Development of a polymerase chain reaction-temporal temperature gradient gel electrophoresis assay for identification of Salmonella enterica subspecies enterica using a hypothetical non-specific endonucleas S. entericae gene sequence

Abstract

Background: Salmonella is one of the major agents of food-borne diseases that cause severe illness in humans. The conventional detection methods of these bacteria are time-consuming with low sensitivity and specificity, which limit their applications. Therefore, developing more rapid and accurate methods is urgently required in food safety programs. Objectives: In this study for the first time, polymerase chain reaction-Temporal temperature gradient gel electrophoresis (TTGE) was optimized for the identification of Salmonella enterica subspecies enterica serovars in processed food samples using a single-copy sequence. Methods: DNA was isolated from pure cultures of Salmonella and non-Salmonella strains. The single copy target sequence was selected and amplified by employing the polymerase chain reaction (PCR) with specific primers, designed in this study, and their specificity and sensitivity were determined. The TTGE parameters, especially the temperature gradient and the time of reaction, were optimized and then this method was applied to investigate spiked food samples. Results: The PCR detection sensitivity was recorded as 12×103 CFU/mL in artificially-contaminated food samples. The best resolution was observed at a temperature gradient from 62.5 to 67.5°C, with a ramp rate of 1°C/hour and electrophoresis for 5 hours at a constant voltage of 130 V. The TTGE patterns obtained from the artificially-contaminated samples were similar with the respected standard bacteria strains. The optimized TTGE protocol resulted in the separation of the same length PCR products into different band positions on the polyacrylamide gel. Conclusions: By optimization of PCR-TTGE and determination of distinct band pattern for standard bacteria, this method can be used as a fast screening test to investigate the presence of S. enterica, subspecies enterica, directly in food samples.