Abstract
BackgroundThere 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.ResultsLoop 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.ConclusionsLAMP-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.
Highlights
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
Given potential differences in sample purity and composition, we suggest that the gel density method is likely to be a more reliable indication of comparative genomic DNA yield than spectrometry
real-time polymerase chain reaction (RT-polymerase chain reaction (PCR)) had a lower threshold of detection than Loop mediated isothermal amplification (LAMP)-Bioluminescent Assay in Real-Time (BART) more reproducible data was achieved using the RT-PCR technique regardless of the extraction adopted in this experiment (Figure 3). These results indicate that the DNA extracted using the cetyl tri-methyl ammonium bromide (CTAB) and Phytopure methods are higher in contaminants or sample impurities incompatible with the LAMP-BART chemistry, perhaps reflecting their original development to service PCR
Summary
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. As the world’s agricultural systems endeavour to sustain an expanding population, technologies have become available to increase the yield and viability of cultivated crops including the introduction of novel traits into crops using genetic transformation of foreign DNA to produce GM varieties. Several nucleic acid amplification techniques (NAATs) are available for the detection of GM contamination in plants and food [7,8] of which the polymerase chain reaction (PCR) is by far the most widely used. Since the discovery of DNA polymerases with strand displacement activity, novel amplification methods have been developed which operate under isothermal conditions (iNAAT) and propagate the initial target sequence by promoting strand displacement using enzymes or modified oligonucleotides
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
