PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples.

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

[1]  Cheorl-Ho Kim Catalytic domain of Salmonella typhimurium 2-oxoglutarate dehydrogenase is localized in N-terminal region ☆ , 2003 .

[2]  Tae-Wook Chung,et al.  Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium , 2001, Glycoconjugate Journal.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  M. Howard,et al.  Three-dimensional structure of the lipoyl domain from the dihydrolipoyl succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli. , 1996, Journal of molecular biology.

[5]  T. Popović,et al.  Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. , 1995, International journal of food microbiology.

[6]  S. Pillai,et al.  A rapid method for screening for Salmonella typhimurium in a chicken cecal microbial consortium using gene amplification. , 1994, Avian diseases.

[7]  W. L. Payne,et al.  Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods , 1993, Applied and environmental microbiology.

[8]  P. Feng,et al.  Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters , 1991, Applied and environmental microbiology.

[9]  J. D'aoust Infective dose of Salmonella typhimurium in cheddar cheese. , 1985, American journal of epidemiology.

[10]  M. Darlison,et al.  Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12. , 1984, European journal of biochemistry.

[11]  L. Reed,et al.  α-Keto acid dehydrogenase complexes , 1972 .

[12]  B. Malorny,et al.  Detection of Salmonella spp. , 2003, Methods in molecular biology.

[13]  S. Shin,et al.  Specific detection of enteropathogen Campylobacter jejuni in food using a polymerase chain reaction , 1999 .

[14]  W. Waites,et al.  Biotechnology-based methods for the detection, enumeration and epidemiology of food poisoning and spoilage organisms. , 1990, Biotechnology & genetic engineering reviews.

[15]  R. Fitts Development of A DNA-DNA Hybridization Test For The Presence of Salmonella in Foods , 1985 .