Simultaneous detection of precore/basal core promoter mutations in hepatitis B virus using arrayed primer extension.
Ha, Wai-Yan Lau, Chi-Chiu Yue, Patrick Y. K. Hung, Kaman K. M. Chan, Kelvin C. Chui, Siu-Hon Chui, Albert K. K. Yam, Wing-Cheong Wong, Ricky N. S.
Ha, Wai-Yan
Lau, Chi-Chiu
Yue, Patrick Y. K.
Hung, Kaman K. M.
Chan, Kelvin C.
Chui, Siu-Hon
Chui, Albert K. K.
Yam, Wing-Cheong
Wong, Ricky N. S.
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Other contributors
Affiliation
Epub Date
Issue Date
2006
Submitted date
2007-02-08
Subjects
Alternative
Abstract
BACKGROUND: Hepatitis B is a major disease that causes serious public health problems worldwide. The loss of HBeAg expression due to point mutations or single nucleotide polymorphisms (SNPs) in the precore/basal core promoter region of the hepatitis B virus (HBV) is associated with hepatocellular cirrhosis and carcinoma. Simultaneous screening for these mutations is strongly advocated for monitoring disease development in HBV-infected patients. The aim of this study is to apply arrayed primer extension (APEX) for the detection of HBV SNPs at the precore/basal core promoter. METHODS AND RESULTS: We optimized APEX for simultaneous detection of eight potential sites of SNPs in the precore/basal core promoter region of HBV. The precore/basal core promoter regions of HBV from 36 HBV-infected patients were amplified by PCR. After purification and DNA fragmentation, the short, single-stranded HBV DNA fragments were allowed to hybridize with the oligonucleotides corresponding to the sites of SNPs immobilized on glass slides, followed by incorporation of different fluorescently labeled dideoxynucleotides. This allows fast and unequivocal discrimination between wild-type and mutant genotypes with high dideoxy-nucleotide incorporation efficiency, sensitivity, and specificity. The coexistence of both genotypes was also detected; this was undetected by DNA sequencing. CONCLUSION: The simultaneous detection of SNPs in HBV precore/basal core promoter by APEX enables large-scale diagnostic analysis, which can be extended to the whole HBV genome.
Citation
Molecular Diagnosis & Therapy, 10(2): 125-134
Publisher
Journal
Research Unit
DOI
PubMed ID
16669611
PubMed Central ID
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Type
Journal article
Language
en
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Series/Report no.
ISSN
1177-1062