Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA using RNA as a template and serving as indispensable tools in studies related to RNA. M-MuLV RT and its analogs are the most commonly used RTs. RTs are widely applied in various diagnostics methods, including reverse-transcription loop-mediated isothermal amplification (RT-LAMP). However, the performance of different RTs in LAMP remains relatively unknown. Here, we report on the first direct comparison of various M-MuLV RTs in RT-LAMP, including enzymes with a different number of mutations and fusions with Sto7d. Several parameters were assessed, namely: optimal reaction temperature, enzyme concentration, reverse transcription time, a minimal amount of RNA template, and tolerance to inhibitors. Mutations increased the optimal reaction temperature from 55 °C to 60-65 °C. All of the RTs were suitable for RT-LAMP with RNA templates in the range of 101-106 copies per reaction. Highly mutated enzymes were 1.5-3-fold more tolerant to whole blood, blood plasma, and guanidinium, but they were two-fold more sensitive to high concentrations of NaCl. The comparison of different RTs presented here could be helpful for selecting the optimal enzyme when developing novel LAMP-based diagnostic tests.
Keywords: LAMP; M-MuLV RT; RT-LAMP; Sto7d; fusion protein; isothermal amplification; reverse transcriptase; superscript.