Colorimetric vs. Real-Time LAMP for detection of genetic modifications in soybean (Glycine max)

INTRODUCTION:Genetically modified food is becoming one of the greatest concerns in agriculture, especially owing to the continuously growing diversity of genetic modifications (GM), which is directly contributing to the difficulty of their monitoring and detection. Long-term impacts on human health and environment are yet to be revealed but recent research efforts indicate possible harmful effects.OBJECTIVES:The aim of this study was to compare two approaches of GM detection: a naked eye detection via Colorimetric LAMP method and a fluorescence- based Real-Time LAMP method. In order to imitate GMO samples, we prepared soybean leaf and seed gDNA spiked with gBlocks (P-35S and P-FMV elements which are most commonly included in transgenic constructs of GM plants). Additionally, we analyzed how different approaches for soybean gDNA isolation, i.e. robust Chelex-based method applicable in PON settings and a kit-based isolation suitable for laboratory conditions, influence LAMP amplification dynamics (Fig 1.).MATHERIALS & METHODS Plant material: Soybean (lat. Glycine max) seed and fresh leaf samples.DNA isolation from plant material:Two approaches were used for DNA extraction:• Robust and fast Chelex-based approach was used for DNA extraction from soybean seed and leaf samples. The total DNA sample was diluted to a final concentration of 1ng/μl and used in the experiments.• Kit-based DNA isolation suitable for laboratory conditions was used for DNA isolation from soybean leaf samples (NZY Plant/fungi isolation kit).LAMP reactions: A LAMP reaction was designed to target P-35S and P-FMV regions using gBlocks, synthetic DNA fragments that have an identical sequence with the specific DNA region of interest within a transgenic construct. The Real-Time LAMP detection was conducted using WarmStart LAMP Kit (New England BioLabs) and the Genie® III Instrument (OptiGene) for isothermal nucleic acid amplification. Colorimetric LAMP reaction was conducted using WarmStart Colorimetric LAMP Kit (New England BioLabs) and the TS-100 Thermo-Shaker for microtubes and PCR plates (Biosan). LAMP assay was conducted at 65°C for 30 minutes which was in case of Colorimetric LAMP followed by 80°C for 10 to inactivate the enzyme. All reactions were conducted in volumes of 12.5μl and PCR H2O was used as the no template control. RESULTS:➢ The Colorimetric LAMP reaction shows no insight into the analytical phase of the experiment, since there were no observable differences related to DNA isolation method.