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.

Details

Title
Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)
Author
Hardinge, Patrick 1 ; Kiddle, Guy 2 ; Tisi, Laurence 2 ; Murray, James A H 1 

 Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff, UK 
 ERBA MDX, Bartholomew Walk, Cambridgeshire Business Park, Ely, Cambridgeshire, UK 
Pages
1-17
Publication year
2018
Publication date
Dec 2018
Publisher
Nature Publishing Group
e-ISSN
20452322
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2149888449
Copyright
© 2018. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.