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

Wang, Yawen, Li, Yiping, Yang, Cuiling, Hui, Lingyun, Han, Qunying, Ma, Lieting, Wang, Quanying, Yang, Guangxiao, Liu, Zhengwen
Journal of virological methods 2013 v.191 no.1 pp. 41-47
DNA, DNA viruses, Duck hepatitis A virus, Duck hepatitis B virus, Escherichia coli, Hepatitis B virus, blood, detection limit, ducks, genes, genetic databases, liver, nucleotide sequences, quantitative polymerase chain reaction, China
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 10³ copies/ml and a good linear standard curve (Y=−3.989X+49.086, r²=0.9993) over a wide range of input DHBV DNA (10³ to 10¹⁰ 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.