|Liming, Samantha - POLYTECHNIC, SINGAPORE|
Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 20, 2004
Publication Date: September 1, 2004
Citation: Liming, S.H., Bhagwat, A.A. 2004. Application of molecular beacon -- real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. International Journal of Food Microbiology. 95:177-187. Interpretive Summary: Conventional methods in food may take up to one week to accurately predict the presence of human pathogens. Considering the limited shelf life of produce, rapid methods for pathogen detection are required. Real-time detection of Salmonella strains will broaden our ability to screen large number of samples in a short time. In this study, a DNA hybridization based detection method for Salmonella enterica serovar Typhimurium, based on polymerase chain reaction (PCR) is developed to enable near-instantaneous detection and quantitative analysis. The modified protocol requires less than 24 hours and is compatible for future high throughput sample analyses requirements. Detection of human pathogens from fresh produce is a crucial step in implementing food safety. Both the fresh produce industry and consumers will benefit from the results of this research.
Technical Abstract: An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon; MB) was used in a real-time polymerase chain reaction (PCR) assay to detect the presence of Salmonella species. A fluorogenic MB-probe was designed to recognize the iagA (invasion associated gene), which is highly specific to all Salmonella species that we tested. As few as 1 to 4 colony-forming units (CFU) per PCR reaction could be detected. The capability of the assay to detect Salmonella species from artificially inoculated fresh-cut produce such as cantaloupe, mixed-salad, cilantro, and alfalfa sprouts was demonstrated. In addition, a comparison of two commercially available kits utilizing MB-PCR (iQ-Check, Bio-Rad Laboratories) and conventional AOAC-approved PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. As few as 4 CFU/25 g of produce were detected after 16 h of enrichment in buffered peptone broth. These assays could be carried out entirely in sealed PCR tubes, enabling a rapid and high throughput detection of Salmonella species in a large number of food and environmental samples. This is the first report of the application of MB probe being used for real-time detection of Salmonella species in whole and fresh-cut fruits and vegetables.