Developing rapid in-field detection procedure for genetically edited rice based on Recombinase Polymerase Amplification technique

Abstract

     The present study was conducted to apply Recombinase Polymerase Amplification (RPA) technique for rapid detection of genetically edited Khang Dan 18 (KD18) rice directly from leaf samples to develop a in-field detection kit. The KD18 rice variety, with a deletion mutation in the OsDSG1 gene, was quickly processed using various methods to obtain DNA for RPA reactions with specific primers. This study demonstrated that the SDS treatment combined with rapid purification using a phenol/chloroform/isoamyl alcohol mixture at a ratio of 25:24:1 is suitable for DNA extraction for RPA reactions. Optimization of the sample processing procedure showed that finely ground rice leaf samples with 20 % SDS solution at 65 °C for 15 minutes, followed by rapid purification, allowed the RPA reaction to take place at a fixed temperature of 37 °C for 20 minutes. The RPA reaction results can be directly recorded by the naked eye through a color reaction with Erichrome Black T. The study established a rapid detection method for genetically edited rice in-field, thus it can be applied for developing a simple, convenient, and quick procedure to determine the presence of genetically edited crops directly in the cultivation fields.