Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.
In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.
Kothapalli
Ravi
R
Hematologic 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
Danyluck
Gregory M
GM
Bailey
Ratna D
RD
Loughran
Thomas P
TP
D006801:Humans; D017909:Levivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase