Development of SYBR Green real-time PCR for Salmonella Detection

Abstract

Salmonella is recognized worldwide as a major causative agent of food-borne illness. Transmission of Salmonella infections occurs through consumption of the contaminated food products. Salmonella outbreaks have been reported in many countries including Thailand, therefore using a rapid and accuracy diagnostic process contributes to decrease morbidity and mortality rate of patients, and also prevents an outbreak in the future. The aim of this study was to develop SYBR Green real-time PCR targeting enterotoxin (stn) gene for the detection of Salmonella. Moreover, stn primers designed from this study (stn170-508) was compared with those designed from Makino and staff (stn101-111). The limit of detection and specificity of both primer sets for real-time PCR and conventional PCR (cPCR) were compared. The limits of detection of Salmonella through cPCR and real-time PCR using stn170-508 primers were 10⁶CFU/ml and 10⁴ CFU/ml, respectively which were better than those using stn101-111 primers (10⁸CFU/ml and 10⁶ CFU/ml, respectively). To determine the specificity of both primer sets, a total of 174 bacterial strains including Salmonella 87 strains and non-Salmonella 87 strains were tested. Both primer sets detected all Salmonella strains only (100% specificity). SYBR Green real-time PCR developed in this study are able to detect Salmonella from pure culture and specifically differentiate Salmonella from other bacterial strains. This technique will be performed to detect Salmonella in food and clinical samples directly.