title: GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use.
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.
authors.affiliation: Lumora Ltd, Bartholomew Walk, Cambridgeshire Business Park, Ely, Cambridgeshire CB7 4EA, UK. g.kiddle@Lumora.co.uk
authors.forename: Guy
authors.initials: G
authors.lastname: Kiddle
authors.forename: Patrick
authors.initials: P
authors.lastname: Hardinge
authors.forename: Neil
authors.initials: N
authors.lastname: Buttigieg
authors.forename: Olga
authors.initials: O
authors.lastname: Gandelman
authors.forename: Clint
authors.initials: C
authors.lastname: Pereira
authors.forename: Cathal J
authors.initials: CJ
authors.lastname: McElgunn
authors.forename: Manuela
authors.initials: M
authors.lastname: Rizzoli
authors.forename: Rebecca
authors.initials: R
authors.lastname: Jackson
authors.forename: Nigel
authors.initials: N
authors.lastname: Appleton
authors.forename: Cathy
authors.initials: C
authors.lastname: Moore
authors.forename: Laurence C
authors.initials: LC
authors.lastname: Tisi
authors.forename: James A H
authors.initials: JA
authors.lastname: Murray
mesh_terms: D003001:Cloning, Molecular; D018744:DNA, Plant / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D005504:Food Analysis; D030841:Food, Genetically Modified; D018745:Genome, Plant; D008163:Luminescent Measurements / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012044:Regression Analysis; D012680:Sensitivity and Specificity; D003313:Zea mays / Q000235:genetics
chemical_list: D018744:DNA, Plant