Quantification of hepatitis C amino acid substitutions 70 and 91 in core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction
Quantification of hepatitis C amino acid substitutions 70 and 91 in core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction
复制标题
通过实时扩增耐药突变系统逆转录聚合酶链式反应对核心编码区丙型肝炎氨基酸取代70和91进行定量
DOI：
10.1080/00365520902937362
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
O. Yokosuka
中科院分区：
文献类型：
--
作者：
S. Nakamoto;T. Kanda;Y. Yonemitsu;M. Arai;K. Fujiwara;K. Fukai;F. Kanai;F. Imazeki;O. Yokosuka
ObjectiveThe effects of hepatitis C virus (HCV) sequence variations on the success of antiviral therapy or the development of hepatocellular carcinoma (HCC) are complex for many reasons. Recently, there have been several reports on the effects of genotype 1b HCV core amino acid substitutions 70 and/or 91 on the outcome of antiviral therapies and the clinical course. The purpose of this study was to establish real-time amplification refractory mutation system (ARMS) reverse transcription (RT)-polymerase chain reaction (PCR) assays for easy detection of these HCV mutations.Material and methodsPlasmids p-core-W, including the wild-type HCV core coding region (70R and 91L), and p-core-M, including the mutant-type HCV core (70Q and 91M), were constructed by cloning and PCR-based mutagenesis for control vector of the wild-type core and that of the mutant core, respectively. Using serially diluted forms of these vectors, SyBr Green-based real-time ARMS RT-PCR detection with each of the specific primer pairs was performed.ResultsEach primer could clearly distinguish the difference between p-core-W and p-core-M at the same copy numbers. Concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-W versus p-core-M could be distinguished, while for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay.ConclusionsThis method could be a useful alternative for the detection of genotype 1b HCV core amino acid substitutions 70 and 91 and be reliably applied for rapid screening.
