title: 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=-3.989X+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.
authors.affiliation: Department of Laboratory Medicine, First Affiliated Hospital, School of medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China.
authors.forename: Yawen
authors.initials: Y
authors.lastname: Wang
authors.forename: Yiping
authors.initials: Y
authors.lastname: Li
authors.forename: Cuiling
authors.initials: C
authors.lastname: Yang
authors.forename: Lingyun
authors.initials: L
authors.lastname: Hui
authors.forename: Qunying
authors.initials: Q
authors.lastname: Han
authors.forename: Lieting
authors.initials: L
authors.lastname: Ma
authors.forename: Quanying
authors.initials: Q
authors.lastname: Wang
authors.forename: Guangxiao
authors.initials: G
authors.lastname: Yang
authors.forename: Zhengwen
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D001769:Blood / Q000821:virology; D002681:China; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D004372:Ducks; D018347:Hepadnaviridae Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D015611:Hepatitis B Virus, Duck / Q000235:genetics / Q000302:isolation & purification*; D006524:Hepatitis, Viral, Animal / Q000175:diagnosis / Q000821:virology; D008099:Liver / Q000821:virology; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes