猴B病毒通用核酸检测体系的建立与评价

摘要

To establish and evaluate a universal real-time fluorescent quantitative PCR method for identification and quantification of 5 Monkey B virus (macacine alphaherpesvirus 1) genotypes, a set of suitable universal primers and probes were designed from the conserved region of the gB gene available in the NCBI nucleotide database.Then, plasmids were constructed and used to generate standard curves. The quantitative PCR method was developed and assessed for its specificity, sensitivity and reproducibility. A linear relationship was observed from 3×102copies to 3×107copies of Monkey B virus. The qPCR developed here did not amplify Herpes simplex virus 1, Herpes simplex virus 2, Epstein-barr virus, Varicella zoster virus and Cytomegalovirus. The detection limit of qPCR assay was 30 copies for 5 genotypes of Monkey B virus. Analyses of the inter-and intra-assay variabilities showed good reproducibility. The establishment of the universal real-time qPCR method for the detection of Monkey B virus DNA would facilitate diagnosis of Monkey B virus infection in humans and make great significance to the generation of high-quality experimental monkeys.%建立能同时检测猴B病毒5种基因型的通用核酸检测体系,并对其进行方法学评价.首先,对猴B病毒5种基因型代表毒株全序列进行比对,选取其保守区域(gB基因)设计猴B病毒的通用引物和探针.其次,构建参考品质粒,并以其为模板建立核酸检测体系的标准曲线.最后,对该核酸检测体系分别进行特异性、通用性、敏感性和重复性评价.建立的标准曲线在3×107~3×102copies模板范围内相关系数|r|=0.999.方法学评价结果显示,该核酸检测体系与人单纯疱疹病毒1型、人单纯疱疹病毒2型、水痘带状疱疹病毒、EB病毒、巨细胞病毒均无交叉反应;可同时检测猴B病毒5种基因型,检测下限为30 copies;批内和批间实验重复性良好.建立的猴B病毒通用核酸检测体系对于疑似猴B病毒感染病例的辅助诊断,以及高质量实验猴群的建立均有着重要意义.