Determination of hepatitis B virus genotype by flow-through reverse dot blot

BackgroundThe clinical outcome and response to therapy of hepatitis B virus infection differ depending upon viral genotype. Most methods of determining the viral genotype are relatively time-consuming and costly. Moreover, the results of some methods are influenced by single nucleotide mutations.ObjectivesTo develop a novel HBV genotyping process insensitive to single nucleotide mutations using an improved reverse dot blot method employing the principle of “flow-through hybridization”.Study designThe flow through reverse dot blot (FT-RDB) method was developed using DNA from different HBV genotypes. HBV sequences from Genebank were used to design primers and probes. Specificity and sensitivity of the method were evaluated with clinical samples in which the HBV viral load was quantified by real-time PCR. Results were compared to those of multiplex PCR and sequencing. Another 59 clinical samples were used to test the clinical applicability of the method.ResultsWe showed that FT-RDB could be made insensitive to single nucleotide mismatch by adjusting the hybridization temperature. All HBV-negative samples showed no signals in the assay. The detection sensitivity of the method was found to be between 103 and 104 DNA copies/ml. The results of FT-RDB were 84% concordant with those of multiplex PCR, and 96% concordant with sequencing results in 101 cases. The genotype all 59 clinical samples was accurately identified.ConclusionsWe demonstrated that the FT-RDB method was rapid, reliable, accurate and inexpensive. It appears to be useful for routine clinical HBV genotyping even in non-specialized hospital laboratories.