Department of Laboratory Medicine, First Affiliated Hospital, School of medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China.
J Virol Methods. 2013 Jul;191(1):41-7. doi: 10.1016/j.jviromet.2013.03.025. Epub 2013 Apr 1.
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.
为了开发一种用于普遍检测鸭乙型肝炎病毒 (DHBV) DNA 的定量检测方法，我们基于 Genbank 中可用的 44 个 DHBV 基因组中核心区域核苷酸 241-414 位的高度同源序列，设计了一种 Taqman 实时荧光定量聚合酶链反应 (FQ-PCR) 检测方法，该方法使用了基于基因组序列的引物和探针。从 pGEM-T 中克隆的 DHBV Core 基因被用来生成 DHBV DNA 标准品。该检测方法的最低检测限为 10(3) 拷贝/ml，在广泛的 DHBV DNA 输入范围内（10(3) 到 10(10) 拷贝/ml）具有良好的线性标准曲线 (Y=-3.989X+49.086, r(2)=0.9993)。内和间检测的标准偏差分别为 0.01-0.06 和 0.05-0.16，变异系数分别为 1.3-1.8%。该检测方法的特异性通过使用鸭肝炎病毒 1 型、乙型肝炎病毒和大肠杆菌 DNA 进行了验证。ABI 7300 和 Bio-Rad iQ5 PCR 仪器的比较结果非常一致。该检测方法在中国西安的鸭的外周血和肝脏组织中 DHBV DNA 的阳性率为 63.8%（51/80）。开发的 FQ-PCR 具有高度敏感、特异性、可重复性和多功能性，可用于普遍检测不同 DHBV 株的 DHBV DNA。
