Multiplex Real-Time Loop-Mediated Isothermal Amplification (LAMP) Based on the Annealing Curve Analysis: Toward an On-Site Multiplex Detection of Transgenic Sequences in Seeds and Food Products

Abstract

Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.