Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
Laboratory of Molecular Virology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
J Virol Methods. 2018 Jun;256:100-106. doi: 10.1016/j.jviromet.2018.03.001. Epub 2018 Mar 4.
For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV.
为了定量检测乙型肝炎病毒 DNA（HBV DNA），通常使用血清或血浆样本进行商业检测，但由于采集方便，口腔液样本也可以作为 HBV 诊断的替代样本。本研究旨在使用合成曲线开发用于血清和口腔液样本的实时 PCR 检测方法，以定量检测 HBV DNA。从 103 名个体（55 名 HBsAg 反应性和 HBV DNA 反应性，商业检测阳性，48 名无 HBV 标志物）中采集样本，并提交给两种基于不同标准曲线的实时 PCR 检测方法进行 HBV 前 S/S 区检测：qPCR 质粒和 qPCR 合成。qPCR 质粒法共检测到 27 份血清样本 HBV DNA 阳性，qPCR 合成法检测到 40 份（分别为 72%和 85%的一致性）。定量 PCR 合成的效率为 99%，灵敏度为 2log10 拷贝/mL。在口腔液样本中，分别使用 qPCR 质粒和合成法检测到 5 份和 10 份样本。本研究表明，使用血清样本的 qPCR 合成法由于其灵敏度，可以作为 HBV DNA 定量检测的替代方法。此外，还可以定量检测口腔液样本中的 HBV DNA，提示该样本在 HBV 分子诊断中的潜在应用。
