Aberham C, Pendl C, Gross P, Zerlauth G, Gessner M
Molecular Biology, Baxter AG, Industriestr. 20, A-1221, Vienna, Austria.
J Virol Methods. 2001 Apr;92(2):183-91. doi: 10.1016/s0166-0934(00)00292-5.
Parvovirus B19 is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild, to more severe outcomes in, for example, immune-compromised patients. B19 is spread primarily via the respiratory route, but it can also be transmitted via blood and blood products. Viral loads in blood or plasma donations amount up to 10(11) genome equivalents/ml. Therefore, screening of plasma for fractionation for the presence of B19 and removal of highly loaded donations is a way to limit considerably the input of B19 into production pools and to improve further the safety of plasma products. An assay for the quantitative detection of B19 DNA, based on real-time PCR using ABI Prism SDS7700 (TaqMan) is described here. This assay allows precise quantitation of viral loads over 7 orders of magnitude. An exogenous internal control (internal quality marker) is included in each individual sample to prevent false negative results. A linearized plasmid is used as an internal quality marker that contains the identical sequence of the B19 target sequence but with an altered probe hybridization site. This allows co-amplification of B19 and internal quality marker and co-detection of FAM (6-carboxyfluorescein) or VIC labeled probes respectively. The assay is validated according to current guidelines (of the International Conference on Harmonization, Paul Ehrlich Institute, and the Council of Europe) and is optimized for high throughput screening.
细小病毒B19是一种红细胞病毒，可引起多种临床表现，从无症状或轻微症状到例如免疫功能低下患者出现的更严重后果。B19主要通过呼吸道传播，但也可通过血液和血液制品传播。血液或血浆捐献中的病毒载量可达10(11)基因组当量/毫升。因此，对用于分馏的血浆进行B19检测并去除高病毒载量的捐献，是一种可大幅限制B19进入生产池并进一步提高血浆制品安全性的方法。本文描述了一种基于使用ABI Prism SDS7700（TaqMan）进行实时PCR的B19 DNA定量检测方法。该方法可对7个数量级以上的病毒载量进行精确定量。每个样品中都包含一个外源性内控物（内部质量标志物），以防止出现假阴性结果。使用一种线性化质粒作为内部质量标志物，它包含与B19靶序列相同的序列，但探针杂交位点有所改变。这使得B19和内部质量标志物能够共同扩增，并分别对FAM（6-羧基荧光素）或VIC标记的探针进行共同检测。该方法已根据现行指南（国际协调会议、保罗·埃利希研究所和欧洲委员会的指南）进行了验证，并针对高通量筛选进行了优化。
