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

  • Monika Singh
  • Rajesh K. Bhoge
  • Sonia Nain
  • Gurinderjit Randhawa
Short Communication
  • 14 Downloads

Abstract

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.

Keywords

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

Abbreviations

CRM

Certified reference material

mas

Mannopine synthase

ocs

Octopine synthase

P-35S

Cauliflower Mosaic Virus 35S promoter

P-FMV

Figwort Mosaic Virus promoter

P-nos

Nopaline synthase promoter

P-ract

Rice actin promoter

pTA29

Pollen-specific promoter

P-ubi

Ubiquitin promoter

T-nos

Nopaline synthase terminator

Notes

Acknowledgements

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)

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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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