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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

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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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Acute Infectious Diseases Control and Prevention, Jiangsu Provincial Center for Disease Control and Prevention, 172, Jiangsu Rd., Nanjing, 210009, Jiangsu Province, China

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

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  1. Zhifeng Li
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  2. Xian Qi
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  3. Minghao Zhou
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  4. Changjun Bao
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  5. Jianli Hu
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  6. Bin Wu
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  7. Shenjiao Wang
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  8. Zhongmin Tan
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  9. Jianguang Fu
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  10. Jun Shan
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  11. Yefei Zhu
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  12. Fenyang Tang
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Corresponding author

Correspondence to Fenyang Tang.

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Li, Z., Qi, X., Zhou, M. et al. 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. Arch Virol 158, 1857–1863 (2013). https://doi.org/10.1007/s00705-013-1677-8

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  • DOI: https://doi.org/10.1007/s00705-013-1677-8

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