Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations
Quantitative detection of hepatitis B virus DNA in two international reference plasma preparations
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DOI：
10.1128/jcm.37.1.68-73.1999
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发表时间：
1999-01-01
影响因子：
9.4
通讯作者：
Thomssen, R
中科院分区：
文献类型：
--
作者：
Heermann, KH;Gerlich, WH;Thomssen, R
定量检测血清或血浆中的B型肝炎病毒（HBV），对监测治疗、判断预后、判断传染性和控制诊断质量具有重要意义。不幸的是，各种市售的HBV DNA检测试剂盒产生了相互矛盾的定量结果，差异高达120倍。Eurohep病理生物学小组已经建立了两个HBV携带者血浆参考样本，并尽可能准确地确定了这些样本中HBV DNA分子的数量。分别从HBV基因型A（HBV表面抗原亚型adw 2）和基因型D（ayw 2/3）的两个单一高病毒血症携带者收集血浆捐献物，并由Eurohep病理生物学组的成员分析这些样本的编码稀释液。报告一致结果的七个实验室的定量结果最初是不同的。有限稀释法和巢式PCR法存在DNA提取不完全的问题。杂交测定使用不准确定量的克隆DNA作为参考。两种杂交检测不能直接用克隆的HBV DNA校准，因为病毒体衍生的DNA反应效率低得多。在识别和消除这些问题后，三个实验室的有限稀释测定和两个生产商的杂交测定产生了一致和一致的结果：2.7 x 10（9）HBV DNA分子/ml A基因型携带者血浆中HBV DNA分子浓度范围为2.1 × 10（9）~ 3.4 × 10（9）个/ml，（范围，2.1 × 10（9）至3.0 × 10（9）HBV DNA分子/ml）。这两个Eurohep参考血浆样本已经用于检测试剂盒的标准化和质量控制试验，来自基因型A携带者的血浆可能会成为世界卫生组织参考样本的基础。
Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up to a factor of 120, The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype adw2) and genotype D (ayw2/3), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion-derived DNA reacted much less efficiently. After identification and elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 x 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.4 x 10(9) HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 X 10(9) HBV DNA molecules/ml (range, 2.1 x 10(9) to 3.0 x 10(9) HBV DNA molecules/ml) in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organisation reference sample.