Validation and comparison of different PCR-based methods for detection of hepatitis B virus precore region mutants

Hepatitis virus variants detection is useful in clinical practice; however, methods that are used for their identification may influence the results significantly. Three PCR-based assays for quantitation of G1896A precore HBV mutants: two allele specific PCRs, single tube (single-AS-PCR) with enzymatic restriction or separate tubes (twin-AS-PCR) and one oligohybridization assay (OA) with three probes were developed and standardized. Wild type and mutant plasmids and 10 sera were used as reference. All methods had sensitivity limits of 104 copies/ml and their specificity encompassed 3 logs (104-107 copies/ml) with dynamic ranges of logs for OA, twin-AS-PCR and single-AS-PCR, respectively. Single-AS-PCR and OA detected minor viral populations when their relative prevalence was at least 10% of the overall viral population whereas their detection by twin-AS-PCR ranged from 0.1 to 10% for samples with 107 and 105 copies/ml viral loads, respectively. Twin-AS-PCR was the most sensitive to detect the minor viral population, whereas single-AS-PCR and OA were more accurate to quantify the relative proportions of the two viral populations independently of the overall viral load. In conclusion, an accurate characterization of HBV precore heterogeneity should be warranted by a careful choice of the most appropriate assay according to the aim of the study.