Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolat es. The results were compared with those of conventional methods. The TaqMa n assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification o f a spiked nonrelevant template (116 bp) in the sample tube. The PCR test c orrectly identified all the Salmonella strains by resulting in positive end -point fluorescence (FAM) signals for the samples and positive control (TET ) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specifici ty of the method was assessed using 120 non-Salmonella strains, which all r esulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough Salmonella strains tested resulted in positive FAM and TET signal s. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thu s, the diagnostic TaqMan assay developed can be a useful and simple alterna tive method for identification of Salmonella, particularly in Large referen ce laboratories.