Real time RT-PCR panel pro identifikaci dvanácti virů hemoragických horeček

English version

Autoři: M. Fajfr ^1,2; V. Neubauerová ³; P. Pajer ³; P. Kubíčková ³; D. Růžek ^4,5
Působiště autorů: Faculty of Military Health Sciences, University of Defense, Hradec Králové ¹; Institute of Clinical Microbiology, University Hospital in Hradec Králové, Hradec Králové ²; Central Military Institute of Health, Army of the Czech Republic, Prague ³; Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice ⁴; Department of Virology, Veterinary Research Institute, Brno ⁵
Vyšlo v časopise: Epidemiol. Mikrobiol. Imunol. 63, 2014, č. 3, s. 238-244
Kategorie: Souhrnná sdělení, původní práce, kazuistiky

Souhrn

Úvod:
Virové hemoragické horečky jsou způsobovány zástupci čtyř virových čeledí a patří mezi choroby s velmi vysokou smrtností. Pro jejich diagnostiku není dostupný žádný komerční kit.

Výsledky:
Vytvořili jsme real time RT-PCR panel pro detekci a identifikaci virů Ebola, Marburg, Lassa, Guanarito, Machupo, Sabiá, Seoul, Puumala, Hantaan, virus krymsko-konžské hemoragické horečky a horečky Rift Valley. Protokol funguje na principu jednotných reakčních podmínek a byl testován na různých PCR přístrojích jak kapilárních, tak i destičkových včetně polní verze termocykleru Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) (Idaho Technology, Inc.).

Závěr:
V kombinaci s adaptovanými protokoly z již publikovaných prací představujeme jednoduchý detekční systém pro rychlou identifikaci původců virových hemoragických horeček z čeledí filoviru, arenavirů a bunyavirů s dostatečnou senzitivitou a specificitou pro použití laboratořích prvního kontaktu a pro diagnostiku v polních podmínkách.

Klíčová slova:
hemoragické horečky – filoviry – arenaviry – real time RT-PCR –diagnostika

Zdroje

1. Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, Jahrling PB, et al. Hemorrhagic Fever Viruses as Biological Weapons (Medical and Public Health Management). JAMA, 2002;287(18):2391–2405.

2. Gibb TR, Norwood DA Jr, Woollen N, Henchal EA. Development and evaluation of a fluorogenic 5´ nuclease assay to detect and differentiate between Ebola virus subtype Zaire and Sudan. J Clin Microbiol, 2001;39(11):4125–4130.

3. Weidmann M, Mühlberger E, Hufert FT. Rapid detection protocol for filoviruses. J Clin Virol, 2004;30(1):94–99.

4. Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, Vincent M, 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(8):4330–4341.

5. Towner JS, Khristova ML, Sealy TK, Vincent MJ, Erickson BR, Bawiec DA, et al. Marburgvirus Genomics and Association with a Large Hemorrhagic Fever Outbreak in Angola. J Virol, 2006;80(13):6497–6516.

6. Drosten C, Göttig S, Schilling S, Asper M, Panning M, Schmitz H, Günther S. 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(7):2323–2330.

7. Lunkenheimer K, Hufert FT, Schmitz H. Detection of Lassa virus RNA in specimens from patients with Lassa fever by using the polymerase chain reaction. J Clin Microbiol, 1990; 28:2689–2692.

8. Lozano ME, Enría D, Maiztegui JI, Grau O, Romanowski V. Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based essay. J Clin Microbiol, 1995; 33(5):1327–1332.

9. Aitichou M, Saleh SS, McElroy AK, et al. Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR. J Virol Method, 2005;124(1–2):21–26.

10. Bird BH, Bawiec DA, Ksiazek TG, et al. Highly sensitive and broadly reactive quantitative reverse transcription-PCR assay for high-throughput detection of Rift Valley fever virus. J Clin Microbiol, 2007;45(11):3506–3513.

11. Garrison AR, Alakbarova S, Kulesh DA, et al. Development of a TaqMan®-Minor Groove Binding Protein Assay for the Detection and Quantification of Crimean-Congo Hemorrhagic Fever Virus. Am J Trop Med Hyg, 2007;77(3):514–520.

12. Trombley AR, Wachter L, Garrison J, Buckley-Beason VA, Jahrling J, Hensley LE, et al. Comprehensive panel of Real-time TaqManTM 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.

13. Panning M, Laue T, Olschlager S, Eickmann M, Becker S, Raith S, et al. Diagnostic Reverse-Transcription Polymerase Chain Reaction Kit for Filoviruses Based on the Strain Collections of all European Biosafety Level 4 Laboratories. J Inf Dis, 2007, 196(Suppl. 2):S199–204.

14. Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. RT-PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg, 2007;101(12):1253–1264.

15. Vieth S, Drosten C, Charrel R, Feldmann H, Günther S. Establishment of conventional and fluorescence resonance energy transfer-based real-time PCR assays for detection of pathogenic New World arenaviruses. J Clin Virol, 2005;32(3):229–235

16. Drosten C, Kummerer BM, Schmitz H, et al. Molecular diagnostics of viral hemorrhagic fevers. Antiviral Res, 2003;57(1–2):61–87.

17. Jabado OJ, Palacios G, Kapoor V, et al. Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments. Nucleic Acids Res, 2006;34(22):6605–6611.

18. Palacios G, Briese T, Kapoor V et al. MassTag Polymerase Chain Reaction for Differential Diagnosis of Viral Hemorrhagic Fevers. Emerg Infect Dis, 2006;12(4):692–695.

19. Towner JS, Rollin PE, Bausch DG, et al. Rapid diagnosis of Ebola Hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral loads as a predictor of outcome. J Virol, 2004;78(8):4330–4341.

20. Papa A, Zelená H, Barnetová D, Petroušová L. Genetic detection of Dobrava/Belgrade virus in a Czech patient with Haemorrhagic fever with renal syndrome. Clin Microbiol Infect, 2010;16(8):1187–1190.

21. Vacková M, Douda P, Beran J, et al. Serologic detection of hantavirus antibodies. Epidemiol Mikrobiol Imunol, 2002;51(2):74–77.

22. Vapalathi O, Mustonen J, Lundkvist A, et al. Hantavirus in Europe. Lancet Infect Dis, 2003;3:653–661.

23. Fajs L, Jakupi X, Ahmeti S, et al. Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in Kosovo. PLoS Negl Trop Dis, 2014;8(1):e2647.

24. Barr DA, Aitken C, Bell DJ, et al. First confirmed case of Crimean-Congo hemorrhagic fever in the UK. Lancet, 2013;282(9902):1458.

25. Frank C, Höhle M, Stark K, Lawrence J. More reason to dread rain on vacation? Dengue fever in 42 German and United Kingdom Madeira tourists during autumm 2012. Euro Surveill, 2013;18(14):20446.