Probe-Free Thioflavin T-Assisted Fluorescent LAMP for Preliminary Screening of Retained Cas9 Sequences in Genome-Edited Tomato-Derived Plant Materials

CRISPR/Cas9 genome editing is increasingly used in crop improvement, creating a need for practical tools to screen plant-derived materials for retained editing components. We developed a probe-free fluorescent loop-mediated isothermal amplification (LAMP) assay that uses thioflavin T (ThT) to detect the Cas9 coding sequence in genome-edited tomato materials. Under the tested conditions, the fluorescence calibration yielded an operational plasmid-based threshold estimate of 4.34 copies/μL; this value is not a validated limit of detection, and additional low-copy replicates are required. Cas9-positive tomato DNA was distinguished from wild-type tomato, four tested non-target plant DNA samples, and a no-template control, and four independent Cas9-positive tomato lines generated positive signals. Analysis of G-rich amplicon motifs and sequence-matched oligonucleotides suggested sequence-dependent contributions to ThT fluorescence, although G-quadruplex formation in LAMP products was not directly confirmed. The assay uses the same Cas9 target and primer framework as a previous HNB-based colorimetric method; its contribution is an instrument-recorded, probe-free fluorescence readout rather than broader target coverage or demonstrated superiority over existing fluorescent chemistries. The method is therefore a proof-of-concept approach for preliminary screening. Validation in processed foods, formal threshold development, low-copy replication, and closed-tube implementation remain necessary before routine use.