Fluorescence Assay for Specific Detection of the CRISPR-Associated Cas9 Gene in Genome-Edited Plants via Loop-Mediated Isothermal Amplification with Thioflavin T

Genome editing using CRISPR/Cas9 allows precise modifications within plants genomes, however, it also poses biosafety and biosecurity concerns, particularly regarding potential off-target mutations. Therefore, establishing robust detection methods is essential. A loop-mediated isothermal amplification (LAMP) assay coupled with Thioflavin T (ThT) fluorescence detection was developed for the sensitive and specific identification of the Cas9 coding sequence in genome-edited plants. Following optimization of reaction conditions—including ThT concentration, Mg²⁺ levels, and incubation time—the assay achieved a detection limit of approximately 4.34 copies/µL based on fluorescence analysis. Two G-rich sequences predicted from the LAMP amplicons were evaluated individually, revealing that one produced significantly higher fluorescence, suggesting a potential G-quadruplex (G4)-like conformation enhancing ThT signal. The method avoids colorimetric subjectivity and does not require real-time monitoring or complex instrumentation. Amplification products were confirmed via agarose gel electrophoresis, and fluorescence measurements were consistent across replicates. This study provided a isothermal assay to utilize ThT for Cas9 detection and offers a cost-effective, reliable platform for screening genome-edited organisms, particularly in settings with limited resources. The system shows strong potential for future application in biosafety and traceability.