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Detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain
David G Besselsen1, April M Wagner, Jessie K Loganbill
1Department of University Animal Care, The University of Arizona, Tucson, Arizona 85721-0101, USA.
Abstract:
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.
Insights
A new fluorogenic nuclease reverse transcriptase-polymerase chain reaction (fnRT-PCR) assay effectively detects Lymphocytic choriomeningitis virus (LCMV) in mice and biological materials. This diagnostic tool offers high-throughput screening for LCMV contamination.
Area of Science:
- Virology
- Molecular Biology
- Diagnostic Assays
Background:
- Lymphocytic choriomeningitis virus (LCMV) causes persistent infections in mice and poses zoonotic risks.
- LCMV contamination is a concern in research animal facilities and biological materials.
- Existing diagnostic methods may have limitations in specificity or throughput.
Purpose of the Study:
- To develop and evaluate a specific fluorogenic nuclease reverse transcriptase-polymerase chain reaction (fnRT-PCR) assay for LCMV detection.
- To assess the sensitivity and specificity of the fnRT-PCR assay for LCMV.
- To determine the utility of the assay for detecting LCMV in various sample types from infected mice.
Main Methods:
- Development of an fnRT-PCR assay targeting a unique region of the LCMV nucleocapsid (NP) gene.
- Testing the assay's specificity against other rodent RNA viruses.
- Quantification of LCMV RNA detection limits and comparison with the mouse antibody production test.
- Application of the assay to various tissues, feces, and cage swipe samples from experimentally infected and mock-infected mice.
Main Results:
- The fnRT-PCR assay specifically detected LCMV without cross-reacting with other rodent RNA viruses.
- The assay detected as little as one picogram of LCMV RNA, though it was less sensitive than the mouse antibody production test.
- Viral RNA was successfully detected in multiple tissues and environmental samples from LCMV-inoculated mice.
- No viral RNA was detected in samples from age- and strain-matched mock-infected control mice.
Conclusions:
- The developed LCMV fnRT-PCR assay is specific and capable of detecting LCMV in mice.
- The assay can identify LCMV RNA in diverse biological and environmental samples.
- This fnRT-PCR assay presents a potentially valuable high-throughput diagnostic tool for LCMV surveillance and contamination control.