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A Multiplex, Fluorescent, and Isothermal Method for Detecting Genetically Modified Maize

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Abstract

Detecting, monitoring, and mapping transgenic sequences in the environment has been a long-standing challenge in ecology. Given the level of social, economic, and scientific interest in them, transgenic (GM) DNA sequences stand as paradigmatic; such sequences further have the unique property of functioning as unequivocal markers in the environment, clearly showing the paths of gene flow between crops and their wild relatives, and across industrial and traditional forms of agriculture. True mapping of transgenic DNA at the landscape level requires a method of DNA amplification that is robust and isothermal in order to be cost effective and field-based. We present here a method with these characteristics. Our multiplex fluorescent method is for the detection of the p35s CaMV promoter frequently used in transgenic plants as well as the alpha-zein protein specific to maize, but can be applicable to any other DNA sequence. The method uses loop-mediated isothermal amplification (LAMP) with visualization achieved using a fluorophore-quencher system of DNA hybridizing probes. We demonstrate the applicability of this tri-color method to transgenic corn, non-transgenic corn, and non-transgenic/non-corn species.

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References

  • Abdul-Ghani R, Al-Mekhlafi AM, Karanis P (2012) Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? Acta Trop 122:233–240

    Article  CAS  Google Scholar 

  • Aonuma H, Yoshimura A, Kobayashi T, Okado K, Badolo A, Nelson B, Kanuka H, Fukumoto S (2010) A single fluorescence-based LAMP reaction for identifying multiple parasites in mosquitoes. Exp Parasitol 125:179–183

    Article  CAS  Google Scholar 

  • Ball CS, Light YK, Koh C-Y, Wheeler SS, Coffey LL, Meagher RJ (2016) Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses. Anal Chem 88:3562–3568

    Article  CAS  Google Scholar 

  • Bektaş A, Chapela I (2014) Loop-mediated isothermal amplification of single pollen grains. J Integr Plant Biol 56:741–748

    Article  Google Scholar 

  • Bektaş A, Chapela I (2016) Efficiency of a fluorescent, non-extraction LAMP DNA amplification method: toward a field-based specific detection of maize pollen grains. Aerobiologia 32:481–488

    Article  Google Scholar 

  • Chen P-H, Pan Y-B, Chen R-K (2008) High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants. J Integr Plant Biol 50:375–383

    Article  CAS  Google Scholar 

  • Chou P-H, Lin Y-C, Teng P-H, Chen C-L, Lee P-Y (2011) Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes. J Virol Methods 173:67–74

    Article  CAS  Google Scholar 

  • Curtis KA, Rudolph DL, Owen SM (2009) Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1. J Med Virol 81:966–972

    Article  CAS  Google Scholar 

  • Dehghan Esmatabadi MJ, Bozorgmehr A, Motalebzadeh H, Bodaghabadi N, Farhangi B, Babashah S, Sadeghizadeh M (2015) Techniques for evaluation of LAMP amplicons and their applications in molecular biology. Asian Pac J Cancer Prev APJCP 16:7409–7414

    Article  Google Scholar 

  • Deiman B, van Aarle P, Sillekens P (2002) Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 20:163–179

    Article  CAS  Google Scholar 

  • Demidov VV (2002) Rolling-circle amplification in DNA diagnostics: the power of simplicity. Expert Rev Mol Diagn 2:542–548

    Article  CAS  Google Scholar 

  • Dukes JP, King DP, Alexandersen S (2006) Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus. Arch Virol 151:1093–1106

    Article  CAS  Google Scholar 

  • Feinberg M, Fernandez S, Cassard S, Bertheau Y (2005) Quantitation of 35S promoter in maize DNA extracts from genetically modified organisms using real-time polymerase chain reaction, part 2: interlaboratory study. J AOAC Int 88:558–573

    CAS  Google Scholar 

  • Fischbach J, Xander NC, Frohme M, Glökler JF (2015) Shining a light on LAMP assays—a comparison of LAMP visualization methods including the novel use of berberine. BioTechniques 58:189–194

    Article  CAS  Google Scholar 

  • Forte VT, Di Pinto A, Martino C, Tantillo GM, Grasso G, Schena FP (2005) A general multiplex-PCR assay for the general detection of genetically modified soya and maize. Food Control 16:535–539

    Article  CAS  Google Scholar 

  • Fukuta S, Mizukami Y, Ishida A, Ueda J, Hasegawa M, Hayashi I, Hashimoto M, Kanbe M (2004) Real-time loop-mediated isothermal amplification for the CaMV-35S promoter as a screening method for genetically modified organisms. Eur Food Res Technol 218:496–500

    Article  CAS  Google Scholar 

  • Gill P, Ghaemi A (2008) Nucleic acid isothermal amplification technologies—a review. Nucleosides Nucleotides Nucleic Acids 27:224–243

    Article  CAS  Google Scholar 

  • Huang H-Y, Pan T-M (2004) Detection of genetically modified maize MON810 and NK603 by multiplex and real-time polymerase chain reaction methods. J Agric Food Chem 52:3264–3268

