Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, c an be transmitted to humans through intact chicken eggs when the contents h ave not been thoroughly cooked. Infection in chickens is asymptomatic; ther efore, simple, sensitive, and specific detection methods are crucial for ef forts to limit human exposure. Suppression subtractive hybridization was us ed to isolate DNA restriction fragments present in Salmonella serovar Enter itidis but absent in other bacteria found in poultry environments. Oligonuc leotide primers to candidate regions were used in polymerase chain reaction s to test 73 non-Enteritidis S. enterica isolates comprising 34 different s erovars, including Dublin and Pullorum, two very close relatives of Enterit idis. A primer pair to one Salmonella difference fragment (termed Sdf I) cl early distinguished serovar Enteritidis from all other serovars tested, whi le two other primer pairs only identified a few non-Enteritidis strains. Th ese primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Ente ritidis were not detected. By treating total DNA with an exonuclease that d egrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4, 060-bp region was defined. These results provide a basis for developing a r apid, sensitive, and highly specific detection system for Salmonella serova r Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.