Rapid Detection of Salmonella spp. by PCR Amplification of Salmonella Specific Region in gatD gene

Abstract

A polymerase chain reaction (PCR) using primers specific to the internal region of the Salmonella gatD gene encoding galactitol-1-phosphate dehydrogenase was developed for a rapid, specific and sensitive detection of Salmonella spp. Four 20 mer oligonucleotides, named P1, P2, P3 and P4, were used in combinations of Pl-P3, P2-P4 and P3-P4 for amplification of the Salmonella specific region of the gatD gene. Single PCR products with about 780, 140 and 920 by were generated with all DNAs prepared from 24 Salmonella strains and P1-P3, P2-P4 and P3-P4 GatD primer sets, respectively. No PCR products were amplified from the DNA samples of 24 non-Salmonella bacteria using any of the GatD primer sets . These PCR bands were detected by ethidium bromide staining even when genomic DNA prepared from 180 cells of S. Typhimurium was used for the PCR. By using PCR with the primer sets following 6 h preenrichment in Enterobacteriaceae enrichment mannitol broth and 18 h selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin medium, 1.7 cfu of S. Typhimurium per 25g of autoclave-sterilized chicken meat were detected even in the presence of E. coli at 2.1×10⁶ cfu/25g.