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Logo of bmcbiotBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Biotechnology
 
BMC Biotechnol. 2012; 12: 15.
Published online Apr 30, 2012. doi:  10.1186/1472-6750-12-15
PMCID: PMC3388468
GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use
Guy Kiddle,corresponding author1 Patrick Hardinge,2 Neil Buttigieg,2 Olga Gandelman,1 Clint Pereira,1 Cathal J McElgunn,1 Manuela Rizzoli,1 Rebecca Jackson,1 Nigel Appleton,1 Cathy Moore,1 Laurence C Tisi,1 and James AH Murray2
1Lumora Ltd, Bartholomew Walk, Cambridgeshire Business Park, Ely, Cambridgeshire CB7 4EA, UK
2Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff CF10 3AX, UK
corresponding authorCorresponding author.
Guy Kiddle: g.kiddle/at/Lumora.co.uk; Patrick Hardinge: hardingep/at/cardiff.ac.uk; Neil Buttigieg: buttigiegnd/at/Cardiff.ac.uk; Olga Gandelman: O.gandelman/at/lumora.co.uk; Clint Pereira: c.pereira/at/lumora.co.uk; Cathal J McElgunn: c.mcelgunn/at/lumora.co.uk; Manuela Rizzoli: manuriz/at/inwind.it; Rebecca Jackson: r.harrall/at/lumora.co.uk; Nigel Appleton: n.appleton/at/lumora.co.uk; Cathy Moore: catamour17/at/hotmail.com; Laurence C Tisi: l.tisi/at/lumora.co.uk; James AH Murray: murrayja1/at/cardiff.ac.uk
Received November 1, 2011; Accepted April 30, 2012.
Abstract
Background
There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.
Results
Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.
Conclusions
LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.
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