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Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.
Ravi Kothapalli1, Gregory M Danyluck, Ratna D Bailey
1Hematologic Malignancies, Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Internal Medicine, University of South Florida, College of Medicine, Tampa, FL 33612, USA. kothapar@moffitt.esf.edu
Journal of Virological Methods
|April 25, 2003
Summary
Product enhanced reverse transcriptase activity (PERT) assays using MS2 RNA as a template may yield false positives due to contaminating DNA in reagents. Careful reagent evaluation is crucial for accurate retroviral detection.
Area of Science:
- Molecular Biology
- Virology
- Hematology
Background:
- Product enhanced reverse transcriptase activity (PERT) assays are used to detect reverse transcriptase activity.
- This activity is associated with retroviral infections and is investigated in conditions like large granular lymphocyte leukemia.
Purpose of the Study:
- To identify reverse transcriptase activity in samples from large granular lymphocyte leukemia patients.
- To validate the reliability of PERT assays using bacteriophage MS2 RNA as a template.
Main Methods:
- Performed PERT assays on sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of leukemia patients and healthy individuals.
- Investigated potential sources of false positive results by examining reagents like Taq DNA polymerase and RNase inhibitor.
- Utilized DNase I treatment and alternative RNA templates (encephalomyocarditis virus RNA) to assess assay specificity.
Main Results:
- All tested samples, including those from virus-infected cell lines, leukemia patients, and healthy individuals, showed positive results.
- Contamination with indigenous amplifiable DNA in essential reagents (Taq DNA polymerase, RNase inhibitor) was identified as the cause of false positives.
- DNase I treatment significantly reduced assay product, and using encephalomyocarditis virus RNA eliminated false positives.
Conclusions:
- The presence of amplifiable DNA in PERT assay reagents can lead to inaccurate detection of reverse transcriptase activity.
- Bacteriophage MS2 RNA is not a reliable template for PERT assays due to its susceptibility to contamination.
- Caution and rigorous reagent validation are necessary when using PERT assays for detecting retroviral infections or discovering novel retroviruses.