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

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.