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Detection of Genetically Modified Organisms (GMOs) Using Isothermal Amplification of Target DNA Sequences

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Detection of Genetically Modified Organisms (GMOs) Using Isothermal Amplification of Target DNA Sequences

David Lee et al. BMC Biotechnol.

Abstract

Background: The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions.

Results: We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP.

Conclusion: This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Figures

Figure 1
Figure 1
Right border sequences of MS8 and RF3. Sequences of the plant (above) and transgene (below) at the right border junctions for MS8 and RF3. Highlighted sequences are the targets of the LAMP primers. The plant sequences are those shown in Table 1: dark blue bases highlighted are the outer displacement primers, yellow and green sequences are the 5' and 3' ends of the LAMP primers respectively, and light blue sequences depict the loop primers.
Figure 2
Figure 2
Detection of MS8 and RF3 transgene demonstrate specificity of LAMP. Testing of assays on MS8/RF3 oilseed rape DNA (A) and separately using plasmids (B) to demonstrate the specificity of the reactions. +ve and -ve denote the presence or absence of target DNA, respectively. Sizes of marker bands are shown.
Figure 3
Figure 3
Sensitivity assessment of LAMP Detection. A. Sensitivity of LAMP using genomic target. DNA from MS8/RF3 sample (16 ng.μL-1) was serially diluted and amplified by LAMP, in triplicate, using primers to assay for the RF3 junction. The numbers in parenthesis represent the approximate copy numbers of the target assuming that the sample represents RF3 in a hemizygous state (determined using RT-PCR data not shown) for the transgene and using 1 pg as the genome size for oilseed rape. C is the no DNA control and M represents molecular size markers. The smear (*) shows an example of non-specific amplification. B. Sensitivity of LAMP using plasmid target. Serial dilutions of the plasmid pGreenII were amplified using primers for the Pnos target. Numbers represent the calculated copy numbers of the plasmid derived from the DNA value. C is the no DNA control and M represents molecular sized markers.
Figure 4
Figure 4
Sensitivity of LAMP reactions with background DNA. LAMP of samples containing 10 copies of RoundUp ReadyTM soya transgene (S), and in a background of 100 ng of oilseed rape DNA (O). M is molecular size standard.

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References

    1. Lipp M, Anklam E, Stave JW. Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study. J AOAC Int. 2000;83:919–927. - PubMed
    1. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplification of DNA. Nucl Acids Res. 2000;28:e63. doi: 10.1093/nar/28.12.e63. - DOI - PMC - PubMed
    1. Loop animation. Eiken Genome Site.
    1. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16:223–229. doi: 10.1006/mcpr.2002.0415. - DOI - PubMed
    1. Report on the molecular characterisation of the genetic map of event Ms8 × Rf3

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