Real-Time LAMP with SYTO 9 Dye

SYTO dyes have been successfully used in real-time PCR^{50 (link)} and real-time LAMP^{51 (link)} as an alternative to the more commonly used SYBR green dyes. Samples of a 4.9Kb linearised plasmid with the CaMV 35S promoter sequence were used to optimise a real time quantitative LAMP reaction (data not shown). The reaction volume of 20 microlitres contained 1x Isothermal Buffer (NEB), 300 micromolar each dNTP, 0.8 molar betaine, 0.5 micromolar SYTO 9 Green, 0.32 Units per microlitre Bst polymerase v2.0 WarmStart (NEB), 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer and 0.2 micromolar each displacement primer, made up to the final volume with molecular grade water. The real-time fluorescent LAMP reaction was performed on the Corbett Rotor-Gene thermal cycler at 60 degrees C for 60 seconds for a total of 100 cycles acquiring fluorescence data during each cycle. The DNA amplification was followed by amplicon melt temperature recording between 60 and 92 degrees C. The Rotor-Gene 6000 software v1.7 and Microsoft Excel were used to analyse the data.