Isothermal amplification methods can typically be used for RNA targets with one-step reverse transcription reactions. Addition of WarmStart RTx (NEB #M0380), AMV (NEB #M0277) or Protoscript II MMuLV (NEB #M0368) Reverse Transcriptase is compatible with the buffer and temperature used for many isothermal amplification reactions, simplifying the reaction workflow. A separate cDNA synthesis step can first be performed, if desired, but for most detection reactions speed and simplicity are preferred. In some cases, the DNA polymerase itself can prove sufficient for single-enzyme RT isothermal amplification. Bst DNA Polymerase, Large Fragment has been observed to have a low level of RT activity, particularly in the high magnesium concentrations of isothermal amplification reactions (~8 mM), but it is highly variable and cannot match the performance of two-enzyme mixes. Bst 2.0 displays increased reverse transcriptase activity and can be used as an RT in some cases, but Bst 3.0 has high reverse transcriptase activity up to 72 °C, enabling single-enzyme RT-LAMP reactions and cDNA synthesis through difficult secondary structures. For general RT-LAMP reactions, we recommend a dedicated reverse transcriptase (WarmStart RTx, NEB #M0380), in addition to the DNA-dependent DNA polymerase, but when a single-enzyme system is desired Bst 3.0 is the enzyme of choice.
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