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Abstract: Refractive surgery can unmask or accelerate transforming growth factor beta-induced (TGFBI)-related corneal dystrophies that are undetectable by routine slit-lamp examination, creating a clear need for a rapid, standardized, preoperative genetic screening. We developed a multiplex, allele-specific quantitative polymerase chain reaction (qPCR) panel targeting five high-frequency TGFBI hotspots (R124C/L/H, R555W/Q) and built a statistics-driven analytical framework to optimize assay decisions. Receiver-operating-characteristic (ROC) analysis defined locus-specific cycle threshold (Ct) cutoffs that were harmonized to a single decision threshold (Ct=36) to simplify deployment. Analytical sensitivity was established by Probit modeling of serial two-fold dilutions, and confirmed by ≥20 replicates per level. In a 158-sample validation set (38 mutation-positive; 120 negative), qPCR agreed perfectly with Sanger sequencing (Cohen's Kappa coefficient (Κ)=1.0). Probit analysis yielded locus-specific limit-of-detection (LoD) values ranging from 0.035-0.20 ng µL^-1; at 0.20 ng µL^-1 the detection rate was over 95%. Repeatability and intermediate precision were high (Ct coefficient of variation (CV) 0.34-1.21%). No cross-reactivity was observed against non-target TGFBI variants or other ophthalmic genes, and interference from blood, oral flora/rinse, or toothpaste produced small, bounded shifts (≈?7.8% to +2.8%). Calibration with serial dilutions demonstrated linear Ct-log(copy) relationships suitable for routine quality control. Prospective screening of 10,055 refractive surgery candidates identified six TGFBI carriers (0.06%) harboring R124H (including one homozygote), R124L, R124C, or R555W mutations, all confirmed by Sanger sequencing. This study established a clinically applicable, statistically optimized multiplex qPCR platform that integrates ROC-derived cutoffs and Probit-defined LoD with rigorous evaluations of precision, specificity and robustness, enabling large-scale population implementation. Positive screening results guide clinical decision-making through a standardized post-screening workflow, and the targeted hotspot screening strategy serves as a cost-effective first-tier high-throughput approach for preoperative risk assessment. The framework provides a transparent, reproducible path to standardize preoperative TGFBI screening and reduce iatrogenic risk in refractive surgery candidates.