title: A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.
abstract: A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Vanessa
authors.identifier: 0000-0001-9798-3477
authors.initials: V
authors.lastname: Suin
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Florence
authors.initials: F
authors.lastname: Nazé
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Francart
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Lamoral
authors.affiliation: Toxoplasma Laboratory, Food-borne Pathogens, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Stéphane
authors.initials: S
authors.lastname: De Craeye
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Michael
authors.initials: M
authors.lastname: Kalai
authors.affiliation: National Reference Centre of Rabies, Viral Diseases, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Engeland Street 642, 1180 Brussels, Belgium.
authors.forename: Steven
authors.identifier: 0000-0002-8150-9372
authors.initials: S
authors.lastname: Van Gucht
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000821:virology; D002415:Cats; D002685:Chiroptera; D003198:Computer Simulation; D004285:Dogs; D005455:Fluorescent Antibody Technique; D006801:Humans; D057230:Limit of Detection; D018114:Lyssavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018353:Rhabdoviridae Infections / Q000175:diagnosis* / Q000821:virology*; D016415:Sequence Alignment
chemical_list: D012367:RNA, Viral