兽用疫苗中BVDV、PCV2和PPV污染三重荧光定量PCR检测方法的建立

为建立可以快速同时检测兽用疫苗中牛病毒性腹泻病毒(BVDV)、猪圆环病毒2型(PCV2)和猪细小病毒(PPV)3种病原的方法,通过研究比对BVDV 5'-UTR、PCV2 Rep以及PPV NS1基因序列,分别设计合成了3对引物和3条荧光探针,在优化反应条件和反应程序后,建立了一种可同时检测BVDV、PCV2以及PPV的三重荧光定量PCR方法,并对其敏感性、特异性进行了评估。结果显示,建立的三重荧光定量PCR方法灵敏度高,对3种病原核酸的最低检测限均为10 copies/μL;特异性强,与其他相关病原(猪瘟病毒、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、非洲猪瘟病毒)均无交叉反应。结果表明,本试验建立的三重荧光定量PCR适于疫苗中外源病毒的检测,可用于疫苗等生物制品质量把控。

Establishment of a Triple Fluorescent Quantitative PCR for Detecting BVDV,PCV-2 and PPV in Veterinary Vaccines

In order to establish a rapid method for simultaneously detecting bovine viral diarrhea virus(BVDV),porcine circovirus type 2(PCV2)and porcine parvovirus(PPV)in veterinary vaccines,three pairs of primers and three fluorescent probes were designed and synthesized through comparing the sequences of BVDV 5'-UTR,PCV2 Rep and PPA NS1 genes,followed by the optimization of reaction conditions and procedures,a triple fluorescent quantitative PCR was established for simultaneously detecting BVDV,PCV2 and PPV,then its sensitivity and specificity were evaluated.The results showed that the established method was with high sensitivity and specificity,its minimum detection limits for the three pathogen nucleic acids were 10 copies/μL,and failed to react with other relevant pathogens,including classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),African swine fever virus(ASFV).It was concluded that the method was applicable for detection of exogenous virus in vaccines,and could be used to control the quality of vaccines and other biological products.