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
- 965 Downloads
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 107 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.
KeywordsDengue Virus Japanese Encephalitis Virus Viral Hemorrhagic Fever Hemorrhagic Fever With Renal Syndrome Japanese Encephalitis Virus
This study was financially supported by the Innovation Platform for Public Health Emergency Preparedness and Response (Grant number: ZX201109) and the Jiangsu Province Health Development Project with Science and Education (Grant number: RC2011085 and RC2011084).
Conflict of interest
The authors declare that they have no competing interests.
- 1.Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H, Günther S (2002) Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 40:2323–2330PubMedCrossRefGoogle Scholar
- 2.Yu XJ, Liang MF, Zhang SY, Liu Y, Li JD, Sun YL, Zhang L, Zhang QF, Popov VL, Li C, Qu J, Li Q, Zhang YP, Hai R, Wu W, Wang Q, Zhan FX, Wang XJ, Kan B, Wang SW, Wan KL, Jing HQ, Lu JX, Yin WW, Zhou H, Guan XH, Liu JF, Bi ZQ, Liu GH, Ren J, Wang H, Zhao Z, Song JD, He JR, Wan T, Zhang JS, Fu XP, Sun LN, Dong XP, Feng ZJ, Yang WZ, Hong T, Zhang Y, Walker DH, Wang Y, Li DX (2011) Fever with thrombocytopenia associated with a novel Bunyavirus in China. N Engl J Med 364:1523–1532PubMedCrossRefGoogle Scholar
- 3.Zhang YZ, He YW, Dai YA, Xiong YW, Zheng H, Zhou DJ, Li J, Sun Q, Luo XL, Cheng YL, Qin XC, Tian JH, Chen XP, Yu B, Jin D, Guo WP, Li W, Wang W, Peng JS, Zhang GB, Zhang S, Chen XM, Wang Y, Li MH, Li Z, Lu S, Ye C, Jong DM, Xu J (2012) Hemorrhagic fever caused by a novel bunyavirus in China: pathogenesis and correlates of fatal outcome. Clin Infect Dis 54:527–553PubMedCrossRefGoogle Scholar
- 15.Sun Y, Liang M, Qu J, Jin C, Zhang Q, Li J, Jiang X, Wang Q, Lu J, Gu W, Zhang S, Li C, Wang X, Zhan F, Yao W, Bi Z, Wang S, Li D (2011) Early diagnosis of novel SFTS bunyavirus infection by quantitative real-time RT-PCR assay. J Clin Virol 12:22Google Scholar
- 16.Lambert AJ, Martin DA, Lanciotti RS (2009) A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses B. In: Hoffmann K, Depner H, Schirrmeier, Beer M (eds) J Virol Methods 136:200–209Google Scholar
- 17.He J, Bose ME, Beck ET, Fan J, Tiwari S, Metallo J, Jurgens LA, Kehl SC, Ledeboer N, Kumar S, Weisburg W, Henrickson KJ (2009) Rapid multiplex reverse transcription-PCR typing of influenza A and B virus, and subtyping of influenza A virus into H1, 2, 3, 5, 7, 9, N1 (human), N1 (animal), N2, and N7, including typing of novel swine origin influenza A (H1N1) virus, during the 2009 outbreak in Milwaukee, Wisconsin. J Clin Microbiol 47:2772–2778PubMedCrossRefGoogle Scholar
- 18.Beck ET, Jurgens LA, Kehl SC, Bose ME, Patitucci T, LaGue E, Darga P, Wilkinson K, Witt LM, Fan J, He J, Kumar S, Henrickson KJ (2009) Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin. J Mol Diagn 12:74–81PubMedCrossRefGoogle Scholar