焦磷酸测序法敏感性检测HBV耐药突变。

StephenW

J Med Virol. 2018 Feb 28. doi: 10.1002/jmv.25066. [Epub ahead of print]
Pyrosequencing Method for Sensitive Detection of HBV Drug Resistance Mutations.Lee GH1,2, Inoue M3, Chong RH2, Toh J3, Wee SY3, Loh KS1, Lim SG1,2.
Author information
1Department of Medicine, National University Health System.2Yong Loo Lin School of Medicine, National University of Singapore.3Experimental Therapeutics Centre.

AbstractBackground & Hypothesis: Hepatitis B (HBV) drug resistance assay is important for guiding therapy after the development of virologic breakthrough for patients receiving nucleoside/-tide analog therapy. However, the existing genotyping tools are either costly or lack sensitivity to detect mixed genotypes, and an improved method of resistant mutation detection is needed.
METHODS: An assay protocol for clinical application using pyrosequencing method was developed, capable of detecting all known validated HBV polymerase gene mutations that impart resistance to lamivudine, adefovir, tenofovir, and entecavir. Sixty-eight serum samples with known HBV resistance genotypes, previously tested with either Sanger sequencing assay or commercial line probe assay, were used for validation. Where there were discrepancies between the two methods, clonal sequencing by Sanger's method was used for confirmation.
RESULTS: The modified pyrosequencing method accurately identified all the cloned polymerase genotypes and was able to distinguish as little as 5% of the mutant populations. This assay can be performed on serum sample with HBV DNA as low as 13.5 IU/ml. The cost per test was less than existing commercial assay. Discussion & Conclusions: HBV drug resistance pyrosequencing assay was accurate, more sensitive and cheaper compared with the existing methods. It can detect minor populations of drug-resistant clones earlier, before the drug resistant clones become dominant, allowing the opportunity for an earlier change of therapy. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

KEYWORDS: drug resistance mutations; hepatitis B virus; line probe assay; nucleoside analog; pyrosequencing assay

PMID:29488627DOI:10.1002/jmv.25066

StephenW

J Med Virol。 2018年2月28日。doi：10.1002 / jmv.25066。 [电子版提前打印]
焦磷酸测序法敏感性检测HBV耐药突变。
Lee GH1,2，Inoue M3，Chong RH2，Toh​​ J3，Wee SY3，Loh KS1，Lim SG1,2。
作者信息

1
    国立大学卫生系医学系。
2
    新加坡国立大学Yong Loo Lin医学院。
3
    实验治疗中心。

抽象

背景和假设：乙型肝炎（HBV）耐药性分析对于接受核苷类似物治疗的患者发展病毒学突破后指导治疗非常重要。然而，现有的基因分型工具要么昂贵，要么缺乏检测混合基因型的灵敏度，需要改进抗性突变检测方法。
方法：

开发了使用焦磷酸测序法进行临床应用的测定方案，其能够检测所有已知的经验证的HBV聚合酶基因突变，其赋予对拉米夫定，阿德福韦，替诺福韦和恩替卡韦的抗性。使用先前用Sanger测序测定法或商业线探针测定法测试的具有已知HBV抗性基因型的68个血清样品用于验证。如果两种方法之间存在差异，则使用Sanger方法的克隆测序来确认。
结果：

改进的焦磷酸测序方法准确鉴定了所有克隆的聚合酶基因型，并能够区分仅5％的突变群体。该测定可以在血清样品上进行，HBV DNA低至13.5IU / ml。每次测试的成本低于现有商业化验。讨论与结论：与现有方法相比，HBV耐药焦磷酸测序法准确，灵敏且便宜。在耐药性克隆占优势之前，它可以早期检测到少数耐药性克隆，从而有机会尽早改变治疗方案。本文受版权保护。版权所有。

本文受版权保护。版权所有。
关键词：

耐药突变;乙型肝炎病毒;线性探针分析;核苷类似物;焦磷酸测序法

结论：
    29488627
DOI：
    10.1002 / jmv.25066