A highly integrated system with rapid DNA extraction, recombinase polymerase amplification, and lateral flow biosensor for on-site detection of genetically modified crops

Abstract

With the large-scale planting of genetically modified (GM) crops, the development of a rapid and convenient method for on-site monitoring GM crops is needed. In this study, a duplex recombinase polymerase amplification (DRPA)-based, quick and simple detection system is presented for on-site detection of GM crops. In this system, a rapid DNA extraction method, a DRPA, and a lateral flow biosensor (LFB) are integrated to allow for rapid DNA extraction and amplification, and fast visualization of the detection results. Using the rapid DNA extraction method, high-quality DNA suitable for RPA and polymerase chain reaction (PCR) was quickly isolated from various crops within 5 min. Utilizing the optimal DRPA assay, the universal screening sequences (Cauliflower mosaic virus 35S promoter [35S] and Agrobacterium tumefaciens NOS terminator [NOS]) were rapidly and simultaneously amplified with high selectivity and sensitivity. The sensitivity threshold of the DRPA assay was ∼10 copies for GM soybean genomic DNA and 100 ng DNA of 0.1% GM soybean. In combination with the LFB in an enclosed cassette, the entire detection process was performed in approximately 20-30 min and eliminated the carryover contamination. No special or expensive equipment was needed for the detection process. The system was successfully applied and validated for on-site detection of GM rice, demonstrating its suitability for on-site testing of GM crops and high potential for application to other fields, especially in low-resource regions that require rapid detection of DNA targets.

Keywords: Genetically modified crops; Lateral flow biosensor; On-site detection; Recombinase polymerase amplification.