Heermann K H, Gerlich W H, Chudy M, Schaefer S, Thomssen R
Division of Medical Microbiology and National Reference Laboratory for Viral Hepatitis, University of Gottingen, D 37075 Gottingen, Germany.
J Clin Microbiol. 1999 Jan;37(1):68-73. doi: 10.1128/JCM.37.1.68-73.1999.
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 Organization reference sample.
血清或血浆中乙型肝炎病毒（HBV）的定量检测对于监测治疗效果、判断疾病预后、评估传染性以及诊断质量控制均具有重要意义。遗憾的是，各种市售的HBV DNA检测试剂盒给出的定量结果相互矛盾，差异高达120倍。欧洲肝脏病病理生物学小组制备了两份来自HBV携带者的血浆参考样本，并尽可能准确地测定了这些样本中的HBV DNA分子数量。分别采集了两名单一高病毒血症HBV A基因型（HBV表面抗原亚型adw2）和D基因型（ayw2/3）携带者的血浆捐赠样本，欧洲肝脏病病理生物学小组的成员对这些样本的编码稀释液进行了分析。最初，报告结果一致的七个实验室给出的定量结果存在差异。有限稀释法和巢式PCR检测存在DNA提取不完全的问题。杂交检测使用定量不准确的克隆DNA作为参考。两种杂交检测无法直接用克隆的HBV DNA进行校准，因为病毒体衍生的DNA反应效率低得多。在识别并消除这些问题后，三个实验室的有限稀释检测法和两家生产商的杂交检测法产生了一致且相符的结果：A基因型携带者血浆中HBV DNA分子含量为2.7×10⁹/ml（范围为2.1×10⁹至3.4×10⁹ HBV DNA分子/ml），D基因型携带者血浆中为2.6×10⁹ HBV DNA分子/ml（范围为2.1×10⁹至3.0×10⁹ HBV DNA分子/ml）。欧洲肝脏病小组的两份参考血浆样本已用于检测试剂盒的标准化和质量控制试验，A基因型携带者的血浆可能会成为世界卫生组织参考样本的基础。
