Detection of Reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction

Abstract

Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.