Short communicationReal-time and visual loop-mediated isothermal amplification: Efficient GMO screening targeting pat and pmi marker genes

- Visual and real-time loop-mediated isothermal amplification (LAMP) assays targeting pat and pmi were developed.
- LAMP assays for pmi gene have been reported for the first time.
- Limit of detection (LOD) of visual LAMP was up to 0.05% genetically modified (GM) content for pat and 0.1% for pmi.
- Real-time LAMP efficiently detected up to 0.01% GM content within 30 min.
- These assays could be employed to screen 35% of GM events approved globally.

Rapid and efficient screening assays based on loop-mediated isothermal amplification (LAMP) were developed, targeting two marker genes, namely, phosphinothricin-N-acetyltransferase (pat) and phosphomannose isomerase (pmi). These marker genes are being employed in more than 35% of GM events approved globally. Specificity of developed visual and real-time LAMP assays was confirmed using seven GM events of two crops, viz., maize (3272, 59122, Bt11, Bt176, MIR604, TC1507), and cotton (Widestrike™). In visual LAMP, positive amplification can be directly analyzed by the colorimetric change from orange to green, whereas real-time LAMP is based on the monitoring of fluorescence signals as amplification and annealing curves. Visual LAMP was found sensitive enough to detect up to 0.05% GM content for pat and 0.1% for pmi within 40 min. Real-time LAMP efficiently detected up to 0.01% GM content within 30 min. Practical applicability of developed assays was confirmed using proficiency test samples of maize. LAMP assays for pmi gene have been reported for the first time. Due to portability of systems, the developed LAMP assays when combined with a fast DNA extraction method could facilitate on-site GMO screening.