title: Application of coamplification at lower denaturation temperature-PCR sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus.
abstract: Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations.
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong SAR.
authors.forename: Danny Ka-Ho
authors.initials: DK
authors.lastname: Wong
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR.
authors.forename: Ottilia
authors.initials: O
authors.lastname: Tsoi
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR.
authors.forename: Fung-Yu
authors.initials: FY
authors.lastname: Huang
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong SAR.
authors.forename: Wai-Kay
authors.initials: WK
authors.lastname: Seto
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong SAR.
authors.forename: James
authors.initials: J
authors.lastname: Fung
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong SAR.
authors.forename: Ching-Lung
authors.initials: CL
authors.lastname: Lai
authors.affiliation: Department of Medicine, The University of Hong Kong, Hong Kong SAR State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong SAR mfyuen@hku.hk.
authors.forename: Man-Fung
authors.initials: MF
authors.lastname: Yuen
mesh_terms: D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009691:Nucleic Acid Denaturation / Q000528:radiation effects*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature