title: A nucleotide sequence variation detection system for the core region of hepatitis C virus-1b.
abstract: Amino-acid substitution at positions 70 and 91 of the hepatitis C virus (HCV)-1b core region is a factor that contributes to a non-virological response in treatment using interferon/ribavirin combination. In this study, a system was developed for detection of nucleotide sequence variation in the core region that is simpler and less expensive than the current direct sequencing method. A PCR detection method using mutation-specific primers was developed, and amino acids at positions 70 and 91 were identified. The protein type was determined based on band intensity in electrophoresis, and classified into wild (70:R, 91:L), mutant (70:Q/H, 91:M) and competitive types. The detection rate, sensitivity and reproducibility were investigated in 108 patients with HCV-1b who were treated with interferon/ribavirin combination therapy, and correlation with the results of direct sequencing was examined. The detection rate was 94.4%, the sensitivity was 10 KIU/mL, the reproducibility was high, and in detectable cases the consistency with direct sequencing was 97.1%; inconsistency was noted in two competitive-type cases and in one case with an unusual amino-acid substitution. The results suggest that the system described in this paper provides an effective, simple and low-cost approach to detection of nucleotide sequence variation in the core region of HCV-1b.
authors.affiliation: SRL Inc., Tokyo, Japan. katsura@srl.srl-inc.co.jp
authors.forename: Katsura
authors.initials: K
authors.lastname: Okamoto
authors.forename: Norio
authors.initials: N
authors.lastname: Akuta
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Kumada
authors.forename: Mariko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Matsuo
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Tazawa
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000627:therapeutic use; D001483:Base Sequence; D004359:Drug Therapy, Combination; D004586:Electrophoresis; D014644:Genetic Variation; D016174:Hepacivirus / Q000145:classification / Q000235:genetics* / Q000502:physiology; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D012254:Ribavirin / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D012709:Serum Albumin; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D012254:Ribavirin; D007372:Interferons