Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation

Abstract

Aim: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.

Methods: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection.

Results: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10 micromol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 10(3) copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg.

Conclusion: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Figure 1

Optimization of the concentration of dNTPs. A: optical absorption density, A_m and A_w: A values of wells with horseradish peroxidase-labeled anti-DIG (A_m) and anti-FITC (A_w), respectively, 1: 200 μmol/L of dNTPs, 2: 50 μmol/L of dNTPs, 3: 10 μmol/L of dNTPs, 4: 2 μmol/L of dNTPs.

Figure 2

Detection of BCP mutation in recombinant plasmid DNA using optimized CD-PCR. A: pHB-BCP2 as template, B: pTZ19U-HBV as template, C: A_m/A_w ratio as criteria for the result analysis, D: detection BCP mutation among mixture of pHB-BCP2 and pTZ19U-HBV. A: optical absorption density, A_m and A_w: A values of wells with horseradish peroxidase-labeled anti-DIG (A_m) and anti-FITC (A_w), respectively, 0-100%: copy ratio of pHB-BCP2 with total recombinant plasmid DNA in mixture.