Development of sensitive and specific detection of salmonella by polymerase chain reaction

Abstract

Salmonella, food-borne pathogenic bacteria of man and warm blooded animals is of increasing concern to many countries including Thailand which exported over a billion US dollar of food products since it is a causative agent of typhoid fever and diarrhea. The conventional culture method for Salmonella detection involving multiple subcultural steps are labour intensive, low sensitivity and time consuming, taking 4-5 days for complete detection assay for Salmonella. In this study, genomic library of S. Weltevreden was constructed in Bluescribe M13- plasmid and cloned into E. coli JM107. The candidates were screened by hybridization with genomic DNA of S. Weltevreden. The 0.2 Kb inserted fragment from clone#6 was selected and sequenced because it conserved among other serovars. A set of PCR primers was designed to amplify a 199 bp Salmonella -specific DNA fragment derived from a clone of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross reaction was observed with other non-Salmonella organisms. The limit of detection was 0.1 fg of purified target DNA and 5 bacteria from pure culture. The detection of artificially contaminated food performed following l2-16 h enrichment step was 3 bacteria g(-l) and could be obtained within 4 h. Comparative study of PCR and microbiological method for detection of Salmonella in 182 frozen chicken samples was performed. The sensitivity and specificity of PCR based on microbiological method were 100% and 61% respectively. The disagreement of 2 method was 33%.