National Center for Clinical Laboratories, Beijing Hospital, People's Republic of China.
Virol J. 2011 May 14;8:227. doi: 10.1186/1743-422X-8-227.
Quantification of hepatitis B virus (HBV) DNA can be used for diagnosing HBV infection and monitoring the effect of antiviral therapy. However, probably because of mismatches between the template and primer/probe, HBV DNA in some HBV infections could not be detected using currently available commercial assays with single primer/probe. By aligning the HBV sequences, we developed a duplex real-time polymerase chain reaction (PCR) assay using two sets of primers/probes and a specific armored DNA as internal control (IC).
The limit of the duplex real-time PCR assay was 29.5 IU/ml, whereas the specificity was 100%. The within-run precision coefficient of variation (CV) ranged from 1.02% to 2.73%, while the between-run CV ranged from 0.83% to 1.25%. The optimal concentration of armored DNA IC in the HBV DNA duplex real-time PCR assay was 1 000 copies/ml. Data from 69 serum samples with HBV infection showed that the performance of the duplex real-time PCR assay was comparable to that of the COBAS Ampliprep/Cobas Taqman (CAP/CTM) HBV assay and was superior to those of the domestic commercial HBV assays.
The duplex real-time PCR assay is sufficiently sensitive, specific, accurate, reproducible and cost-effective for the detection of HBV DNA. It is suitable for high throughput screening and frequent HBV DNA level monitoring.
乙型肝炎病毒 (HBV) DNA 的定量可用于诊断 HBV 感染和监测抗病毒治疗的效果。然而，由于目前可用的单引物/探针商业检测方法与模板和引物/探针之间不匹配，某些 HBV 感染中可能无法检测到 HBV DNA。通过对 HBV 序列进行比对，我们开发了一种使用两套引物/探针和一种特定装甲 DNA 作为内部对照 (IC) 的双实时聚合酶链反应 (PCR) 检测方法。
双实时 PCR 检测方法的检测限为 29.5 IU/ml，而特异性为 100%。批内精密度变异系数 (CV) 范围为 1.02%至 2.73%，而批间 CV 范围为 0.83%至 1.25%。HBV DNA 双实时 PCR 检测方法中装甲 DNA IC 的最佳浓度为 1000 拷贝/ml。来自 69 份 HBV 感染血清样本的数据表明，双实时 PCR 检测方法的性能与 COBAS Ampliprep/Cobas Taqman (CAP/CTM) HBV 检测方法相当，优于国内商业 HBV 检测方法。
双实时 PCR 检测方法具有足够的灵敏度、特异性、准确性、重复性和成本效益，适用于 HBV DNA 的高通量筛查和频繁的 HBV DNA 水平监测。
