Quantification of hepatitis C amino acid substitutions 70 and 91 in the core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction.
OBJECTIVE
The 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.


METHODS
Plasmids 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.


RESULTS
Each 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.


CONCLUSIONS
This 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.
Nakamoto
Shingo
S
Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba, Japan.
Kanda
Tatsuo
T
Yonemitsu
Yutaka
Y
Arai
Makoto
M
Fujiwara
Keiichi
K
Fukai
Kenichi
K
Kanai
Fumihiko
F
Imazeki
Fumio
F
Yokosuka
Osamu
O
D019943:Amino Acid Substitution / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014758:Viral Core Proteins / Q000235:genetics
D003062:Codon; D017931:DNA Primers; D012367:RNA, Viral; D014758:Viral Core Proteins