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Applied and Environmental Microbiology, November 2001, p. 4984-4991, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.4984-4991.2001

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Peter G. Agron,1 Richard L. Walker,2 Hailu Kinde,3 Sherilyn J. Sawyer,2 Dawn C. Hayes,2 Jessica Wollard,1 and Gary L. Andersen1,*

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 945511; California Animal Health and Food Safety Laboratory-Davis Branch, School of Veterinary Medicine, University of California, Davis, California 956162; and California Animal Health and Food Safety Laboratory-San Bernardino Branch, School of Veterinary Medicine, University of California, Davis, San Bernardino, California 924083

Received 2 May 2001/Accepted 2 August 2001

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These 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 Enteritidis were not detected. By treating total DNA with an exonuclease that degrades 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 rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.

* Corresponding author. Mailing address: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Ave., L-441, Livermore, CA 94550. Phone: (925) 423-2525. Fax: (925) 422-2282. E-mail: andersen2{at}llnl.gov.

Applied and Environmental Microbiology, November 2001, p. 4984-4991, Vol. 67, No. 11
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.11.4984-4991.2001


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