Performance of a Group II Intron Reverse Transcriptase from Anoxybacillus flavithermus in Duplex RT-LAMP for SARS-CoV‑2 Detection

Under these conditions, the optimal temperature range and time of reverse transcription were similar for both enzymes, 55–60 °C and 15 min, respectively. However, high concentrations of Afl RT (>50 U/reaction) inhibited RT-LAMP. Notably, RT-LAMP with Afl RT demonstrated higher sensitivity for detecting both in vitro-transcribed RNA fragments and SARS-CoV-2–positive samples.

In validation against RT-qPCR using nasal swabs (200 SARS-CoV-2-positive and 200 negative samples) from patients with respiratory symptoms, the observed sensitivity of RT-LAMP with Afl RT was 100% (200/200; 95% CI, 98.17–100%), whereas with M-MuLV RT, sensitivity was 97.0% (194/200; 95% CI, 93.58–98.89%). Specificity was 100% for both RTs (200/200; 95% CI, 98.17–100%). These observations demonstrate that Afl RT is suitable for RT-LAMP and may increase the sensitivity of LAMP-based diagnostics.