),respectively.In this way61 samples of Chongqing Municipality were tested and the positive rates of PRRSV,PCV2,APP and M.hyo are 22.9%(14/61),18.0%(11/61),8.2%(5/61)and 18.0%(11/61),respectively.So the establishment of the method can provide a powerful technical means for the rapid simultaneous diagnosis of the4 pathogens in clinical.

关 键 词：猪繁殖与呼吸综合征病毒 猪圆环病毒2型 胸膜肺炎放线杆菌 猪肺炎支原体 多重PCR 

分 类 号：S852.6[农业科学—基础兽医学]