地鼠多瘤病毒PCR检测方法的建立

摘要

Objective To develop a PCR assay for detecting nucleic acid sequence of hamster polyomavirus (HaPyV) in hamster kidney cells used for manufacture of live attenuated Japanese encephalitis vaccine.Methods A pair of specific primers for HaPyV VP1 gene was designed.The target fragment was amplified by PCR using cloned plasmid PMD19T-HaPyV688 containing HaPyV VP1 gene.The specificity of PCR assay was evaluated with DNAs extracted from simian vaeuolating virus 40,murine minute virus,porcine parvovirus and pseudorabies virus.The PCR amplified products were identified by dot blot hybridization and sequencing.The sensitivity of PCR assay was determined by amplification of tenfold serial dilution of plasmid PMD19T-HaPyV688.Murine polyomavirus (MuPyV) was used for consistency validation of PCR assay for detection of plasmid DNA and virus DNA.HaPyV DNA in hamster kidney cell suspension was determined by the developed PCR method.Results A 600 bp fragment was amplified only from the samples containing PMD19T-HaPyV688 and MuPyV DNAs and proved to be VP1 specific.The minimum DNA copies of PMD19THaPyV688 and MuPyV which could be detected were 5300 and 590,respectively.No amplification signals of HaPyV were observed in7 sub-lots of hamster kidney cell suspension.Conclusion The PCR assay developed is a specific and sensitive method for detection of HaPyV DNA in hamster kidney cells used for manufacture.%目的 建立地鼠多瘤病毒(hamster polyomavirus,HaPyV)PCR检测方法,并应用于乙型脑炎减毒活疫苗生产过程中地鼠肾细胞的感染检测.方法 设计针对HaPyV衣壳蛋白VP1基因片段的特异引物,以含HaPyV VP1片段的质粒PMD19T-HaPyV688为模板进行PCR扩增并测序确认扩增产物.以多种病毒DNA为模板进行PCR扩增,并对扩增产物进行斑点杂交鉴定以验证PCR方法的特异性.将定量的质粒PMD19T-HaPyV688 10倍系列稀释后进行PCR敏感性检测.以小鼠多瘤病毒(murine polyomavirus,MuPyV)检验PCR法对质粒DNA和病毒DNA检测的一致性.应用建立的方法对生产用地鼠肾细胞悬液进行HaPyV DNA检测.结果 仅含有质粒PMD19T-HaPyV688和MuPyV DNA的样品能够扩增出600 bp的片段,经斑点杂交及测序证明其分别为HaPyV和MuPyV的VP1特异基因片段.PCR所能检出的PMD19T-HaPyV688和MuPyV的最少DNA拷贝数分别为5300和590.7个亚批生产用地鼠肾细胞悬液的HaPyV检测结果均为阴性.结论 建立的PCR方法具有较高的敏感性和特异性,可用于生产用地鼠肾细胞HaPyV感染的检测.