Rtx reverse transcriptase

The set of primers designed was first evaluated for colorimetric visualisation in RT-LAMP reactions mixtures (25 µL) containing: 1.6 µM FIP/BIP primers, 0.2 µM F3/B3 primers, 0.4 µM LF/LB primers, 0.4 µM of dNTPs, 6 mM MgSO₄, 1X Isothermal Amplification Buffer, 1 µL Bst polymerase 2.0 WarmStart (New England Biolabs Ltd., Ipswich, MA, USA) and 0.5 µL RTx Reverse Transcriptase (New England Biolabs Ltd., Ipswich, MA, USA), with 3 µL of template RNA (C+, for positive control; RNase-free water instead RNA for negative control). The RT-LAMP reactions were performed in 0.5-mL micro-centrifuge tubes by incubation in a heating block at 63 °C for 60 min followed at 80 °C for 10 min to stop the reaction. The RT-LAMP amplification results were visually inspected by adding 2 µL of 1:10 diluted 10,000× g concentration fluorescent dye SYBR Green I (Invitrogen, Waltham, MA, USA), in each reaction tube. Green fluorescence was observed in the successful RT-LAMP reaction and original orange in the negative reaction. The tubes were briefly centrifuged and carefully opened before adding the dye to avoid possible cross-contamination with amplified products.