Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus.
Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy.
Virology Pole, Institut Pasteur of Dakar, 220 Dakar, Senegal.
Martin
M
Faye
Institut Pasteur, Lyssavirus Dynamics and Host Adaptation Unit, National Reference Centre for Rabies, WHO Collaborating Center for Reference and Research on Rabies, Paris, France.
Laurent
L
Dacheux
Institute of Aquaculture University of Stirling, FK9 4LA Stirling, Scotland, UK.
Manfred
M
Weidmann
Service of Infectious Diseases, Hospital FANN, Dakar, Senegal.
Sylvie Audrey
SA
Diop
G4 Biostatistics, Bioinformatics and Modelization, Institute Pasteur of Dakar, 220 Dakar, Senegal.
Cheikh
C
Loucoubar
Institut Pasteur, Lyssavirus Dynamics and Host Adaptation Unit, National Reference Centre for Rabies, WHO Collaborating Center for Reference and Research on Rabies, Paris, France.
Hervé
H
Bourhy
Virology Pole, Institut Pasteur of Dakar, 220 Dakar, Senegal.
Amadou Alpha
AA
Sall
Virology Pole, Institut Pasteur of Dakar, 220 Dakar, Senegal. Electronic address: ofaye@pasteur.sn.
Ousmane
O
Faye
D000349:Africa; D000818:Animals; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011818:Rabies / Q000175:diagnosis* / Q000662:veterinary; D011820:Rabies virus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
Africa; Broad detection; Molecular techniques; Rabies virus (RABV); Real-time RT-qPCR assays