    Article  CAS  Google Scholar 

  • Hurst CD, Knight A, Bruce IJ (1999) PCR detection of genetically modified soya and maize in foodstuffs. Mol Breed 5:579–586

    Article  CAS  Google Scholar 

  • James D, Schmidt A, Wall E, Green M, Masri S (2003) Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis. J Agric Food Chem 51:5829–5834

    Article  CAS  Google Scholar 

  • Kiddle G, Hardinge P, Buttigieg N, Gandelman O, Pereira C, McElgunn CJ, Rizzoli M, Jackson R, Appleton N, Moore C et al (2012) GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol 12:15

    Article  CAS  Google Scholar 

  • Kim J-H, Kim S-A, Seo Y-J, Lee W-Y, Park S-H, Kim H-Y (2008) Multiplex PCR detection of the MON1445, MON15985, MON88913, and LLcotton25 varieties of GM cotton. Food Sci Biotechnol 17:829–832

    CAS  Google Scholar 

  • Köppel R, Bucher T (2015) Rapid establishment of droplet digital PCR for quantitative GMO analysis. Eur Food Res Technol 241:427–439

    Article  Google Scholar 

  • Köppel R, Bucher T, Frei A, Waiblinger H-U (2015) Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin. Eur Food Res Technol 241:521–527

    Article  Google Scholar 

  • Mahony J, Chong S, Bulir D, Ruyter A, Mwawasi K, Waltho D (2013) Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity. J Clin Virol 58:127–131

    Article  CAS  Google Scholar 

  • Marvier M, Carrière Y, Ellstrand N, Gepts P, Kareiva P, Rosi-Marshall E, Tabashnik BE, Wolfenbarger LL (2008) Harvesting data from genetically engineered crops. Science 320:452–453

    Article  CAS  Google Scholar 

  • Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51:263–273

    Article  CAS  Google Scholar 

  • Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63–e63

    Article  CAS  Google Scholar 

  • Piñeyro-Nelson A, Van Heerwaarden J, Perales HR, Serratos-Hernández JA, Rangel A, Hufford MB, Gepts P, Garay-Arroyo A, Rivera-Bustamante R, Álvarez-Buylla ER (2009) Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations. Mol Ecol 18:750–761

    Article  Google Scholar 

  • Querci M, Van den Bulcke M, Žel J, Van den Eede G, Broll H (2009) New approaches in GMO detection. Anal Bioanal Chem 396:1991–2002

    Article  Google Scholar 

  • Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA (2011) Evaluation of digital PCR for absolute DNA quantification. Anal Chem 83:6474–6484

    Article  CAS  Google Scholar 

  • Seidler RJ and Hern S (1988) Special report: the release of ice minus recombinant bacteria at California test sites. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/3-88/060

  • Setterquist RA, Smith GK (1996) Ready to use agarose encapsulated PCR reagents. Nucleic Acids Res. 24(8):1580–1581

  • Shao Y, Zhu S, Jin C, Chen F (2011) Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk. Int J Food Microbiol 148:75–79

    Article  CAS  Google Scholar 

  • Singh CK, Ojha A, Bhatanagar RK, Kachru DN (2007) Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs—standard single, multiplex and construct-specific PCR assays. Anal Bioanal Chem 390:377–387

    Article  Google Scholar 

  • Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3:877–882

    Article  CAS  Google Scholar 

  • Urdea M, Penny LA, Olmsted SS, Giovanni MY, Kaspar P, Shepherd A, Wilson P, Dahl CA, Buchsbaum S, Moeller G et al (2006) Requirements for high impact diagnostics in the developing world. Nature 444:73–79

    Article  Google Scholar 

  • Walker GT, Fraiser MS, Schram JL, Little MC, Nadeau JG, Malinowski DP (1992) Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res 20:1691–1696

    Article  CAS  Google Scholar 

  • Yamazaki W, Mioulet V, Murray L, Madi M, Haga T, Misawa N, Horii Y, King DP (2013) Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus. J Virol Methods 192:18–24

    Article  CAS  Google Scholar 

  • Zhen Z, Zhang M, Yu Y, Gao X, Zhu Y, Yan Y, Zhang R (2016) Establishment of a loop-mediated isothermal amplification (LAMP) detection method for genetically modified maize MON88017. Eur Food Res Technol 242:1787–1793

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Environmental Sciences, Policy and Management, University of California Berkeley, Berkeley, CA, 94720, USA

    Ali Bektaş

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  1. Ali Bektaş
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Correspondence to Ali Bektaş.

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Ali Bektaş declares that he has no conflict of interest.

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Bektaş, A. A Multiplex, Fluorescent, and Isothermal Method for Detecting Genetically Modified Maize. Food Anal. Methods 11, 686–692 (2018). https://doi.org/10.1007/s12161-017-1041-9

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  • DOI: https://doi.org/10.1007/s12161-017-1041-9

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