Development of a SYBR Green I based one-step real-time PCR assay for the detection of Hantaan virus

Abstract

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.

Keywords: Hantaan virus; Hemorrhagic fever with renal syndrome; Real-time PCR.