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.

Figure 1

The oligonucleotides used in this study. HBV wild type (WT) DNA sequence (GeneBank Accession #X04615) from positions 1631 to 1830 is listed. The position and the direction of the oligonucleotides are shown with line arrows aligned to the HBV WT sequence. The sequence of the LNA-containing oligonucleotide is listed with the LNA monomers in upper-case letters.

Figure 2

Differentiation of the HBV 1762T/1764A double mutation from the WT by melting curve analysis and the WT clamp. (1) HBV 1762T/1764A (300 copies); (2) WT HBV (300 copies); (3) H2O only; (4) WT HBV plus LNA clamp; (5) WT:HBV1762T/1764A equal molar mix; (6) WT:HBV1762T/1764A mix plus LNA clamp. Details of real time PCR and melting curve analysis are described in materials and methods section.

Figure 3

Sensitivity of LNA-mediated clamped real time PCR for the detection of HBV 1762T/1764A double mutation. Panel A, melting curves of (1) 10 copies of HBV 1762T/1764A plus LNA clamp; (2) 30,000 copies of WT HBV; (3) 30,000 copies of WT HBV plus LNA clamp; (4) 10 copies of HBV 1762T/1764A plus 30,000 copies of WT HBV; (5) 10 copies of HBV 1762T/1764A plus 30,000 copies of WT HBV plus LNA clamp; (6) H2O only. Panel B, amplification curves of HBV 1762T/1764A double mutation at (1) 3,000 copies; (2) 300 copies; (3) 30 copies; (4) 10 copies; (5) 5 copies; (6) 0 copy (H2O only). The PCR was carried out in the presence of 30,000 copies of WT HBV DNA and 2 μM WT clamp.

Figure 4

Analysis of HCC tumor DNA by PCR followed by direct DNA sequencing. The Seq fwd and Seq rev primers (see Figure 1) were used for amplification and sequencing. The wild type (WT) is from GeneBank Accession #X04615. Bases identical to the WT sequence are indicated by the colon signs. N indicates a base that cannot be determined automatically by the software. In panel B, the chromatogram file for the sample 219K is shown.

Figure 5

Analysis of HCC tumor DNA by LNA-mediated clamped real time PCR and melting curve analysis. Shown here are the melting curves for the HCC tissue samples with the sample names indicated along the curves. Reactions from samples 131K and 150K did not have the LNA clamp.

Figure 6

Effect of LNA-mediated PCR clamping on DNA sequencing. Regular PCRs were performed with or without the LNA-containing WT HBV clamp (+ or − LNA clamp), and were subjected to direct sequencing. Results for the HCC samples 169K and 174K are shown in panel A. Results from the plasmid mixtures are shown in panel B. DM/WT 1:4 indicates a mixture of the double mutation and WT plasmids at a ratio of 1:4. The chromatogram files are shown with the readout sequence on the top. TTAAAGGTCTT is the WT sequence with 1762A and 1764G underlined. TTAATGATCTT contains 1762T and 1764A double mutation. Note that 1762 and 1764 positions may contain mixed bases.