Semi-quantitative discrimination of HBV mutants using allele-specific oligonucleotide hybridization with Handy Bio-Strand(METHODS)
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概要
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The analysis of hepatitis B virus (HBV) mutations is important for understanding HBV progression and for deciding on appropriate clinical treatments. However, it is difficult to determine the quantitative abundance of various mutants in heterogeneous mixtures by conventional methods such as direct sequencing or the TaqMan assay. In this study, we investigated the possibility of using both allele-specific oligonucleotide hybridization (ASOH) and allele-specific oligonucleotide competitive hybridization (ASOCH) with the Handy Bio-Strand system for the quantitative identification of three well-defined HBV variants: the basal core promoter (BCP) mutations (nt1762 and nt1764), the pre-core (PC) mutation (nt1896), and variance at nt1858. Using standardized mixtures of wild-type and mutant DNA, optimal hybridization conditions for ASOH and ASOCH were determined. Next, the performance of these methods was evaluated using actual serum DNAs from HBV patients. Excellent reproducibility was obtained both in the analysis of internal positive controls and in the semi-quantitative categorization of heterogeneous viral mixtures into five abundance groups (0%, 25%, 50%, 75%, and 100% mutant virus). Combined with real-time PCR to determine the HBV viral load, this hybridization method offers a new tool with applications both in HBV clinical research and treatment.
著者
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Ginya Harumi
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology
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Asahina Junko
Precision System Science Co., Ltd.
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Nakao Rumiko
Clinical Research Center, National Nagasaki Medical Center
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Tamada Yohko
Clinical Research Center, National Nagasaki Medical Center
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Takahashi Masaaki
Precision System Science Co., Ltd.
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Yohda Masafumi
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology
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Yatsuhashi Hiroshi
Clinical Research Center, National Nagasaki Medical Center
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Asahina Junko
Precision System Science Co. Ltd.
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Asahina Junko
東京農工大学 生命工学専攻
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Yatsuhashi Hiroshi
Clinical Research Center National Nagasaki Medical Center
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Nakao Rumiko
国立病院長崎医療センター 臨床研究センター
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Nakao Rumiko
Clinical Research Center National Nagasaki Medical Center
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Ginya Harumi
Department Of Biotechnology And Life Science Tokyo University Of Agriculture And Technology:precisio
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Ginya Harumi
東京農工大学 生命工学専攻
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Tamada Yohko
Clinical Research Center National Nagasaki Medical Center
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Yohda M
Tokyo Univ. Agriculture And Technol. Tokyo
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Yohda Masafumi
Department Of Biotechnology And Life Science Graduate School Of Engineering Tokyo University Of Agri
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Yohda M
Inst. Physical And Chemical Res. (riken) Saitama Jpn
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Miyashita Yukiko
Department Of Biotechnology And Life Science Tokyo University Of Agriculture And Technology
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Yatsuhashi Hiroshi
Clinical Research Center National Hospital Organization (nho) Nagasaki Medical Center
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Yoshida Mamiko
Precision Systems Science Co. Ltd.
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Takahashi Masaaki
Precision System Science Co. Ltd.
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Ginya Harumi
Department Of Biotechnology And Life Science Tokyo University Of Agriculture And Technology:precisio
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