Detection of Sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis

• Published in: Comparative medicine Vol. 53; no. 2; pp. 173 - 177
• Main Authors: Wagner, A.M, Loganbill, J.K, Besselsen, D.G
• Format: Journal Article
• Language: English
• Published: United States American Association for Laboratory Animal Science 01-04-2003
• Subjects: accuracy; Animals; Fluorescent Dyes; fluorescent labeling; Immunocompromised Host; laboratory animals; Mice; Mice, Inbred ICR; Mice, SCID; microbial contamination; Murine pneumonia virus; Murine pneumonia virus - genetics; Murine pneumonia virus - isolation & purification; Murine pneumonia virus - pathogenicity; Pneumovirus Infections - transmission; Pneumovirus Infections - veterinary; Pneumovirus Infections - virology; respiratory tract diseases; Respirovirus Infections - transmission; Respirovirus Infections - veterinary; Respirovirus Infections - virology; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - veterinary; RNA, Viral - analysis; Sendai virus; Sendai virus - genetics; Sendai virus - isolation & purification; Sendai virus - pathogenicity; Sensitivity and Specificity; Severe Combined Immunodeficiency - veterinary; Severe Combined Immunodeficiency - virology
• ISSN: 1532-0820; 2769-819X

Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM in immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer and probe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virus and PVM, respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRTPCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, and the Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.