https://scholars.lib.ntu.edu.tw/handle/123456789/568496
標題: | High-resolution melting and real-time PCR for quantification and detection of drug-resistant HBV mutants in a single amplicon | 作者: | Hsiao C.-C. Chang J. Wu J.-Y. Liu W.-H. Han S.-Y. PEI-JER CHEN Shiou-Hwei Yeh |
公開日期: | 2012 | 出版社: | International Medical Press Ltd | 卷: | 17 | 期: | 2 | 起(迄)頁: | 291-303 | 來源出版物: | Antiviral Therapy | 摘要: | Background: Antiviral therapy by nucleoside/nucleotide analogues (NAs) effectively reduces HBV replication in chronic hepatitis B (CHB) patients. Because long-term NA treatments will eventually select for drugresistant mutants, early detection of mutants and frequent monitoring of viral loads is crucial for successful NA therapy. Because no efficient test for one-tube quantification and qualification of various HBV-resistant mutants exists, we propose to use high-resolution melting (HRM) analysis in combination with real-time PCR to achieve this unmet need. Methods: We developed a single amplicon for detecting HBV mutants resistant to lamivudine (LMV), adefovir (ADV) and entecavir (ETV), which are commonly used for CHB treatment. Our design consists of two steps: real-time PCR for viral quantification, and hybridization probe HRM analysis for detection of specific drug-resistant mutants. Results: Assay quantification was accurate (R=0.98) for viral loads from 103 to 109 copies/ml. HRM analysis produced distinct melting temperatures that clearly distinguished the mutants, rtM204V/I (LMV), rtA181V and rtN236T (ADV), and rtT184G and rtM250V (ETV), from their respective wild types. The assay detected mutants at only 10-25% of the HBV population. The clinical applicability of this assay was tested in a pilot study with serial samples from patients receiving LMV treatment. Conclusions: Flexibility, speed and cost-efficiency are additional benefits unique to our assay. The clinical sample results further support the feasibility of applying our design to frequent and long-term monitoring of CHB patients receiving NA treatments in the clinical setting. ? 2012 International Medical Press. |
URI: | https://www.scopus.com/inward/record.uri?eid=2-s2.0-84984585740&doi=10.3851%2fIMP2022&partnerID=40&md5=f2853b9dc368b8bf58483aac063cb2cc https://scholars.lib.ntu.edu.tw/handle/123456789/568496 |
ISSN: | 1359-6535 | DOI: | 10.3851/IMP2022 | SDG/關鍵字: | adefovir; entecavir; lamivudine; accuracy; amplicon; antiviral resistance; article; clinical article; controlled study; cost effectiveness analysis; DNA determination; feasibility study; hepatitis B; Hepatitis B virus; high resolution melting analysis; human; hybridization; molecular probe; nonhuman; nucleotide sequence; patient monitoring; pilot study; priority journal; quantitative analysis; real time polymerase chain reaction; virus detection; virus load; virus mutant; wild type |
顯示於: | 臨床醫學研究所 |
在 IR 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。