Nucleic Acid-Based Cross-Linking Assay for Detection and Quantification of Hepatitis B Virus DNA
Nucleic Acid-Based Cross-Linking Assay for Detection and Quantification of Hepatitis B Virus DNA
复制标题
用于乙型肝炎病毒 DNA 检测和定量的核酸交联测定
DOI：
--
复制
发表时间：
1999
影响因子：
9.4
通讯作者：
Ching‐lung Lai
中科院分区：
文献类型：
--
作者：
V. Lai;R. Guan;M. Wood;S. Lo;M. Yuen;Ching‐lung Lai
摘要 使用核酸光交联技术开发了一种定量血清中乙型肝炎病毒 (HBV) DNA 水平的直接测定方法。合成了与主要 HBV 亚型的病毒基因组互补的交联修饰 DNA 探针，并将其用于可在 6 小时内完成的测定。通过测试 Eurohep HBV 参考标准品和克隆 HBV DNA 的系列稀释液确定，该测定的定量范围为 5 × 105 至 3 × 109 HBV DNA 分子/ml 血清。该测定的运行内和运行间变异系数 (CV) 分别为 4.3 和 4.0%。该检测用于测定 302 份血清样本中的 HBV DNA 水平，并将结果与使用 Chiron 分支 DNA (bDNA) 检测 HBV DNA 检测相同样本后获得的结果进行比较。在测试的样本中，218 个样本的 HBV DNA 检测结果均呈阳性，72 个样本的结果低于两项检测的临界值。其余 12 个样本中，仅通过交联检测，10 个样本的 HBV DNA 呈阳性；其他 2 个样本仅通过 bDNA 检测呈阳性。 28 个样品必须通过 bDNA 测定重新测试（CV，每个样品重复测试获得的结果之间的 20% 以上），而只有 3 个样品需要通过交联测定重新测试。两项测试测得的 HBV DNA 水平之间的相关性非常高 (r = 0.902；P = 0.01)。我们的结论是，交联测定是检测和定量血清中 HBV DNA 水平的一种灵敏且可重复的方法。
ABSTRACT A nucleic acid photo-cross-linking technology was used to develop a direct assay for the quantification of hepatitis B virus (HBV) DNA levels in serum. Cross-linker-modified DNA probes complementary to the viral genomes of the major HBV subtypes were synthesized and used in an assay that could be completed in less than 6 h. The quantification range of the assay, as determined by testing serial dilutions of Eurohep HBV reference standards and cloned HBV DNA, was 5 × 105 to 3 × 109 molecules of HBV DNA/ml of serum. Within-run and between-run coefficients of variation (CVs) for the assay were 4.3 and 4.0%, respectively. The assay was used to determine HBV DNA levels in 302 serum samples, and the results were compared to those obtained after testing the same samples with the Chiron branched-DNA (bDNA) assay for HBV DNA. Of the samples tested, 218 were positive for HBV DNA by both assays and 72 gave results below the cutoff for both assays. Of the remaining 12 samples, 10 were positive for HBV DNA by the cross-linking assay only; the 2 other samples were positive by the bDNA assay only. Twenty-eight samples had to be retested by the bDNA assay (CV, >20% between the results obtained from the testing of each sample in duplicate), whereas only three samples required retesting by the cross-linking assay. The correlation between the HBV DNA levels, as measured by the two tests, was very high (r = 0.902; P = 0.01). We conclude that the cross-linking assay is a sensitive and reproducible method for the detection and quantification of HBV DNA levels in serum.