目的：建立能同时检测人细小病毒B19（简称B19病毒）3种基因型的通用核酸检测（ NAT）体系，并进行方法学评价。方法对B19病毒3种基因型代表毒株全序列进行比对，选取其保守区域（ NS1区）设计B19病毒的通用引物和探针。为避免假阴性结果，在体系中引入竞争性噬菌体内标。将适宜浓度的内标添加到一系列梯度稀释的参考品质粒中，建立B19病毒实时荧光定量PCR（ qPCR）标准曲线。对建立的B19病毒通用NAT体系分别进行敏感性、特异性、重复性等方法学评价。结果建立的B19病毒实时qPCR检测体系的标准曲线在1×109～1× 103拷贝（copies）／μl模板浓度范围内相关性良好。方法学评价显示，体系的最低检测下限为10拷贝／μl；与其他血源传播病毒等无交叉反应；批内和批间实验重复性良好。结论建立了B19病毒3种基因型全检的通用NAT体系，不仅可用于B19病毒感染的诊断，还可用于血液和血液制品中B19病毒的NAT筛查，对于保障输血和血液制品的病毒安全性具有重要意义。%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 .
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