Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
J Virol Methods. 2014 Feb;196:145-51. doi: 10.1016/j.jviromet.2013.11.004. Epub 2013 Nov 21.
Hantaan virus (HTNV), which belongs to the genus Hantavirus, causes hemorrhagic fever with renal syndrome (HFRS) mainly in China. The diagnosis of HFRS depends on clinical manifestations and serological tests. A SYBR Green I based one-step real-time PCR assay was established in this study to detect HTNV. The HTNV standard curves were generated by plotting mean cycle threshold (Ct) values versus 10-fold serial dilutions of a previous titrated HTNV stock over a wide range of concentrations (1×10(7) to 1PFU/ml). The minimum detection limit of the assay was 1PFU/ml, and it was 100-fold more sensitive than conventional RT-PCR. Melting curve analysis indicated that there were no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Seoul virus (SEOV). The specificity of the asssay was also verified by nuleotide sequencing of the PCR products. Intra- and inter-assay variability data were analyzed to examine the reproducibility of the assay. HTNV viral loads in HFRS patients were also investigated with the assay. These results indicated that the one-step real-time PCR assay is useful for detecting HTNV and for monitoring the viral loads.
汉坦病毒（HTNV）属于汉坦病毒属，主要在中国引起肾综合征出血热（HFRS）。HFRS 的诊断取决于临床表现和血清学检测。本研究建立了一种基于 SYBR Green I 的一步实时 PCR 检测方法来检测 HTNV。HTNV 标准曲线通过绘制平均循环阈值（Ct）值与先前滴定的 HTNV 库存的 10 倍系列稀释相对于广泛浓度范围（1×10(7)至 1PFU/ml）。该检测方法的最小检测限为 1PFU/ml，比常规 RT-PCR 灵敏 100 倍。熔解曲线分析表明该检测方法中没有引物二聚体和非特异性产物。与汉城病毒（SEOV）没有交叉反应。通过 PCR 产物的核苷酸测序也验证了该检测方法的特异性。通过分析室内和室间变异性数据来检查该检测方法的重复性。还使用该检测方法研究了 HFRS 患者的 HTNV 病毒载量。这些结果表明，一步实时 PCR 检测方法可用于检测 HTNV 并监测病毒载量。
