Loop-mediated isothermal amplification: a rapid detection method for rice actin and nopaline synthase promoters in genetically modified crops

  • Monika SinghEmail author
  • Rajesh K. Bhoge
  • Sonia Nain
  • Gurinderjit Randhawa
Short Communication


The commonly employed promoters in transgenic constructs such as Cauliflower Mosaic Virus 35S and Figwort Mosaic Virus promoters are not necessarily present in all the approved/upcoming genetically modified (GM) events. GM events of cotton and maize with rice actin (P-ract) and nopaline synthase (P-nos) promoters have been approved globally. The scope of screening assays is expanded to a diversified range of promoters being employed in GM crops. Loop-mediated isothermal amplification (LAMP) is a method of choice for screening of genetically modified organisms (GMOs) due to ease-of-use, rapidity and applicability to crude DNA samples. An efficient LAMP-based screening method was developed targeting P-ract and P-nos for better coverage of GM events approved globally. LAMP reactions were isothermally incubated at 63 °C for 30 min for P-ract and 65 °C for 60 min for P-nos. Positive amplification products were visualized by change in colour from orange to green on addition of SYBR® Green 1 dye. Specificity was confirmed using defined sets including both the targets and non-targets. The developed assays showed acceptable specificity and sensitivity with limit of detection of 0.05% for P-ract and 0.01% for P-nos. Practical applicability of assays was confirmed using proficiency test samples of maize for P-ract and spiked samples of cotton for P-nos. These assays could be utilized for efficient GMO testing with less time and low cost inputs.


GM crops GM detection Loop-mediated isothermal amplification nos Promoter Rice actin promoter 



Certified reference material


Mannopine synthase


Octopine synthase


Cauliflower Mosaic Virus 35S promoter


Figwort Mosaic Virus promoter


Nopaline synthase promoter


Rice actin promoter


Pollen-specific promoter


Ubiquitin promoter


Nopaline synthase terminator



The grant provided by the Department of Biotechnology, Government of India is duly acknowledged. We also acknowledge the Indian Council of Agricultural Research, New Delhi and the Director, ICAR-National Bureau of Plant Genetic Resources, New Delhi for providing necessary facilities.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2018_479_MOESM1_ESM.docx (2.9 mb)
Supplementary material 1 (DOCX 2952 kb)


  1. Bhoge RK, Chhabra R, Randhawa GJ, Sathiyabama M, Singh M (2015) Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification. Food Cont 55:18–30CrossRefGoogle Scholar
  2. Chen L, Guo J, Wang Q, Kai G, Yang L (2011) Development of the visual LAMP assays for seven genetically modified maize events and their application in practical samples analysis. J Agric Food Chem 59(11):5914–5918CrossRefPubMedGoogle Scholar
  3. Debode F, Janssen E, Berben G (2013) Development of 10 new screening PCR assays for GMO detection targeting promoters (pFMV, pNOS, pSSuAra, pTA29, pUbi, pRice actin) and terminators (t35S, tE9, tOCS, tg7). Eur Food Res Technol 236(4):659–669CrossRefGoogle Scholar
  4. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1(4):19–21CrossRefGoogle Scholar
  5. Guan X, Guo J, Shen P, Yang L, Zhang D (2010) Visual and rapid detection of two genetically modified soybean events using loop-mediated isothermal amplification method. Food Anal Methods 3(4):313–320CrossRefGoogle Scholar
  6. Huang X, Chen L, Xu J, Ji HF, Zhu S, Chen H (2014) Rapid visual detection of phytase gene in genetically modified maize using loop-mediated isothermal amplification method. Food Chem 156:184–189CrossRefPubMedGoogle Scholar
  7. Kiddle G, Hardinge P, Buttigieg N, Gandelman O, Pereira C, McElgunn C, Rizzoli M, Jackson R, Appleton N, Moore C, Tisi L, Murray J (2012) GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnol 12(1):15CrossRefPubMedPubMedCentralGoogle Scholar
  8. Lipp M, Bluth A, Eyquem F, Kruse L, Schimmel H, Van den Eede G, Anklam E (2001) Validation of a method based on polymerase chain reaction for the detection of genetically modified organisms in various processed foodstuffs. Eur Food Res Technol 212(4):497–504CrossRefGoogle Scholar
  9. McElroy D, Blowers AD, Jenes B, Wu R (1991) Construction of expression vectors based on the rice actin 1 (Act1) 5′ region for use in monocot transformation. Mol Gen Genet 231:150–160CrossRefPubMedGoogle Scholar
  10. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucl Acids Res 28(12):e63CrossRefPubMedGoogle Scholar
  11. Randhawa GJ, Singh M, Morisset D, Sood P, Žel J (2013) Loop-mediated isothermal amplification: Rapid visual and real-time methods for detection of genetically modified crops. J Agric Food Chem 61(47):11338–11346CrossRefPubMedGoogle Scholar
  12. Singh M, Randhawa GJ, Sood P, Bhoge RK (2015) Loop-mediated isothermal amplification targeting insect resistant and herbicide tolerant transgenes: monitoring for GM contamination in supply chain. Food Cont 51:283–292CrossRefGoogle Scholar
  13. Singh M, Bhoge RK, Randhawa GJ (2017) Real-time and visual loop-mediated isothermal amplification: efficient GMO screening targeting pat and pmi marker genes. Food Cont 71:248–254CrossRefGoogle Scholar
  14. Singh M, Bhoge RK, Randhawa G (2018) Loop-mediated isothermal amplification for detection of endogenous Sad1 gene in cotton: an internal control for rapid onsite GMO testing. J AOAC Int 101(5):1657–1660CrossRefPubMedGoogle Scholar
  15. Waiblinger HU, Ernst B, Anderson A, Pietsch K (2008) Validation and collaborative study of a P-35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products. Eur Food Res Technol 226(5):1221–1228CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2018

Authors and Affiliations

  1. 1.Division of Genomic ResourcesIndian Council of Agricultural Research - National Bureau of Plant Genetic ResourcesNew DelhiIndia

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