Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food

DNA sequences of an internal transcribed spacer (ITS) region for 40 Salmonella serovars were determined and compared with ITS sequences of Salmonella spp., and non-Salmonella spp. already available on the GenBank database. From such comparison, two Salmonella-specific ITS based PCR primers, ITSF and ITSR, were designed. When Salmonella strains with various serotypes were PCR assayed with primers ITSF/ITSR, all generated PCR products with molecular weight bands equal to 312 bp. On the other hand, 48 non-Salmonella isolates, including strains of Enterobacteriaceae and other food pathogens generated negative results. Detection limits of this PCR method was 1-9 CFU per assay. These PCR primers were used for the detection of Salmonella cells in artificially contaminated foods, including chicken meat and whole milk. The detection limit was 1-9×103 CFU per assay. With an 8-h enrichment step performed prior to the PCR assay, however, the detection limit became 1-9 CFU per gram of the food sample.