TY - JOUR T1 - A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test. JF - Biomed Res Int Y1 - 2014 A1 - Vanessa Suin A1 - Nazé, Florence A1 - Aurélie Francart A1 - Lamoral, Sophie A1 - Stéphane De Craeye A1 - Kalai, Michael A1 - Steven Van Gucht KW - Animals KW - Base Sequence KW - brain KW - Cats KW - Chiroptera KW - Computer Simulation KW - Dogs KW - Fluorescent Antibody Technique KW - Humans KW - Limit of Detection KW - Lyssavirus KW - mice KW - Molecular Sequence Data KW - Real-Time Polymerase Chain Reaction KW - Reproducibility of Results KW - Reverse Transcriptase Polymerase Chain Reaction KW - Rhabdoviridae Infections KW - RNA, Viral KW - Sequence Alignment AB -

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.

VL - 2014 U1 - http://www.ncbi.nlm.nih.gov/pubmed/24822188?dopt=Abstract M3 - 10.1155/2014/256175 ER -