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oai:figshare.com:article/34027415 2026-09-29T19:12:47Z category_7 category_12 category_13 category_14 category_272 category_45 category_46 category_734 category_61 category_64 portal_63 item_type_6 month_year_09_2026

Performance of a Group II Intron Reverse Transcriptase from Anoxybacillus flavithermus in Duplex RT-LAMP for SARS-CoV‑2 Detection Igor P. Oscorbin (25138679) Elizaveta D. Baranova (25138682) Maxim L. Filipenko (25138685) Medicine Cell Biology Genetics Molecular Biology Environmental Sciences not elsewhere classified Sociology Immunology Biological Sciences not elsewhere classified Developmental Biology Cancer optimal temperature range mediated isothermal amplification retroviral rnase h demonstrate superior thermostability deficient reverse transcriptase transcribed rna fragments reverse transcription loop 58 – 98 95 ° c 200 negative samples lamp targeting sars reverse transcription reverse transcriptases retroviral counterparts observations demonstrate rna viruses respiratory symptoms proven advantageous may increase internal control high concentrations care diagnostics betaine increased based diagnostics anoxybacillus flavithermus 89 %). 15 min Reverse transcriptases from bacterial group II introns (GII RTs) demonstrate superior thermostability and processivity compared with retroviral counterparts. While GII RTs have proven advantageous for RNA-seq, their performance in other applications, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), remains poorly studied. RT-LAMP is a widely used method suitable for point-of-care diagnostics of RNA viruses and other pathogens. In this study, we compared the performance in RT-LAMP of GII RT from the thermophilic bacterium Anoxybacillus flavithermus (Afl RT) with that of the retroviral RNase H-deficient reverse transcriptase M-MuLV. A duplex RT-LAMP targeting SARS-CoV-2 and MS2 phage genomic RNA (internal control) served as a model. Preheating at 95 °C and 1.5 M betaine increased the efficiency of RT-LAMP with both reverse transcriptases. 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. 2026-09-29T00:00:00Z Text Journal contribution 10.1021/acsinfecdis.6c00293.s001 https://figshare.com/articles/journal_contribution/Performance_of_a_Group_II_Intron_Reverse_Transcriptase_from_Anoxybacillus_flavithermus_in_Duplex_RT-LAMP_for_SARS-CoV_2_Detection/34027415 CC BY-NC 4.0