Sun Pyo Hong1, Nam Keun Kim2, Seong Gyu Hwang2, 3, Hyun Jae Chung1, Sukjoon Kim1, Jin Hee Han2, Hyung Tae Kim3, Kyu Sung Rim2, 3, Myung Seo Kang4, Wangdon Yoo1, Soo-Ok Kim1
1 GeneMatrix Inc., Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea
2 Institute for Clinical Research, Pundang CHA General Hospital, College of Medicine, Pochon CHA University, Seongnam, South Korea
3 Department of Internal Medicine, Pundang CHA General Hospital, College of Medicine, Pochon CHA University, Seongnam, South Korea
4 Department of Laboratory Medicine, Pundang CHA General Hospital, College of Medicine, Pochon CHA University, Seongnam, South Korea
Corresponding author : Seong Gyu Hwang. Address: Department of Internal Medicine, Division of Gastroenterology-Hepatology, Pundang CHA General Hospital, College of Medicine, Pochon CHA University 351, Yatap-dong, Pundang-gu, Sungnam-si, Kyonggi-do 463-712, South Korea.
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.
The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif.
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.
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.
Keywords : Hepatitis B virus; Lamivudine; YMDD variant; Genotyping; Mass spectrometry