2.5. Sensitivity Validation of the RPA-LFD and RPA-CRISPR/Cas12a Reaction Systems

Dilutions ranging from 100 ng/μL to 100 fg/μL were used for validation. Serially diluted P. pini gDNA was used for the sensitivity analysis of the RPA-LFD system, which made use of P. pini-specific RPA-LFD primer sets. Similarly, the same dilutions of P. pini gDNA were used as templates to validate the sensitivity of the RPA-CRISPR/Cas12a assay. A blue LED transilluminator was used to observe fluorescence at 470 nm, and full-wavelength zymography was used to measure fluorescence intensity. Three repetitions of each set of experiments were conducted, with a positive control (100 ng/μL P. pini gDNA) and a negative control (ddH₂O). The data were processed as described above. The experimental group and control group were compared via one-way analysis of variance (ANOVA), and p < 0.05 (*) was considered significant.