Mutant of Moloney murine leukemia virus reverse transcriptase exhibits higher resistance to common RT-qPCR inhibitors

Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV RT) enzymes was investigated. IC(50) values were determined for potential RNA contaminants, including guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related acidic polysaccharides. Sensitivity (as judged by MMLV RI IC50 values) was directly correlated to the outcome of mock reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sensitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RI pentuple mutant (E69K/E302R/W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resistance was also noted in RT-qPCR comparisons employing crude cell lysates. (C) 2010 Elsevier Inc. All rights reserved.