title: 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.
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Ping-zhong
authors.initials: PZ
authors.lastname: Wang
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Hai-tao
authors.initials: HT
authors.lastname: Yu
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhao
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Du
authors.affiliation: Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China. Electronic address: xfbai2011@163.com.
authors.forename: Xue-fan
authors.initials: XF
authors.lastname: Bai
mesh_terms: D052160:Benzothiazoles; D002681:China; D003959:Diamines; D018063:Hantaan virus / Q000302:isolation & purification*; D006480:Hemorrhagic Fever with Renal Syndrome / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009930:Organic Chemicals / Q000032:analysis*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D014773:Virology / Q000379:methods
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: Hantaan virus; Hemorrhagic fever with renal syndrome; Real-time PCR