Syto 9 green

Experiments were done with 4 repeats and each LAMP reaction contained the following: 1 µL 10X custom isothermal buffer, 0.6 µL MgSO4 (stock at 100 mM), 1.4 µL dNTPs (stock at 10 mM each), 0.5 µL BSA (20 mg/ml), 1.60 µL Betaine (5 M), 0.25 µL SYTO 9 Green (20 µM stock), 0.40 µL of Bst 2.0 DNA Polymerase (8,000 U/µL) (New England Biolabs, UK), 0.25 µL of AMV Reverse Transcriptase (25 U/µL) (Promega Corporation, US), 0.1 µL of RNase inhibitor (stock at 20 U/µL), 1 µL of 10X primer mixture, 1 µL of genomic RNA (clinical samples) or synthetic RNA template solution ranging from 10 to 10⁶ copies/µL, and enough nuclease-free water to bring the volume to 10 µL. The RT-qLAMP reactions were performed in a LightCycler 96 Real-Time PCR system (LC96) (Roche Diagnostics) at 63^oC for 25 min. Quantification metric is based on the C_t method setting the threshold at 0.1 after normalization.

DNA samples were amplified and detected in real time on a Qiagen (Hilden, Germany) Rotor-Gene thermal cycler acquiring to the green channel. Unless otherwise stated, all reagents are supplied by Sigma Aldrich (Poole, UK). The reaction chemistry for LAMP and amplification detection was 1X isothermal buffer (NEB, Massachusetts, United States), 300 micromolar each dNTP, 0.8 micromolar Betaine, 0.5 micromolar SYTO9 Green, 0.32 units per microlitre Bst polymerase v2.0 warm start (NEB), 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer, 0.2 micromolar each displacement primer and molecular grade water for a reaction volume of 20 microlitre. The parameters were set for 60 cycles of 60 seconds at 60 degrees C unless otherwise stated. Melt temperature analysis provided data between 60 and 95 degrees C. LAMP amplification detection with SYTO9 was substituted with LAMP molecular beacons and supplemented with molecular grade water if required.