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Erschienen in: Archives of Virology 9/2013

01.09.2013 | Original Article

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

verfasst von: Zhifeng Li, Xian Qi, Minghao Zhou, Changjun Bao, Jianli Hu, Bin Wu, Shenjiao Wang, Zhongmin Tan, Jianguang Fu, Jun Shan, Yefei Zhu, Fenyang Tang

Erschienen in: Archives of Virology | Ausgabe 9/2013

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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 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.
Literatur
1.
Zurück zum Zitat 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 40:2323–2330PubMedCrossRef 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 40:2323–2330PubMedCrossRef
2.
Zurück zum Zitat 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 364:1523–1532PubMedCrossRef 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 364:1523–1532PubMedCrossRef
3.
Zurück zum Zitat 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 54:527–553PubMedCrossRef 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 54:527–553PubMedCrossRef
5.
Zurück zum Zitat Wu W, Bai Z, Zhou H, Tu Z, Fang M, Tang B, Liu J, Liu L, Liu J, Chen W (2011) Molecular epidemiology of dengue viruses in southern China from 1978 to 2006. Virol J 8:322PubMedCrossRef Wu W, Bai Z, Zhou H, Tu Z, Fang M, Tang B, Liu J, Liu L, Liu J, Chen W (2011) Molecular epidemiology of dengue viruses in southern China from 1978 to 2006. Virol J 8:322PubMedCrossRef
6.
Zurück zum Zitat Zhang YZ, Zou Y, Fu ZF, Plyusnin A (2010) Hantavirus infections in humans and animals, China. Emerg Infect Dis 16:1195–1203PubMedCrossRef Zhang YZ, Zou Y, Fu ZF, Plyusnin A (2010) Hantavirus infections in humans and animals, China. Emerg Infect Dis 16:1195–1203PubMedCrossRef
7.
Zurück zum Zitat Zou Y, Hu J, Wang ZX, Wang DM, Yu C, Zhou JZ, Fu ZF, Zhang YZ (2008) Genetic characterization of hantaviruses isolated from Guizhou, China: evidence for spillover and reassortment in nature. J Med Virol 80:1033–1041PubMedCrossRef Zou Y, Hu J, Wang ZX, Wang DM, Yu C, Zhou JZ, Fu ZF, Zhang YZ (2008) Genetic characterization of hantaviruses isolated from Guizhou, China: evidence for spillover and reassortment in nature. J Med Virol 80:1033–1041PubMedCrossRef
8.
Zurück zum Zitat Wu X, Hong H, Yue J, Wu Y, Li X, Jiang L, Li L, Li Q, Gao G, Yang X (2010) Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells. Virol J 7:270PubMedCrossRef Wu X, Hong H, Yue J, Wu Y, Li X, Jiang L, Li L, Li Q, Gao G, Yang X (2010) Inhibitory effect of small interfering RNA on dengue virus replication in mosquito cells. Virol J 7:270PubMedCrossRef
9.
Zurück zum Zitat Aitichou M, Saleh SS, McElroy AK, Schmaljohn C, Ibrahim MS (2005) Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR. J Virol Methods 124:21–26PubMedCrossRef Aitichou M, Saleh SS, McElroy AK, Schmaljohn C, Ibrahim MS (2005) Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR. J Virol Methods 124:21–26PubMedCrossRef
10.
Zurück zum Zitat Kang X, Li Y, Liu H, Lin F, Cai X, Sun T, Chang G, Zhu Q, Yang Y (2010) A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses. Virol J 7:284PubMedCrossRef Kang X, Li Y, Liu H, Lin F, Cai X, Sun T, Chang G, Zhu Q, Yang Y (2010) A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses. Virol J 7:284PubMedCrossRef
11.
Zurück zum Zitat Linssen B, Kinney RM, Aguilar P, Russell KL, Watts DM, Kaaden OR, Pfeffer M (2000) Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses. J Clin Microbiol 38:1527–1535PubMed Linssen B, Kinney RM, Aguilar P, Russell KL, Watts DM, Kaaden OR, Pfeffer M (2000) Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses. J Clin Microbiol 38:1527–1535PubMed
12.
Zurück zum Zitat Huhtamo E, Hasu E, Uzcátegui NY, Erra E, Nikkari S, Kantele A, Vapalahti O, Piiparinen H (2010) Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology. J Clin Virol 47:49–53PubMedCrossRef Huhtamo E, Hasu E, Uzcátegui NY, Erra E, Nikkari S, Kantele A, Vapalahti O, Piiparinen H (2010) Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology. J Clin Virol 47:49–53PubMedCrossRef
13.
Zurück zum Zitat Hoffmann B, Beer M, Schelp C, Schirrmeier H, Depner KM (2005) Validation of areal-time RT-PCR assay for sensitive and specific detection of classical swine fever. J Virol Methods 130:36–44PubMedCrossRef Hoffmann B, Beer M, Schelp C, Schirrmeier H, Depner KM (2005) Validation of areal-time RT-PCR assay for sensitive and specific detection of classical swine fever. J Virol Methods 130:36–44PubMedCrossRef
14.
Zurück zum Zitat Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh A, Shah PS, Cecilia D (2009) Development of real time PCR for detection and quantitation of Dengue viruses. Virol J 6:10PubMedCrossRef Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh A, Shah PS, Cecilia D (2009) Development of real time PCR for detection and quantitation of Dengue viruses. Virol J 6:10PubMedCrossRef
15.
Zurück zum Zitat 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 12:22 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 12:22
16.
Zurück zum Zitat 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 136:200–209 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 136:200–209
17.
Zurück zum Zitat 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 47:2772–2778PubMedCrossRef 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 47:2772–2778PubMedCrossRef
18.
Zurück zum Zitat 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 12:74–81PubMedCrossRef 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 12:74–81PubMedCrossRef
Metadaten
Titel
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
verfasst von
Zhifeng Li
Xian Qi
Minghao Zhou
Changjun Bao
Jianli Hu
Bin Wu
Shenjiao Wang
Zhongmin Tan
Jianguang Fu
Jun Shan
Yefei Zhu
Fenyang Tang
Publikationsdatum
01.09.2013
Verlag
Springer Vienna
Erschienen in
Archives of Virology / Ausgabe 9/2013
Print ISSN: 0304-8608
Elektronische ISSN: 1432-8798
DOI
https://doi.org/10.1007/s00705-013-1677-8

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