Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)

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.

Full Dynamic Range Quantification using Loop-mediated Amplification (LAMP) by Combining Analysis of Amplification Timing and Variance between Replicates at Low Copy Number

“…Linearised plasmid. Linearised plasmid DNA was re-suspended from aliquots of a stock of quantified pART7 14 lyophilised with carrier DNA as previously described 15 . The defined copy number concentration was calculated from initial quantification by NanoDrop spectrophotometry and Agilent Bioanalyzer.…”

“…www.nature.com/scientificreports www.nature.com/scientificreports/ Transgenic genomic template. Calculations for genomic DNA and transgenic GM maize reference materials follow the guidelines described in Hardinge et al 2018 15 .…”

“…Spectrophotometry. Initial quantification of the template DNA by NanoDrop 1000 (Fisher Scientific, UK) spectrophotometer followed the manufacturer's guidelines for double stranded DNA as described 15 . The extracts were assessed for purity using the NanoDrop using the wavelength ratios 260:280 nm and 260:230 nm.…”

“…The Agilent Bioanalyser (Agilent; California, USA) was used to initially quantify the linearised plasmid pART7. Agilent Tapestation genomic tapes were used to quantify maize genomic DNA from the events Bt11 and Mon810, following the protocol for genomic tapes as described 15 . Briefly 1 microlitre of either genomic DNA or ladder was combined with 10 microlitres of genomic DNA sample buffer, vortexed and loaded into the instrument.…”

Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection

“…A standard method for the detection of LAMP is the measurement of the turbidity caused by precipitated magnesium pyrophosphate, 23,24 as well as endpoint detection with the naked eye. [25][26][27] Other target sequence-independent detection methods rely on gel electrophoresis, 28 metal indicators for calcium, 29 colorimetric LAMP, 30 coffee-ring effect on colloidcrystal substrates, 31 paper-based rapid detection of LAMPmagnetic bead aggregates, 32 melting and annealing curve analysis, 33 intercalating uorescent dyes such as SYBR green, [34][35][36] bioluminescence through pyrophosphate conversion 37,38 or electrochemiluminescence. 39,40 LAMP stands out in terms of its high analytical specicity towards the target analyte due to the utilization of six primers.…”