牛多瘤病毒PCR检测方法的建立及验证Development and verification of a PCR assay for bovine polyomavirus
牛步青;胡术然;高有;张海浩;陈蕾西;常亚军;易力;姚宇峰;任芳芳;
摘要(Abstract)：
目的建立牛多瘤病毒(bovine polyomavirus,BPyV)的PCR检测方法,并进行验证。方法根据NCBI中登录的BPyV主要衣壳蛋白基因序列(BPyV_gp4 VP1,NC_001442.1)设计2对引物:9PF、9PR和3PF、3PR。合成BPyV主要衣壳蛋白基因序列,并与pUC57质粒构建重组质粒。以重组质粒为模板进行PCR扩增,优化退火温度。验证方法的灵敏度及特异性,并使用该方法对Vero、MDBK、KMB17和BT细胞的培养上清液进行检测。结果建立的PCR法可以BPyV-frag重组质粒为模板扩增出特异性条带,最适退火温度为60℃。2对引物的灵敏度均达2 fg/μL(558 copies/μL),引物对9PF、9PR的灵敏度略高于3PF、3PR;引物3PF、3PR和9PF、9PR以高浓度的BPV DNA(101.6 ng/μL)和BAV DNA(97.5 ng/μL)为模板时,均无扩增条带,微量BPyV-frag重组质粒产生扩增条带。Vero、MDBK、KMB17和BT细胞培养上清液中均未检出BPyV。结论成功建立了BPyV的PCR检测方法,该方法灵敏度高,特异性强,可用于检测细胞培养上清液中的BPyV。
关键词(KeyWords)： 牛多瘤病毒;聚合酶链反应;细胞培养上清液
基金项目(Foundation): 云南省科技厅创新人才计划（2019HC006）
作者(Authors): 牛步青;胡术然;高有;张海浩;陈蕾西;常亚军;易力;姚宇峰;任芳芳;
DOI: 10.13200/j.cnki.cjb.003454
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