Validation of Real-Time PCR and Enzyme-Linked Fluorescent Assay-Based Methods for Detection of Salmonella spp. in Chicken Feces Samples

Abstract

A comparative study of enzyme-linked fluorescent assay (ELFA)-based methods and real-time polymerase chain reaction (PCR)-based methods using three and two different sample preparation protocols, respectively, and the standard culture-based method EN ISO 6579:2002/Amd1:2007, for the detection of Salmonella spp. in chicken feces, was performed on 20 artificially and 68 naturally contaminated chicken feces. Selectivity, relative specificity, relative accuracy, relative sensitivity, and relative detection level were determined. According to criteria established in the methods comparison study included in EN ISO 16140:2003 for validation of alternative microbiological methods, the ELFA-based methods (V1 and V2) as well as a real-time PCR method (PCR2) were comparable to the reference method for the detection of Salmonella in chicken feces. They provided results in 48 h and presented a high sensitivity (97% for all of them). The three methods showed a relative specificity of 94%, V1 being the method which presented the highest relative accuracy (96%). While detection level for V2 and reference method was between 3 and 13 CFU/25 g, PCR2 method was able to detect down to 3 CFU/25 g. In conclusion, both the real-time PCR and the ELFA-based assays can be used as rapid and user-friendly screening methods for detection of Salmonella spp. in chicken feces.