军事医学Issue(3)：174-178,5.DOI:10.7644/j.issn.1674-9960.2015.03.004
人细小病毒B19三种基因型通用核酸检测体系的建立与评价
Establishment and evaluation of a universal nucleic acid test method for detecting human parvovirus B19
摘要
Abstract
Objective To establish and evaluate a universal real-time fluorescent quantitative PCR(qPCR)method for identifying and quantifying three human parvovirus B 19 ( B19V) genotypes.Methods Firstly, following a bioinformatic analysis of a subset of B19V genomic sequences available in the NCBI nucleotide database ,representative of genotypes 1 to 3,a set of suitable universal primers and TaqMan probes was designed from the NS 1 gene of B19V.Aplasmid was used as a quantitative standard that contained the identical sequence of the B 19 target sequence .An internal control ( IC ) was included to prevent false negative results .Then,serial 1-log dilutions of quantitative standards were prepared and used in the qPCR assays for generation of a standard curve .Finally,the specificity,sensitivity and reproducibility of the assay were assessed.Results A linear relationship of the real-time PCR method for detecting B19V from 1 ×109copies/μl to 1 ×103 copies/μl was observed .The developed qPCR protocols allowed for the detection of genotypes 1 to 3 with a limit of detection ( LOD) of 10 copies/μl.Furthermore, the assay did not amplify other blood-borne viruses.The inter-and intra-assay variability analyses showed good reproducibility of the assay .Conclusion A universal real-time qPCR method for the detection of B19V DNA is established,which will facilitate the diagnosis of B19V infections and the screening of blood and plasma-derived products , thereby improving the viral safety of transfusion and plasma-derived products .关键词
人细小病毒B19/TaqMan荧光定量PCR/核酸检测Key words
human parvovirus B19/TaqMan real-time fluorescent quantitative PCR/nucleic acid test分类
医药卫生引用本文复制引用
贾俊婷,郭逸,赵雄,马玉媛,章金刚..人细小病毒B19三种基因型通用核酸检测体系的建立与评价[J].军事医学,2015,(3):174-178,5.基金项目
北京市自然科学基金资助项目 （）
