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      Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences

      research-article
      1 , , 1 , 2 , 3 , 1 , 4
      BMC Biotechnology
      BioMed Central

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          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.

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          Most cited references10

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          Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

          The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity. Copyright 2001 Academic Press.
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            Loop-mediated isothermal amplification for detection of African trypanosomes.

            While PCR is a method of choice for the detection of African trypanosomes in both humans and animals, the expense of this method negates its use as a diagnostic method for the detection of endemic trypanosomiasis in African countries. The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions with only simple incubators. An added advantage of LAMP over PCR-based methods is that DNA amplification can be monitored spectrophotometrically and/or with the naked eye without the use of dyes. Here we report our conditions for a highly sensitive, specific, and easy diagnostic assay based on LAMP technology for the detection of parasites in the Trypanosoma brucei group (including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, and T. evansi) and T. congolense. We show that the sensitivity of the LAMP-based method for detection of trypanosomes in vitro is up to 100 times higher than that of PCR-based methods. In vivo studies in mice infected with human-infective T. brucei gambiense further highlight the potential clinical importance of LAMP as a diagnostic tool for the identification of African trypanosomiasis.
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              Loop-mediated isothermal amplification reaction using a nondenatured template.

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                Author and article information

                Journal
                BMC Biotechnol
                BMC Biotechnology
                BioMed Central
                1472-6750
                2009
                2 February 2009
                : 9
                : 7
                Affiliations
                [1 ]John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge, UK
                [2 ]Dipartimento di Scienze del Suolo, della Pianta, dell' Ambiente e delle Produzioni Animali, University of Naples, Federico II, Via Universita 100, 80055, Portici, Italy
                [3 ]Central Science Laboratory, Sand Hutton, York, UK
                [4 ]Institute of Biological, Environmental & Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, Ceredigion, UK
                Article
                1472-6750-9-7
                10.1186/1472-6750-9-7
                2656497
                19187544
                116b7001-0c9f-4275-94b9-556658e1920e
                Copyright © 2009 Lee et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 4 June 2008
                : 2 February 2009
                Categories
                Research Article

                Biotechnology
                Biotechnology

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