為建立可以快速同時檢測獸用疫苗中牛病毒性腹瀉病毒（BVDV）、豬圓環(huán)病毒2型（PCV2）和豬細(xì)小病毒（PPV）3種病原的方法，通過研究比對BVDV 5'-UTR、PCV2 Rep以及PPV NS1基因序列，分別設(shè)計合成了3對引物和3條熒光探針，在優(yōu)化反應(yīng)條件和反應(yīng)程序后，建立了一種可同時檢測BVDV、PCV2以及PPV的三重?zé)晒舛縋CR方法，并對其敏感性、特異性進行了評估。結(jié)果顯示，建立的三重?zé)晒舛縋CR方法靈敏度高，對3種病原核酸的最低檢測限均為10 copies/μL；特異性強，與其他相關(guān)病原（豬瘟病毒、豬繁殖與呼吸綜合征病毒、偽狂犬病病毒、非洲豬瘟病毒）均無交叉反應(yīng)。結(jié)果表明，本試驗建立的三重?zé)晒舛縋CR適于疫苗中外源病毒的檢測，可用于疫苗等生物制品質(zhì)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.
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