Toppinen M, Norja P, Aaltonen L-M, Wessberg S, Hedman L, Söderlund-Venermo M, Hedman K
University of Helsinki, Department of Virology, Haartmaninkatu 3, 00290 Helsinki, Finland.
University of Helsinki, Department of Virology, Haartmaninkatu 3, 00290 Helsinki, Finland.
J Virol Methods. 2015 Jun 15;218:40-5. doi: 10.1016/j.jviromet.2015.03.006. Epub 2015 Mar 17.
Parvovirus B19 (B19V) is a minute ssDNA virus associated with a wide range of diseases from childhood erythema to fetal death. After primary infection, the viral genomes persist lifelong in solid tissues of most types. Quantification of the viral DNA is important in the timing of primary infection, assessment of tissue persistence and screening of blood donor plasma. In this study, we present a new PCR assay for detection and quantification as well as for differentiation of all three B19V genotypes. A new B19V qPCR was designed to target a 154-bp region of the NS1 area. Serum, plasma and solid tissue samples were suitable for testing in the assay. The WHO International Reference Panel for Parvovirus B19 Genotypes was utilized to validate the assay for detection of different genotypes of B19V in clinical material. Each panel member yielded, by the new qPCR, a quantity similar to the one reported by National Institute for Biological Standards and Control (NIBSC). The qPCR was specific for B19V and amplified and quantified all three genotypes with detection sensitivities of ≤10 copies/reaction. The differentiation of B19V genotypes was performed by Sanger sequencing of the amplified products.
细小病毒B19（B19V）是一种微小的单链DNA病毒，与从儿童期红斑到胎儿死亡等多种疾病相关。初次感染后，病毒基因组在大多数类型的实体组织中终身存在。病毒DNA的定量对于初次感染的时间判断、组织持久性评估以及献血者血浆筛查都很重要。在本研究中，我们提出了一种新的PCR检测方法，用于检测、定量以及区分所有三种B19V基因型。设计了一种新的B19V定量PCR，以NS1区域的一个154碱基对区域为靶点。血清、血浆和实体组织样本适用于该检测方法。利用世界卫生组织细小病毒B19基因型国际参考品系来验证该检测方法在临床材料中检测不同基因型B19V的能力。通过新的定量PCR，每个品系成员得到的数量与国家生物标准与控制研究所（NIBSC）报告的数量相似。该定量PCR对B19V具有特异性，能扩增和定量所有三种基因型，检测灵敏度≤10拷贝/反应。通过对扩增产物进行桑格测序来区分B19V基因型。
