A dual-RPA based lateral flow strip for sensitive, on-site detection of <i>CP4</i>- <i>EPSPS</i> and <i>Cry1Ab</i>/ <i>Ac</i> genes in genetically modified crops

Abstract

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient. In this study, a one-tube dual RPA reaction system for <i>CP4-EPSPS</i> and <i>Cry1Ab/Ac</i> was proposed and combined with a lateral flow immunochromatographic assay, named "Dual-RPA-LFD", to visualize the dual detection of GM crops. In which, the herbicide tolerance gene <i>CP4-EPSPS</i> and the insect resistance gene <i>Cry1Ab/Ac</i> were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits. Gradient diluted plasmids, transgenic standards, and actual samples were used as templates to conduct sensitivity, specificity, and practicality assays, respectively. The constructed method achieved the visual detection of plasmid at levels as low as 100 copies, demonstrating its high sensitivity. In addition, good applicability to transgenic samples was observed, with no cross-interference between two test lines and no influence from other genes. In conclusion, this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 minutes at 37 ℃ in a rapid, equipment-free field manner, providing a new alternative for rapid screening for transgenic assays in the field.