Comparison of Reverse Transcriptase (RT) Activities of Various M-MuLV RTs for RT-LAMP Assays

SIMPLE SUMMARY: Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA with RNA as a template, and are used in almost all studies related to RNA. M-MuLV RT is widely used in diagnostics methods, including in reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The performance of various reverse transcriptases in RT-LAMP remains poorly studied. Here, we report the first direct comparison of various M-MuLV RTs in RT-LAMP. The enzymes studied contained different numbers of mutations or an additional Sto7d protein. Several parameters were assessed: optimal reaction temperature, enzyme concentration, reverse transcription time, a minimal amount of RNA template, and tolerance to inhibitors. Mutations increased the optimal temperature of the reverse transcription up to 5–10 °C. All of the RTs were suitable for RT-LAMP with RNA templates in the range of 10(1)–10(6) copies per reaction. Highly mutated enzymes were more tolerant to most of the inhibitors, but more sensitive to high concentrations of NaCl. The results presented could help select the optimal enzyme for novel LAMP-based diagnostic tests. ABSTRACT: 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 10(1)–10(6) 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.