A Simple, Portable and Multiplex Lamp-Based Crispr/Cas12a Assay for Visually Screening Genetically Modified Crops

Abstract

With the growing controversy over the potential risk of genetically modified (GM) crops to environment and even human health worldwide, there is an urgent need to develop rapid, easy-to-operate, portable, multiplex and cost-effective methods for on-field testing of GM crops. Herein, we develop a novel multiplex loop-mediated isothermal amplification (LAMP)-based CRISPR/Cas12a method for sensitive and accurate screening of GM crops. The novel multiplex LAMP uses several pairs of stem-loop primers (SLPs) according to target genes to increase the formation ability of double stem-loop DNA to enhance LAMP efficiency. By designing the same stem-loop region of SLP pairs, multiple genes can be amplified simultaneously using universal primers to effectively avoid amplification bias. Therefore, as low as 100 aM target genes can be simultaneously amplified and then activate CRISPR/Cas12a system to cleave reporter probe for fluorescence releasing, which can be observed under UV irradiation at an end-point manner. Further detection of real samples reveals that only 0.5% GM maize can be visually differentiated from a large amount of non-GM maize. Therefore, our method shows great promise to be applied in the testing of other GM organisms and monitoring of food authenticity under isothermal condition.

Keywords: stem-loop primer, multiplex LAMP, CRISPR/Cas12a, visual detection, genetically modified crop