Open Access Detection of Rodent Coronaviruses by Use of Fluorogenic Reverse Transcriptase-Polymerase Chain Reaction Analysis

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Abstract:

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proved useful for the detection of mouse hepatitis virus (MHV) and rat coronavirus (RCV) in acutely infected animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. Consequently, a fluorogenic nuclease RT-PCR assay specific for rodent coronaviruses was developed. Primer and probe sequences were selected from the viral genome segment that encodes the membrane (M) protein that is highly conserved among rodent coronaviruses. Use of the fluorogenic nuclease RT-PCR detected all strains of MHV and RCV that were evaluated, but did not detect other RNA viruses that naturally infect rodents. Use of the assay detected as little as two femtograms of in vitro transcribed RNA generated from cloned amplicon, and when compared directly with mouse antibody production tests, had similar sensitivity at detecting MHV-A59 in infected cell culture lysates. Finally, use of the assay detected coronavirus RNA in tissues, cage swipes, and feces obtained from mice experimentally infected with MHV, and in tissues and cage swipes obtained from rats naturally infected with RCV. These results indicate that the fluorogenic nuclease RT-PCR assay should provide a potentially high-throughput, PCR-based method to detect rodent coronaviruses in infected rodents and contaminated biological materials.

Document Type: Research Article

Affiliations: 1: Department of University Animal Care, The University of Arizona, Tucson, Arizona, 85721-0101; Veterinary Science and Microbiology, The University of Arizona, Tucson, Arizona, 85721-0101 2: Department of University Animal Care, The University of Arizona, Tucson, Arizona, 85721-0101

Publication date: April 1, 2002

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  • Comparative Medicine (CM), an international journal of comparative and experimental medicine, is the leading English-language publication in the field and is ranked by the Science Citation Index in the upper third of all scientific journals. The mission of CM is to disseminate high-quality, peer-reviewed information that expands biomedical knowledge and promotes human and animal health through the study of laboratory animal disease, animal models of disease, and basic biologic mechanisms related to disease in people and animals.

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