Development and application of a universal Taqman real-time PCR for quantitation of duck hepatitis B virus DNA

Abstract To develop a quantitative assay for universal detection of duck hepatitis B virus (DHBV) DNA, a Taqman real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) assay was developed using primers and probes based on genomic sequences located at nucleotide 241–414 of the DHBV Core region which possesses the highest homology among the 44 DHBV genomes available in Genbank. The DHBV Core gene cloned in pGEM-T was used to generate DHBV DNA standard. The assay had a lowest detection limit of 10 3 copies/ml and a good linear standard curve ( Y  = −3.989 X  + 49.086, r 2  = 0.9993) over a wide range of input DHBV DNA (10 3 to 10 10 copies/ml). The standard deviation of intra- and inter-assay was 0.01–0.06 and 0.05–0.16, respectively, and the coefficient of variation was 1.3–1.8%. The specificity of the assay was validated using duck hepatitis virus type 1, hepatitis B virus, and E. coli DNA. Comparison of ABI 7300 and Bio-Rad iQ5 PCR instruments yielded highly consistent results. The assay showed a positive rate of 63.8% (51/80) DHBV DNA in peripheral blood and liver tissue from ducks from Xi’an, China. The FQ-PCR developed is highly sensitive, specific, reproducible and versatile, and may be used to universally detect DHBV DNA of different DHBV strains.