Development of molecular confirmation tools for swift and easy rabies diagnostics
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RESEARCH
Open Access
Development of molecular confirmation tools for swift and easy rabies diagnostics Kore Schlottau1, Conrad M. Freuling2, Thomas Müller2, Martin Beer1 and Bernd Hoffmann1*
Abstract Background: As rabies still represents a major public threat with tens of thousands of deaths per year, particularly in developing countries, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. Rabies diagnosis relies on highly sensitive and specific laboratory tests for detection of viral antigens. Among those tests, at present the immunofluorescence antibody test is the “gold standard test” for rabies diagnosis, followed by virus isolation in either mice or cell culture. Because of the advantages of molecular assays in terms of sensitivity and applicability their approval as confirmatory diagnostic test by international organizations (OIE, WHO) is envisaged. Therefore, the objective was to develop and validate novel molecular assays and RNA extraction methods for rabies that reduce the turnaround time but remain highly sensitive and specific. Methods: Here, novel assays, i.e. HighSpeed RT-qPCR and isothermal recombinase polymerase amplification (RPA) were designed and tested. Furthermore, three magnetic bead-based rapid extraction methods for manual or automated extraction were validated and combined with the new downstream assays. Results: While the conventional column based RNA extraction method showed the highest intra-run variations, all magnetic bead-based rapid extraction methods delivered nearly comparable sensitivity and efficiency of RNA recovery. All newly developed molecular tests were able to detect different rabies virus strains in a markedly reduced timeframe in comparison to the standard diagnostic assays. The observed detection limit for the HighSpeed RT-qPCR was 10 genome copies per reaction, and 1000 genome copies per reaction for the RPA assay. Conclusion: Magnetic bead-based rapid RNA extraction methods are highly sensitive and show a high level of reproducibility and therefore, are particularly suitable for molecular diagnostic assays including rabies. In addition, with a detection limit of 10 genome copies per reaction, the HighSpeed RT-qPCR is suitable for rapid ante mortem rabies diagnosis in humans as well as confirmatory test in integrated bite management and subsequent postexposure prophylaxis. Keywords: Rabies, RT-qPCR, HighSpeed, RT-RPA, Nucleic acid extraction
Background Rabies is a lethal zoonotic disease caused by a group of 16 negative-strand RNA viruses of the genus Lyssavirus in the family Rhabdoviridae of the order Mononegavirales [1]. It is a societal tragedy that in the twenty-first century, rabies, a zoonosis that can easily be prevented in humans and controlled in domestic animal species is still neglected and continues to create a significant social and economic burden on a global scale [2–4]. In lowincome countries where control efforts are lacking and * Correspondence: [email protected] 1 Institute of Diagnosti
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