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. 2012 Sep 21;27(5):273–277. doi: 10.1007/s12250-012-3252-y

Rapid detection of filoviruses by real-time TaqMan polymerase chain reaction assays

Yi Huang 1, Hongping Wei 1, Yunpeng Wang 1, Zhengli Shi 1, Herve Raoul 2, Zhiming Yuan 1,✉
PMCID: PMC8218046  PMID: 23001480

Abstract

Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China, while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant, equivalent to 10,000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.

Keywords: Ebola virus, Marburg virus, Nucleoprotein (NP) gene, TaqMan RT-PCR

Footnotes

Foundation items: Supported by Important National Science & Technology Specific Projects (2009ZX10004-504, 2009ZX09301-014); National Natural Science Foundation of China (81072675).

Reference

  • 1.Adrienne R. T., Leslie W., Jeffrey G., et al. Comprehensive Panel of Real-Time TaqMan™ Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses. Am J Trop Med Hyg. 2010;82(5):954–960. doi: 10.4269/ajtmh.2010.09-0636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Centers for Disease Control Prevention. Imported case of Marburg hemorrhagic fever — Colorado, 2008. Mor Morta Wkl Rep. 2009;58(49):1377–1381. [PubMed] [Google Scholar]
  • 3.Centers for Disease Control and Prevention. (2010a). Known cases and outbreaks of Ebola hemorrhagic fever, in chronological order. http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola/ebolatable.htm.
  • 4.Centers for Disease Control and Prevention. (2010b). Known cases and outbreaks of Marburg hemorrhagic fever, in chronological order. http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/marburg/marburgtable.htm.
  • 5.Drosten C., Gottig S., Schilling S., et al. 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. 2002;40:2323e30. doi: 10.1128/JCM.40.7.2323-2330.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Feldmann H., Geisbert T. W., Jahrling P. B., et al. Filoviridae. In: Fauquet C. M., Mayo M. A., Maniloff J., et al., editors. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses. London: Elsevier Academic Press; 2004. pp. 645–653. [Google Scholar]
  • 7.Formenty P., Leroy E. M., Epelboin A., et al. Detection of Ebola virus in oral fluid specimens during outbreaks of Ebola virus hemorrhagic fever in the Republic of Congo. Clin Infect Dis. 2006;42:1521e6. doi: 10.1086/503836. [DOI] [PubMed] [Google Scholar]
  • 8.Feldmann H., Jones S., Klenk H. D., et al. Ebola virus: from discovery to vaccine. Nat Rev Immunol. 2003;3:677–685. doi: 10.1038/nri1154. [DOI] [PubMed] [Google Scholar]
  • 9.Garcia S., Crance J. M., Billecocq A., et al. Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds. J Clin Microbiol. 2001;39:4456–4461. doi: 10.1128/JCM.39.12.4456-4461.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Garin D., Peyrefitte C., Crance J. M., et al. Highly sensitive TaqMan PCR detection of Puumala hantavirus. Microbes Infect. 2001;3:739–745. doi: 10.1016/S1286-4579(01)01424-1. [DOI] [PubMed] [Google Scholar]
  • 11.Gibb T. R., Norwood D. A., Jr, Woollen N., et al. Development and evaluation of a fluorogenic 5’ nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan. J Clin Microbiol. 2001;39:4125e30. doi: 10.1128/JCM.39.11.4125-4130.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Grolla A., Jones S. M., Fernando L. The Use of a Mobile Laboratory Unit in Support of Patient Management and Epidemiological Surveillance during the 2005 Marburg Outbreak in Angola. PLoS Negl Trop Dis. 2011;5(5):e1183. doi: 10.1371/journal.pntd.0001183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Houng H. S. H., Hritz D., Kanesa-Thasan N. Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3′-noncoding sequence. J Virol. 2000;86:1–11. doi: 10.1016/s0166-0934(99)00166-4. [DOI] [PubMed] [Google Scholar]
  • 14.Jeffs B., Roddy P., Weatherill D., et al. The Médecins Sans Frontières intervention in the Marburg hemorrhagic fever epidemic, Uige, Angola, 2005. I. Lessons learned in the hospital. J Infect Dis. 2007;196(Suppl2):S154–S161. doi: 10.1086/520548. [DOI] [PubMed] [Google Scholar]
  • 15.Larkin M. A., Blackshields G., Brown N. P., et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. doi: 10.1093/bioinformatics/btm404. [DOI] [PubMed] [Google Scholar]
  • 16.Roddy P., Weatherill D., Jeffs B., et al. The Médecins Sans Frontières intervention in the Marburg hemorrhagic fever epidemic, Uige, Angola, 2005. Lessons learned in the community. J Infec Dis. 2007;196(Suppl2):S162–S167. doi: 10.1086/520544. [DOI] [PubMed] [Google Scholar]
  • 17.Rotz L. D., Khan A. S., Lillibridge S. R., et al. Public health assessment of potential biological terrorism agents. Emerg Infect Dis. 2002;8:225e30. doi: 10.3201/eid0802.010164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Lab Press; 1989. [Google Scholar]
  • 19.Sanchez A., Geisbert T. W., Feldmann H. Filoviridae: Marburg and Ebola viruses. In: David M. K., Peter M. H., editors. Fields Virology. Philadelphia: Lippincott Williams and Wilkins, /Wolters Kluwer Business; 2006. pp. 1409–1448. [Google Scholar]
  • 20.Timen A., Koopmans M. P., Vossen A. C., et al. Response to imported case of Marburg hemorrhagic fever, the Netherland. Emerg Infect Dis. 2009;15:1171–1175. doi: 10.3201/eid1508.090051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Towner J. S., Rollin P. E., Bausch D. G., et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome. J Virol. 2004;78:4330–4341. doi: 10.1128/JVI.78.8.4330-4341.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Towner J. S., Sealy T. K., Ksiazek T. G., et al. High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings. J Infect Dis. 2007;196(Suppl2):S205e12. doi: 10.1086/520601. [DOI] [PubMed] [Google Scholar]
  • 23.Towner J. S., Sealy T. K., Khristova M. L., et al. Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 2008;4:e1000212. doi: 10.1371/journal.ppat.1000212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Trombley A. R., Wachter L., Garrison J., et al. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses. Am J Trop Med Hyg. 2010;82:954e60. doi: 10.4269/ajtmh.2010.09-0636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.World Health Organization. Ebola haemorrhagic fever. A summary of the outbreak in Gabon. Wkly Epidemiol Rec. 1997;72(1–2):7–8. [PubMed] [Google Scholar]

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