Detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis.
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 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 andprobe 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 virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe 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.
Wagner
April M
AM
University Animal Care, University of Arizona, 1127 E. Lowell Street, Tucson, Arizona 85721-0101, USA.
Loganbill
Jessie K
JK
Besselsen
David G
DG
D000818:Animals; D005456:Fluorescent Dyes; D016867:Immunocompromised Host; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D029122:Murine pneumonia virus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D018186:Pneumovirus Infections / Q000635:transmission / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D010253:Respirovirus Infections / Q000635:transmission / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D029082:Sendai virus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D012680:Sensitivity and Specificity; D016511:Severe Combined Immunodeficiency / Q000662:veterinary / Q000821:virology
D005456:Fluorescent Dyes; D012367:RNA, Viral