بررسي امكان تشخيص حضور باكتري هاي متعلق به گونه Salmonella enterica زيرگونه enterica در مواد غذايي با روش PCR-TTGE

Development of a PCR-Temporal Temperature Gradient Gel Electrophoresis assay for identification of Salmonella enterica subspecies enterica in food samples

Bacterial foodborne pathogens are major cause of morbidity and mortality worldwide. Salmonella is the etiologic agent of Salmonellosis in humans causing severe illness in infants, the elderly, and immunocompromised patients. Cultural and serological detection methods are time-consuming and labor-intensive. Besides, these methods have limited application because of low specificity and sensitivity. Therefore, it is urgently in need to develop simple, rapid, and accurate detection methods to detect Salmonella in order to ensure food safety. In this study PCR-TTGE was optimised for identification of Salmonella enterica subspecies enterica serovers. Bacterial strains used in this study were cultured at 37°C in tryptic soy broth (TSB) and their DNA was isolated from pure culture of each strain. The designed PCR primers could amplify specifically and efficiently a DNA fragment of 214 bp for all of 4 different salmonella serovars tested. There was no amplification with genomic DNA prepared from 5 nonsalmonella species tested to verify the specificity of the assay. Results showed that the detection limit of the PCR assay was 12x10 3 CFU/ml in spiked food product by Salmonella Typhimurium. Analysis of the multiple sequence alignment for the 214 bp fragment from four salmonella serovers demonstrated some interserovers polymorphisms to generate different bands on the TTGE gels. The TTGE protocol resulted in the separation of the PCR products into 4 different band positions. This study shows the potential of the method to be used as a fast screening test to investigate the presence of Salmonella enterica subspecies enterica in food samples