Development of loop-mediated isothermal amplification technique for LM canola T45, 73496, and MON88302 and its field application

Abstract

Brassica is an essential genus in agriculture, and gene flow from living modified (LM) organisms to native relatives or non-modified cultivars of the crucifer family is a critical issue in Asia. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, and accurate method for identifying LM crops. This study aimed to develop a LAMP assay for the laboratory diagnosis and field analysis of three novel LM canola events: T45, 73496, and MON88302. The genomic DNA of each LM canola was amplified by incubating at 63 °C for 30 min with a newly developed 2 × LAMP premix containing Bst DNA polymerase and event-specific primer sets. The sensitivity of the LAMP assay for the three canola events in our condition ranged from 100 pg·μL⁻¹ to 1 ng·μL⁻¹. Genetic elements of the three LM events were identified through a combination of the LAMP assay, a syringe-type DNA extraction kit, and a portable isothermal amplifier. This study proposes a novel, simple system based on isothermal amplification for the DNA detection of the three LM canola events in a survey field.