Specific detection of Salmonella by multiplex polymerase chain reaction.

Abstract

This study evaluates the use of multiplex polymerase chain reaction (PCR) technology for detection of Salmonella species in pure cultures and also in environmental samples. Three sets of oligonucleotide primers were used in the PCR assay: PhoP primers specific to a 299 bp region in the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, E. coli and Citrobacter species, served as a presumptive indicator of enteric bacteria. Subsequently Hin and H-1i primers, which targeted a 236 bp region of the hin/H2 gene, and a 173 bp region of the H-1-i flagellar gene in Salmonella typhimurium respectively, were used for specific detection of salmonellae. Specificity and sensitivity of PCR amplified products were evaluated by ethidium bromide (EtBr) staining or gene probe analysis. Under optimal PCR conditions described in this study, Salmonella species can be specifically detected by these 3 primer sets. The sensitivity of detection in terms of whole cells was a 10⁻⁵ dilution (approximately 10³ cells) of boiled late log phase cultured cells (detection by EtBr) after 25 cycles of PCR and 10⁻⁸ dilution (approximately 10° cells) after a 50 cycle 'double PCR' protocol. A similar level of sensitivity was observed using a gene probe analysis (³²P end-labeling) to detect the PCR amplified products. The multiplex PCR products observed from known environmental isolates allowed identification of several isolates as Salmonella species. These results agreed with conventional analyses. The efficacy of this PCR technology for environmental applications was also evaluated by testing the primers on soil and environmental water samples. All unseeded soil samples showed no PCR amplified products when detected by EtBr staining after 25 and 50 cycles PCR. Only one pond surface water sample resulted in a positive PCR result. This water sample was also positive when tested by conventional methodologies. Seeded soil and well water samples all resulted in PCR products being observed from 25 or 50 cycles. These results indicate that the primers have the potential to specifically detect Salmonella species in environmental samples.