Simultaneous Detection of Precore/Basal Core Promoter Mutations in Hepatitis B Virus Using Arrayed Primer Extension
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ORIGINAL RESEARCH ARTICLE
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Simultaneous Detection of Precore/Basal Core Promoter Mutations in Hepatitis B Virus Using Arrayed Primer Extension Wai-Yan Ha,1 Chi-Chiu Lau,1 Patrick Y.K. Yue,1 Kaman K.M. Hung,1 Kelvin Chan,2 Siu-Hon Chui,3 Albert K.K. Chui,4 Wing-Cheong Yam5 and Ricky N.S. Wong1,6 1 2 3 4 5 6
Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK Diagnostix Medical Centre Ltd, Hong Kong Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong Department of Microbiology, Faculty of Medicine, The University of Hong Kong, Hong Kong Department of Biology, Hong Kong Baptist University, Hong Kong
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 HBVinfected 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 dideoxynucleotide 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 largescale diagnostic analysis, which can be extended to the whole HBV genome.
Hepatitis B is a major disease that causes serious public health problems worldwide. According to the World Health Organization (WHO), about 2 billion people have been infected with the hepatitis B virus (HBV) worldwide. More than 350 million people have been chronically infected; three-quarters of these are Asian.[1] These patients are typically hepatitis B e antigen (HBeAg)-positive and HBV DNA-positive in serum. As HBeAg is thought to be the major humoral and cellular target,[2] some patients developed antibodies to HBeAg (seroconversion), leading to the loss of detectable HBeAg and allowing liver disease remission.[3] However, 25% of chron
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