Mou Yi, Athar Muhammad Ammar, Wu Yuzhen, Xu Ye, Wu Jianhua, Xu Zhenxing, Hayder Zulfiqar, Khan Saeed, Idrees Muhammad, Nasir Muhammad Israr, Liao Yiqun, Li Qingge
State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, China.
J Clin Microbiol. 2016 Nov;54(11):2661-2668. doi: 10.1128/JCM.00439-16. Epub 2016 Aug 17.
Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.
检测抗乙型肝炎病毒（HBV）耐药突变对于慢性乙型肝炎病毒感染的治疗决策至关重要。我们描述了一种基于实时PCR的检测方法，该方法使用多色熔解曲线分析（MMCA），能够准确检测HBV聚合酶基因逆转录酶区域10个氨基酸位置上的24个HBV核苷酸突变。该双反应检测方法的检测限为每个反应5个拷贝，当总体浓度为10拷贝/μl时，在主要野生型群体存在的情况下，能够检测到具有逆转录酶M204V氨基酸突变的次要突变群体（占总群体的5%）。该检测方法可在3小时内完成，每个样本的材料成本低于10美元。使用来自核苷（酸）类似物治疗和未治疗患者的三组样本进行的临床验证研究表明，99.3%（840/846）的样本和99.9%（8454/8460）的氨基酸结果与来自HBV逆转录酶区域的PCR扩增子的桑格测序结果一致（PCR桑格测序）。PCR桑格测序方法未检测到六个含有由次要突变亚群体组成的混合感染样本中的HBV DNA，但MMCA检测到了，并且通过在较低变性温度下的共扩增-PCR桑格测序证实了结果。在接受治疗的患者中，48.6%（103/212）携带显示拉米夫定单耐药、阿德福韦单耐药、恩替卡韦耐药或拉米夫定和阿德福韦耐药的病毒。在未治疗的患者中，中国组含突变的样本比巴基斯坦组更多（3.3%对0.56%）。由于其准确性、快速性、广泛的覆盖范围和成本效益，实时PCR检测方法可能成为资源有限国家检测抗HBV耐药突变的有力工具。
