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Quenched fluorescence LAMP amplification

PH Patrick Hardinge
JM James A. H. Murray
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Oligonucleotide LAMP primers with JOE, FAM or TAMRA fluorophores attached to the 5 prime end were incorporated into the fluorescent LAMP amplification method without the inclusion of SYTO9. For dual detection SYTO9 was included with JOE labelled FIP primers. The LAMP reaction was monitored in real time on the RotorGene thermocycler according the emission wavelength of the fluorophore. The multiplex and dual detection reactions monitored 6′FAM or SYTO9 using the green channel (source 470 +/− 10 nanometers, detection 510 +/− 5 nanometers), JOE on the yellow channel (source 530 +/− 5 nanometers, detection 557 +/− 5 nanometers) and TAMRA with the orange channel (excitation at 585 +/− 5 nanometers, detection aat 610 +/− 5 nanometers) in the same reaction. Melt temperature analysis followed amplification between 60 and 95 degrees C. Results were analysed RotorGene 6000 software v1.7 and Microsoft Excel.

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