Single primer isothermal amplification for real-time and visual on-site detection of genetically modified crops by recognizing CP4-EPSPS gene

Abstract

In response to the potential controversies on increasing circulation of genetically modified organisms (GMOs) in the global markets, the rapid and sensitive detection methods are essential for monitoring of GMOs to maintain consumers' rights. Herein, we first developed a rapid technique for the real-time and visual on-site detection of the genetically modified crops (specifically targeting CP4-EPSPS gene) by using single primer isothermal amplification (SPIA) combined with SYBR Green II. 19 GM crop events and 12 non-GM crop events were used to assess the specificity of the developed SPIA assay, which presented excellent specificity. The limit of detection of the developed SPIA method was as low as 4 copies/μL, which was better than that of the real-time PCR method (20 copies/μL). The SPIA method was further used to assess 30 of actual samples, and the experimental results revealed that 3 positive samples were found, which were in good agreement with those of the real-time PCR methods. Overall, the major advantages of the developed SPIA over real-time PCR are the lower LOD, simplicity, and inexpensiveness. The developed SPIA method provides a potential alternative for the real-time and visual on-site detection of GMOs, which also accelerates the development of highly sensitive nucleic acid testing techniques.