Abstract
Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter/terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives.
Highlights
Isothermal amplification methods have been used for GM detection[6,7] with the potential benefit of lower cost instrumentation, since isothermal amplification of nucleic acid template operates at a single temperature without the requirement for thermocycling equipment
Two distinct types of primer are required for Loop-mediated isothermal amplification (LAMP); hairpin-forming LAMP primers that are integral to the amplification, and displacement primers which are required during amplification initiation to generate the initial dumbbell structure that creates the basic amplicon
The position of the LAMP primers used by Kiddle et al are highlighted and labeled according to type, the primers designed by Zahradnik et al are indicated by dashed lines
Summary
Isothermal amplification methods have been used for GM detection[6,7] with the potential benefit of lower cost instrumentation, since isothermal amplification of nucleic acid template operates at a single temperature without the requirement for thermocycling equipment. Loop-mediated isothermal amplification (LAMP) is a method that uses a strand displacing DNA polymerase and hairpin-forming oligonucleotide primers[11]. Two distinct types of primer are required for LAMP; hairpin-forming LAMP primers that are integral to the amplification, and displacement primers which are required during amplification initiation to generate the initial dumbbell structure that creates the basic amplicon. These primers are designed to target at least six template annealing positions, resulting in a high degree of specificity (Fig. 1a). LAMP amplification has been used successfully with BART due to the high concentrations of inorganic pyrophosphate produced enabling low nucleic acid copy number detection
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