Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay.
Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-based RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.
Maudru
T
T
Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Peden
K
K
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010926:Placental Hormones / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D004791:Enzyme Inhibitors; D010926:Placental Hormones; D012367:RNA, Viral; C022052:placental ribonuclease inhibitor; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D012260:Ribonucleases