Abstract
Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.
Highlights
The Loop-mediated isothermal amplification (LAMP) reaction is initiated by strand invasion of the DNA template by hairpin-forming LAMP primers which anneal and extend catalysed by a strand displacing DNA polymerase
The ‘cauliflower-like’ structures which are rapidly generated in LAMP amplification potentially bring into proximity the 5′ end of a Loop primer with the 5′ end of a LAMP primer (Fig. 1)
We investigated using a FAM labelled LoopF and a JOE labelled FIP to explore whether fluorescence resonance energy transfer (FRET) could be observed between these two fluorophores by excitation of FAM transferring energy to JOE for increased fluorescence (Fig. 2)
Summary
The LAMP reaction is initiated by strand invasion of the DNA template by hairpin-forming LAMP primers which anneal and extend catalysed by a strand displacing DNA polymerase. LAMP assays may use indirect methods of amplification detection and these, whether real-time or end-point, rely on careful primer design, optimal reaction conditions and robustness testing to negate false positives. Such indirect detection strategies include the addition of intercalating dyes for fluorescence or colorimetric determination, turbidity[20] from pyrophosphate precipitation, observations of LAMP ladder patterns with agarose gel electrophoresis[5], quenched calcein[21], hydroxy-naphthol blue[22] as well as a number of fluorescent probe based techniques[15,23,24]. Further multiplex detections were shown with melt LAMP (mLAMP)[38,39] designed to use fluorescent labelled FIP primers for end point detection following real time turbidimetry of multiple targets
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