LNA real-time PCR probe quantification of hepatitis B virus DNA

Abstract

In the present study, we standardized a TaqMan locked nucleic acid (LNA) real-time polymerase chain reaction (PCR) probe for the accurate quantification and detection of hepatitis B virus (HBV) DNA in serum (plasma), and evaluated its methodology. LNA probe technology had a much better detection performance in HBV DNA than the common TaqMan probe. The assay based on the LNA probe had a wider linear detection range, higher sensitivity, stability and amplification efficiency, and a lower concentration of probes than the TaqMan probe. Among the 15 cases with chronic hepatitis B surface antigen (HBsAg) (+) alone, only 4 cases that were detected by TaqMan real-time PCR were negative; however, the same samples were positive by LNA real-time PCR (p<0.05). A positive correlation between viral load measurements for the 35 samples with HBV-positive DNA was detected in both LNA and TaqMan real-time PCR.

Figure 1.

Amplification plots of in-house HBV standards using the TaqMan and LNA real-time PCR probes. The X axis denotes the number of cycles, and the Y axis denotes fluorescent intensity values. The horizontal line across the graph denotes the threshold line. The plots from left to right are 4x10⁷, 4x10⁶, 4x10⁵, 4x10⁴, 4x10³, 4x10², 40 IU/ml and water. HBV, hepatitis B virus; PCR, polymerase chain reaction; LNA, locked nucleic acid; dR, double ratio.

Figure 2.

Standard curve of HBV DNA obtained using the LNA real-time PCR probe. The data show a positive correlation in the region of 40 to 4.0x10⁷ IU/ml. The equation is Y=−3.425 log (X) + 45.72, with a correlation co-efficient of 0.999. HBV, hepatitis B virus; PCR, polymerase chain reaction; LNA, locked nucleic acid; dR, double ratio; Ct, threshold cycle.

Figure 3.

Effect of freezing and thawing on the standard curve. Ct, threshold cycle.

Figure 4.

Comparative study on the change in background fluorescence with freezing and thawing (5 times). Background fluorescence of the LNA probe (16,000 to 34,000 fluorescence units) was higher than the TaqMan probe (12,000 to 20,000), and the LNA probe fluorescence slightly decreased with freezing and thawing (~1%) as compared to the TaqMan probe (~36%) (5 times). LNA, locked nucleic acid; dR, double ratio.

Figure 5.

Correlation between viral load measurements for 35 HBV-positive DNA serum samples within the linear range for both LNA and TaqMan real-time PCR. LNA, locked nucleic acid; HBV, hepatitis B virus; PCR, polymerase chain reaction.