Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers.
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.
Kim
Jong-Kee
JK
Seegene Institute of Life Science, 65-5 Bangyi-Dong, Songpa-Gu, Seoul, Republic of Korea.
Lee
Hyeon-Ji
HJ
Lee
Young-Jo
YJ
Chun
Jong-Yoon
JY
Lee
In-Kyoung
IK
Lim
Young-Suk
YS
Suh
Dong Jin
DJ
Ko
Sun-Young
SY
Kim
Myeong Hee
MH
Oh
Heung-Bum
HB
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
D000998:Antiviral Agents; D017931:DNA Primers; D019259:Lamivudine