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. However, public resistance to commercialization of genetically modified plants is still widespread in Europe [1
]. Existing European regulation limits the extent of GM presence in non-GM foodstuffs, and the increasing introduction of GM products into Europe is likely to result in parallel GM and non-GM ("conventional") supply chains. In addition, the more widespread planting of GM crops in Europe will lead to the need for on-farm confirmation of GM status. Together these factors are likely to lead to a substantial increase in the extent and frequency of testing for the presence of DNA of a GM-derived origin.
The European Union has currently defined the proportion of GM that can be present to be no more than 0.9% GM in a non-GM product [3
]. As a consequence, diagnostic tests must be deployed that can accurately quantify the GM proportion for monitoring [6
]. Careful sampling and handling techniques are required to ensure the analysis is statistically relevant and appropriate controls are also needed to compare the presence of a transgene to a suitable reference gene.
Several nucleic acid amplification techniques (NAATs) are available for the detection of GM contamination in plants and food [7
] of which the polymerase chain reaction (PCR) is by far the most widely used. However PCR requires rapid thermo-cycling to denature the target DNA strands, prior to and during amplification [9
], which imposes specific equipment requirements. 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.
Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid and specific nucleic acid amplification technology. It is characterized by the use of 4 different primers, specifically designed to recognize 6 distinct regions on the target DNA template, and proceeds at a constant temperature driven by invasion and strand displacement [11
]. Amplification and detection of target genes can be completed in a single step at a constant temperature, by incubating DNA template, primers and a strand displacement DNA polymerase. It provides high amplification efficiency, with replication of the original template copy 109
times during a 15-60 min reaction [13
]. The primer pairs used in LAMP are given specific designations; LAMP primers that generate hairpin loops, the outer displacement primers, and LOOP primers that accelerate the reaction by amplifying from the hairpin previously created by the LAMP primers [13
Several methods exist to determine the extent that DNA has been amplified either after or during a given reaction, of which the most frequently used are the incorporation of fluorescent primers into the amplification product or the use of intercalating fluorescent dyes. Other techniques monitor side products of the DNA synthesis responsible for the amplification reaction. For example, turbidity and fluorescence techniques can also used to detect inorganic pyrophosphate liberated during nucleic acid amplification [15
]. A recently described bioluminescence real time assay [BART] [17
] allows the quantitative analysis of iNAATs, in real time. The biochemistry of BART is based on the 'Enzymatic Luminometric Inorganic pyrophosphate Detection Assay, or "ELIDA" [20
] (Figure ). Unlike previous applications of the ELIDA assay (most notably Pyro-sequencing™), BART allows dynamic changes in pyrophosphate levels to be monitored continuously in real-time over extended periods at 60°C for up to 2 hours. During a BART reaction, the level of light output increases to a peak whose timing under the same assay conditions reflects the initial concentration of the targeted DNA. Hence quantification of BART reactions utilises the time to peak light output and is not dependent on absolute light intensity produced, which greatly simplifies data interpretation and the hardware requirements, as well as making assays robust to turbidity and suspended solids [19
Figure 1 Chemistry of the BART bioluminescent coupled assay. PPi liberated during DNA synthesis reacts with adenosine-5'-O-persulfate (APS) in a reaction catalysed by adenosine triphosphate sulfurylase, to form adenosine triphosphate (ATP). A recombinant thermostable (more ...)
The accuracy of molecular diagnostic tests is dependent on appropriate integrity, purity and concentration of the input DNA and therefore on the choice of sample extraction procedure [22
]. Plant tissues contain a variety of well-known compounds that can be inhibitory to molecular amplifications [25
], including acidic polysaccharides, a variety of salts, secondary metabolites and phytochelatins. Most plant genomic DNA extraction technologies are designed to reduce or eliminate these contaminants. Polysaccharides can be removed by exploiting their differential solubilisation in solutions containing detergents, and affinity resins have also been used for the same purpose [26
]. Hydrophobic cell constituents such as lipids and poly-phenols are routinely excluded from DNA extracts by partitioning with organic solvents, such as chloroform and alcohol. Unfortunately, many of the reagents used to extract and stabilize DNA, such as ethylene diamine tetra-acetic acid (EDTA), phenol, and the ionic detergents, sodium dodecyl sulfate (SDS) and cetyl tri-methyl ammonium bromide (CTAB) also tend to affect NAAT performance [28
]. Measures to avoid carrying-over these contaminants can make these protocols labour intensive and time consuming to yield DNA of a sufficient quality for PCR.
Several published reports demonstrate that LAMP amplifications tolerate higher levels of certain inhibitors than PCR [32
]. This suggests that LAMP could have a capacity to amplify polynucleotides from rapidly processed and crude sample matrix derived from plant material [34
]. Other factors that affect the reliable detection and quantification of low target copy polynucleic acids using this technique are likely to include overall DNA loading within a reaction, which can have a impact upon sensitivity, as it possibly influences non-specific primer interactions [36
]. Hence genome size, ploidy and unknown sources of contaminating DNA could affect amplification performance by altering the ratio of target to non-target DNA presence and hence potentially making target quantification and comparisons with reference samples and standards inaccurate. Here we demonstrate the use of LAMP-BART to detect GM events at low copy number levels in samples derived from maize, which has a large genome size and hence a relatively high proportion of non-target DNA. We show that LAMP-BART tolerates crude plant extracts without significant inhibition and examine the characteristics of the sample matrix that impact upon the quantitative nature of this technique and demonstrate its suitability in fieldable systems.