title: Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-PCR coupled with sanger sequencing.
abstract: Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases.
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Can
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Jinpiao
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Shang
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Ye
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China ouqishui@mail.fjmu.edu.cn yangbin2326@163.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China; Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian Province, China ouqishui@mail.fjmu.edu.cn yangbin2326@163.com.
authors.forename: Qishui
authors.initials: Q
authors.lastname: Ou
mesh_terms: D000328:Adult; D000368:Aged; D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D005260:Female; D006515:Hepatitis B virus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D020125:Mutation, Missense; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D044366:Transition Temperature; D055815:Young Adult
chemical_list: D017931:DNA Primers; D012194:RNA-Directed DNA Polymerase