Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments
Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments
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DOI：
10.1016/j.jhep.2004.01.006
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发表时间：
2004-05-01
影响因子：
25.7
通讯作者：
Kim, SO
中科院分区：
文献类型：
--
作者：
Hong, SP;Kim, NK;Kim, SO
背景/目标：在长期治疗过程中出现的拉米夫定耐药B型肝炎病毒（HBV）突变经常导致HBV DNA聚合酶YMDD基序的氨基酸替换。目前检测此类变体的方法是耗时的、劳动密集型的，并且不适合于筛选大量样品。本文介绍了基质辅助激光解吸电离飞行时间质谱的发展（MALDI-TOF MS）基因分型检测方法适合于以敏感和特异的方式检测HBV变异体。方法：该方法基于PCR扩增和含有YMDD基序突变位点的寡核苷酸的质量测量。基于MALDI-TOF MS的基因分型检测试剂盒灵敏度足以检测每毫升血清中低至100个HBV基因组拷贝，并具有确定野生型和变异病毒混合物的上级特异性。当分析来自40名患者的血清时，基于MALDI-TOF MS的测定正确鉴定了已知的病毒变体和先前方法未检测到的其他病毒准种，以及它们的相对丰度。灵敏度、准确性和对高通量分析的适应性使得基于MALDI-TOF MS的测定适合于接受拉米夫定的HBV感染患者的大规模筛查，并且可以帮助提供对疾病进展和对治疗的反应的进一步理解。(C)2004年欧洲肝脏研究协会。Elsevier B. V.出版，保留所有权利。
Background/Aims: Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner.Methods: The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif.Results: The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance.Conclusions: The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy. (C) 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.