title: Rapid and sensitive detection of hepatitis B virus 1762T/1764A double mutation from hepatocellular carcinomas using LNA-mediated PCR clamping and hybridization probes.
abstract: The 1762T/1764A double mutation of the hepatitis B virus (HBV) basal core promoter has been suggested to be a potential biomarker for hepatocellular carcinoma (HCC) among individuals with chronic HBV infection. In this study, a real-time PCR assay is established using the hybridization probes and an oligonucleotide clamp containing locked nucleic acids (LNAs). The LNA-containing oligonucleotide clamp specific for the wild type HBV is able to suppress the amplification of the wild type HBV templates. In addition, the clamp can inhibit the binding of the WT templates to the fluorescence probes thereby suppress the wild type HBV signals during the melting curve analyses. These effects facilitated the detection of HBV double mutation in the presence of 3000-fold excess of the wild type genome. Thus PCR amplification coupled with the melting curve analyses provides a quick, simple, and highly sensitive tool for the detection of this HBV double mutation.
authors.affiliation: Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, PA 18901, USA. ren@ihvr.org
authors.forename: Xiangdong David
authors.initials: XD
authors.lastname: Ren
authors.forename: Selena Y
authors.initials: SY
authors.lastname: Lin
authors.forename: Xiaohe
authors.initials: X
authors.lastname: Wang
authors.forename: Tianlun
authors.initials: T
authors.lastname: Zhou
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Block
authors.forename: Ying-Hsiu
authors.initials: YH
authors.lastname: Su
mesh_terms: D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004279:DNA, Viral