Our study evaluated the sensitivity and repeatability of nested PCR-based assays for directly detecting L. monocytogenes in artificially contaminated human serum, pasteurized milk and physiological saline samples. The detection of the hlyA (267bp) and iap (371bp) gene fragments was compared. The logistic regression (logit model) was used to evaluate the probability of detection of L. monocytogenes at various contamination levels and to calculate the number of test repetitions required to reach necessary detection limits (e.g. 50%, 80%, 90%, and 95%). The reliable limit of detection for both genetic markers, ensuring ≥95% probability of detection, was established at 10² CFU/100μL.