title: Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers.
abstract: Mutations in the YMDD motif of the hepatitis B virus (HBV) polymerase gene increase lamivudine resistance of HBV, highlighting the clinical importance of accurate and sensitive detection of HBV mutants. Using dual-priming oligonucleotide primer technology, an assay that can detect mutations at codons 180 (L528M) and 204 (YVDD, YIDD, and YSDD) by a single-step multiplex PCR was developed. This Seeplex Lami-DR assay was sufficiently sensitive to detect 10(3)HBV/ml and was able to detect minor mutants comprising as little as 2% of the viral population. Mutants were detected in 57 of 65 serum samples (88%) from patients with chronic hepatitis B who had been treated with lamivudine (median, 32 months; range, 1-83 months). The agreement with direct sequencing was only 38.5% (25/65). Discrepancies between these methods resulted from detection of additional mutants by the Seeplex Lami-DR assay, as confirmed by a novel verification analysis. This assay is not only highly accurate and sensitive, but is also simple and cost-effective, requiring no expensive probes, laborious sequencing procedures, or digestion with restriction enzymes. Accordingly, the Seeplex HBV Lami-DR assay should be considered as a first-line, cost-effective tool for detecting viral mutations in patients with chronic hepatitis B receiving lamivudine therapy.
authors.affiliation: Seegene Institute of Life Science, 65-5 Bangyi-Dong, Songpa-Gu, Seoul, Republic of Korea.
authors.forename: Jong-Kee
authors.initials: JK
authors.lastname: Kim
authors.forename: Hyeon-Ji
authors.initials: HJ
authors.lastname: Lee
authors.forename: Young-Jo
authors.initials: YJ
authors.lastname: Lee
authors.forename: Jong-Yoon
authors.initials: JY
authors.lastname: Chun
authors.forename: In-Kyoung
authors.initials: IK
authors.lastname: Lee
authors.forename: Young-Suk
authors.initials: YS
authors.lastname: Lim
authors.forename: Dong Jin
authors.initials: DJ
authors.lastname: Suh
authors.forename: Sun-Young
authors.initials: SY
authors.lastname: Ko
authors.forename: Myeong Hee
authors.initials: MH
authors.lastname: Kim
authors.forename: Heung-Bum
authors.initials: HB
authors.lastname: Oh
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics; D019694:Hepatitis B, Chronic / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology* / Q000627:therapeutic use; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D019259:Lamivudine