title: Ultrasensitive quantification of hepatitis B virus A1762T/G1764A mutant by a SimpleProbe PCR using a wild-type-selective PCR blocker and a primer-blocker-probe partial-overlap approach.
abstract: Hepatitis B virus (HBV) carrying the A1762T/G1764A double mutation in the basal core promoter (BCP) region is associated with HBe antigen seroconversion and increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). Quantification of the mutant viruses may help in predicting the risk of HCC. However, the viral genome tends to have nucleotide polymorphism, which makes it difficult to design hybridization-based assays including real-time PCR. Ultrasensitive quantification of the mutant viruses at the early developmental stage is even more challenging, as the mutant is masked by excessive amounts of the wild-type (WT) viruses. In this study, we developed a selective inhibitory PCR (siPCR) using a locked nucleic acid-based PCR blocker to selectively inhibit the amplification of the WT viral DNA but not the mutant DNA. At the end of siPCR, the proportion of the mutant could be increased by about 10,000-fold, making the mutant more readily detectable by downstream applications such as real-time PCR and DNA sequencing. We also describe a primer-probe partial overlap approach which significantly simplified the melting curve patterns and minimized the influence of viral genome polymorphism on assay accuracy. Analysis of 62 patient samples showed a complete match of the melting curve patterns with the sequencing results. More than 97% of HBV BCP sequences in the GenBank database can be correctly identified by the melting curve analysis. The combination of siPCR and the SimpleProbe real-time PCR enabled mutant quantification in the presence of a 100,000-fold excess of the WT DNA.
authors.affiliation: Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Nie
authors.forename: Alison A
authors.initials: AA
authors.lastname: Evans
authors.forename: W Thomas
authors.initials: WT
authors.lastname: London
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Block
authors.forename: Xiangdong David
authors.initials: XD
authors.lastname: Ren
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D006509:Hepatitis B / Q000821:virology*; D006512:Hepatitis B Core Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D015345:Oligonucleotide Probes