Laboratory Management Section, BML, Inc., Kawagoe-shi, Saitama, Japan.
J Virol Methods. 2011 Jan;171(1):67-73. doi: 10.1016/j.jviromet.2010.10.001. Epub 2010 Oct 13.
Early detection of resistant mutations of hepatitis B virus (HBV) is important for patients on nucleos(t)ide analog therapy. An assay based on the PCR-Invader technology was developed to detect resistant mutations with high sensitivity. The assay specifically detects mutations at codons 180, 181, 184, 202, 204, and 250 of the HBV polymerase reverse transcriptase domain. These mutations result in resistance to lamivudine and entecavir. In mixtures of plasmids containing wild-type and resistant mutants, fold-over-zero values for resistant mutations were detected in 2% of the total. Seventy-five serum samples from patients, whose treatment had been switched from lamivudine to entecavir, were examined by the PCR-Invader assay and direct sequencing. The PCR-Invader assay detected all resistant mutations that were detected by direct sequencing and even detected the presence of mutants that direct sequencing could not. Cloning sequencing confirmed those mutations found by the PCR-Invader assay and not by direct sequencing. The PCR-Invader assay is a useful tool for the early detection of drug-resistant mutations.
早期检测乙型肝炎病毒（HBV）耐药突变对于核苷（酸）类似物治疗的患者非常重要。本研究开发了一种基于 PCR-Invader 技术的检测方法，具有较高的灵敏度，可特异性检测 HBV 聚合酶逆转录酶区 180、181、184、202、204 和 250 密码子的耐药突变。这些突变导致对拉米夫定和恩替卡韦的耐药。在含有野生型和耐药突变体的质粒混合物中，耐药突变的对折零值在总样本的 2%中被检测到。对 75 例已从拉米夫定转换为恩替卡韦治疗的患者血清样本进行了 PCR-Invader 检测和直接测序分析。PCR-Invader 检测方法能够检测到直接测序法检测到的所有耐药突变，甚至还能检测到直接测序法不能检测到的突变体。克隆测序证实了 PCR-Invader 检测方法而不是直接测序法发现的那些突变。该检测方法是一种早期检测耐药突变的有用工具。
