Rapid detection of drug-resistant mutations in hepatitis B virus by the PCR-Invader assay.
Early detection of resistant mutations of hepatitis B virus (HBV) is important for patients on nucleos(t)ide analog therapy. An assay based on the PCR-Invader technology was developed to detect resistant mutations with high sensitivity. The assay specifically detects mutations at codons 180, 181, 184, 202, 204, and 250 of the HBV polymerase reverse transcriptase domain. These mutations result in resistance to lamivudine and entecavir. In mixtures of plasmids containing wild-type and resistant mutants, fold-over-zero values for resistant mutations were detected in 2% of the total. Seventy-five serum samples from patients, whose treatment had been switched from lamivudine to entecavir, were examined by the PCR-Invader assay and direct sequencing. The PCR-Invader assay detected all resistant mutations that were detected by direct sequencing and even detected the presence of mutants that direct sequencing could not. Cloning sequencing confirmed those mutations found by the PCR-Invader assay and not by direct sequencing. The PCR-Invader assay is a useful tool for the early detection of drug-resistant mutations.
Laboratory Management Section, BML, Inc., Kawagoe-shi, Saitama, Japan. tado-k@bml.co.jp
Kenichi
K
Tadokoro
Fumitaka
F
Suzuki
Mariko
M
Kobayashi
Toshikazu
T
Yamaguchi
Makoto
M
Nagano
Toru
T
Egashira
Hiromitsu
H
Kumada
D000998:Antiviral Agents / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D000998:Antiviral Agents; D004279:DNA, Viral; D014764:Viral Proteins; D019259:Lamivudine; C413685:entecavir; D006147:Guanine; D012194:RNA-Directed DNA Polymerase