On the other hand, robust and precise assays are usually correlated with high PCR efficiency. The amplification plot of the experiment using the primer/probe fish set generated a slope of 3.3009 or 100.885% of efficiency, with a correlation coefficient of 0.99. The Cq and efficiency values obtained demonstrate the applicability of the rapid real-time PCR method developed to detect fish in food. LOD is defined as the lowest concentration of fish DNA at which 95% of positive samples are detected (Bustin et al., 2009). In the present assay, a minimum of 0.05 ng of fish was necessary to obtain a positive result.