Establishment and evaluation of a universal nucleic acid test method for detecting human parvovirus B19
10.7644/j.issn.1674-9960.2015.03.004
- VernacularTitle: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
- Author:
Junting JIA
;
Yi GUO
;
Xiong ZHAO
;
Yuyuan MA
;
Jingang ZHANG
- Publication Type:Journal Article
- Keywords:
human parvovirus B19;
TaqMan real-time fluorescent quantitative PCR;
nucleic acid test
- From:
Military Medical Sciences
2015;(3):174-178
- CountryChina
- Language:Chinese
-
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 .
