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Background: in Thailand, an estimated three million people are chronically infected with hepatitis B virus (HBV), mostly with genotypes C and B. Level of HBV replication as measured by quantification of HBV DNA in blood is crucial for management of chronic hepatitis B. However, HBV DNA measurement is not widely used due to limited availability and high cost of the test.
Objectives: To establish an assay for both HBV DNA load and HBV genotyping based on Fluorescence Resonance Energy Transfer (FRET) real-time polymerase chain reaction (PCR) technique and evaluate it using clinical samples.
Materials and methods: Published primers and FRET probes were used. Whole genome clones of HBV genotype B and C were used as reference. HBV DNA was extracted from clinical samples using a commercial platform (Abbott m2000sp system) and was then measured with the LightCyclerâ (Roche Diagnostics Applied Science). Results obtained with the in-house assay were compared to those obtained with COBASâ TaqManâ HBV Test, v2.0 (reference).
Results: We established an in-house assay with a sensitivity of 100 IU/mL of HBV DNA. Measurement of HBV DNA in 36 clinical samples showed a very good correlation between HBV DNA load measured with in-house real-time PCR assay (R = 0.946), compared to the reference assay. This assay identified HBV genotype B and genotype C based on the melting temperature peaks, 61-64oC and 52-56oC, respectively, and identified the presence of mixed HBV B and C genotypes if the minor population represents 10% or more of the whole population. HBV genotyping was possible in 22 of 28 samples with HBV DNA load above 2.00 log10 IU/mL: 4 (18%) were genotype B and 18 (82%) were genotype C.
Conclusion: This in-house real-time PCR assay showed good performance for HBV DNA quantification in clinical samples and could distinguish B or C genotypes. It can represent an alternative to commercial assays for clinical use.
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