Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments.
OBJECTIVE
Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner.


METHODS
The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif.


RESULTS
The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance.


CONCLUSIONS
The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy.
Hong
Sun Pyo
SP
GeneMatrix Inc., Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
Kim
Nam Keun
NK
Hwang
Seong Gyu
SG
Chung
Hyun Jae
HJ
Kim
Sukjoon
S
Han
Jin Hee
JH
Kim
Hyung Tae
HT
Rim
Kyu Sung
KS
Kang
Myung Seo
MS
Yoo
Wangdon
W
Kim
Soo-Ok
SO
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000627:therapeutic use; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D006515:Hepatitis B virus / Q000201:enzymology / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000998:Antiviral Agents; D004279:DNA, Viral; D019259:Lamivudine; D004259:DNA-Directed DNA Polymerase