Detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis.
Lymphocytic choriomeningitis virus (LCMV) induces persistent infections in laboratory mice; is a known contaminant of biological materials, such as transplantable tumor cell lines; and is of great concern in animal facilities due to its zoonotic potential. Fluorogenic nuclease reverse transcriptase-polymerase chain reaction (fnRT-PCR) assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. An fnRT-PCR assay specific for LCMV was, therefore, developed by targeting primer and probe sequences to a unique region of the LCMV nucleocapsid (NP) gene. The LCMV fnRT-PCR assay detected only LCMV and did not detect other RNA viruses that naturally infect rodents. The fnRT-PCR assay detected as little as one picogram of LCMV RNA, but was 100-fold less sensitive when directly compared with the mouse antibody production test. The fnRT-PCR assay was also able to detect viral RNA in numerous tissues and in feces and cage swipe specimens collected from experimentally inoculated BALB/c mice, but did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. In conclusion, the LCMV fnRT-PCR assay offers a potentially high-throughput diagnostic assay to detect LCMV in mice and contaminated biological materials.
Besselsen
David G
DG
Department of University Animal Care, The University of Arizona, Tucson, Arizona 85721-0101, USA.
Wagner
April M
AM
Loganbill
Jessie K
JK
D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D017931:DNA Primers; D005260:Female; D005456:Fluorescent Dyes; D008217:Lymphocytic choriomeningitis virus / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D011247:Pregnancy; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes; D012367:RNA, Viral