title: A two-tube multiplex real-time RT-PCR assay for the detection of four hemorrhagic fever viruses: severe fever with thrombocytopenia syndrome virus, Hantaan virus, Seoul virus, and dengue virus.
abstract: The aim of this study was to develop and evaluate a two-tube multiplex real-time RT-PCR assay for the detection and identification of four viral hemorrhagic fever (VHF) pathogens, severe fever with thrombocytopenia syndrome virus (SFTSV), Hantaan virus (HTNV), Seoul virus (SEOV), and dengue virus (DENV), from human clinical samples. The two-tube multiplex real-time RT-PCR assay we developed has a sensitivity of 10 copies/μL for each of the targets, and the performance was linear within the range of at least 10(7) transcript copies. Moreover, we evaluated the specificity of the assay using other virus RNA as template, and found no cross-reactivity. This new assay is able to detect SFTSV, HTNV, SEOV and DENV in two reactions and brings a cost of 40 % compared to separate reactions. Evaluation of this assay with clinical serum samples from laboratory-confirmed patients and healthy donors showed 100 % clinical diagnostic sensitivity and over 99 % specificity. The assay was applied for scanning 346 clinical samples collected from patients admitted to the hospital with suspected VHF and compared with virus isolation and immunofluorescence assay (IFA). The assay indentified 59 SFTSV-, 12 HTNV-, 11 SEOV- and 9 DENV-positive samples and showed higher sensitivity. This assay thus provides a reliable and cost-effective screening tool for early clinical diagnosis of SFTSV, HTNV, SEOV and DENV in the acute phase.
authors.affiliation: Department of Acute Infectious Diseases Control and Prevention, Jiangsu Provincial Center for Disease Control and Prevention, 172, Jiangsu Rd., Nanjing, 210009, Jiangsu Province, China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Li
authors.forename: Xian
authors.initials: X
authors.lastname: Qi
authors.forename: Minghao
authors.initials: M
authors.lastname: Zhou
authors.forename: Changjun
authors.initials: C
authors.lastname: Bao
authors.forename: Jianli
authors.initials: J
authors.lastname: Hu
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.forename: Shenjiao
authors.initials: S
authors.lastname: Wang
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Tan
authors.forename: Jianguang
authors.initials: J
authors.lastname: Fu
authors.forename: Jun
authors.initials: J
authors.lastname: Shan
authors.forename: Yefei
authors.initials: Y
authors.lastname: Zhu
authors.forename: Fenyang
authors.initials: F
authors.lastname: Tang
mesh_terms: D002044:Bunyaviridae Infections / Q000175:diagnosis / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D018063:Hantaan virus / Q000235:genetics / Q000302:isolation & purification*; D006480:Hemorrhagic Fever with Renal Syndrome / Q000175:diagnosis / Q000821:virology; D006482:Hemorrhagic Fevers, Viral / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D008297:Male; D016851:Orthobunyavirus / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D029263:Seoul virus / Q000235:genetics / Q000302:isolation & purification*; D019595:Severe Dengue / Q000175:diagnosis / Q000821:virology