猪用疫苗中牛病毒性腹泻病毒和非洲猪瘟病毒双重荧光PCR检测方法的建立及验证

Development and verification of a duplex real-time PCR for detection of bovine viral diarrhea virus and African swine fever virus in pig vaccines

目的建立同时检测猪用疫苗中牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)和非洲猪瘟病毒(African swine fever virus,ASFV)2种病原的双重荧光PCR方法,并进行验证及初步应用。方法根据NCBI中收录的BVDV5'UTR和ASFV VP72基因序列,分别设计1对特异性引物和1条TaqMan探针,建立一种能同时检测BVDV和ASFV的双重荧光PCR方法。对建立的方法进行灵敏度、特异性和精密性验证。采用建立的方法分别检测猪伪狂犬病病毒(pseudorabies virus,PRV)活疫苗和猪瘟病毒(classical swine fever virus,CSFV)活疫苗(76批次)以及添加BVDV和重组质粒pMD-VP72的猪PRV活疫苗(各5瓶)中的BVDV和ASFV,并按照《中华人民共和国兽药典》三部(2020版)和中华人民共和国农业农村部公告第172号规定的方法分别对BVDV和ASFV进行复核检测。结果建立的双重荧光PCR法最低可检测浓度为4.2拷贝/μL的BVDV重组质粒和浓度为8.7拷贝/μL的ASFV重组质粒;不与其他常见病原体发生交叉反应;重复性和试验间精密性检测的变异系数(CV)均不高于2%。结论本实验建立的双重荧光PCR方法灵敏度高,特异性强,精密性好,可用于猪用疫苗中外源病毒的快速检测及猪用疫苗等生物制品的质量控制。

Objective To develop and verify a duplex real-time PCR method for simultaneous detection of bovine viral diarrhea virus(BVDV)and African swine fever virus(ASFV)in pig vaccines and apply it preliminarily.Methods According to the sequence of BVDV 5'UTR and ASFV VP72 included in NCBI,a pair of specific primers and a TaqMan probe were designed respectively,and a duplex real-time PCR method for simultaneous detection of BVDV and ASFV was established.The developed method were verified for the sensitivity,specificity and precision,and used to detect BVDV and ASFV of a total of 76 batches of live vaccines against porcine pseudorabies virus(PRV)and classical swine fever virus(CSFV),as well as live vaccines against porcine PRV added with BVDV and recombinant plasmid pMD-VP72,5 bottles for each,respectively.The BVDV and ASFV were rechecked and detected using the methods stipulated in the third part of Veterinary Pharmacopoeia of the People's Republic of China(2020 edition)and Announcement No.172 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China.Results 4.2 copies/μL of BVDV and 8.7 copies/μL of ASFV recombinant plasmids were detected by the developed duplex real-time PCR;No cross reaction with other common pathogens was observed and the coefficient of variation(CV)of repeatability and precision was not more than 2%.Conclusion The duplex real-time PCR method developed in this experiment showed good sensitivity,specificity and precision,which might be used for the rapid detection of exogenous viruses in pig vaccines and the quality control of pig vaccines and other biological products.