Improved Detection of Salmonella spp. in Foods by Fluorescent In Situ Hybridization with 23S rRNA Probes: A Comparison with Conventional Culture Methods

Abstract

This report describes a new technique for the detection and identification of Salmonella species in food with the use of fluorescent in situ hybridization (FISH) with 23S rRNA–targeted oligonucleotide probes. Two species-specific 23S rRNA–targeted oligonucleotide probes (Sal-1 and Sal-3) were selected, and one (Sal-544) was newly designed. The relative specificities of these probes were compared with those of bacterial 23S rRNA sequences from the GenBank database and tested by in situ hybridization with bacterial cell smears of pure cultures. Fifty-one tested reference strains of Salmonella serovars belonging to subspecies I (enterica) hybridized with these probes. No cross-reactions with 46 other strains of the family Enterobacteriaceae or with another 14 bacterial strains from other families were observed. Storage of a Salmonella Panama test strain under various environmental conditions (2, 5, and 15% NaCl; −20°C, 4°C, and room temperature; pHs of 3.3 to 7.4) did not adversely affect the FISH method. No matrix effects were observed with 18 different kinds of foods. FISH was able to detect Salmonella spp. in 52 (probe Sal-1), 56 (probe Sal-3), and 35 (probe Sal-544) of 225 naturally contaminated food samples after 16 h of incubation in a preenrichment broth. When conventional culture and detection methods were used, Salmonella could be isolated from only 30 of these 225 samples. In contrast, FISH failed to identify Salmonella in only two of the culture-positive samples when Sal-1 and Sal-3 were used and in only three of the culture-positive samples when Sal-544 was used.