Broad-Spectrum Detection And Identification Of Foodborne Pathogens With Multicolor Real-time PCR

Foodborne bacterial pathogens are recognized as one of the major etiological agents that cause foodborne disease. The availability of rapid, specific and high throughput assays to detect the presence or absence, or even the degree of contamination of pathogens, has become increasingly important for the food industry. This dissertation describes three solutions for the broad-spectrum detection and identification of foodborne pathogens.In chapter one, the methods for the detection of foodborne pathogens were reviewed. Three categories of methods, namely traditional culture-based methods, immunological methods and molecular methods were summarized briefly. The progress and future perspectives were also discussed. To break the bottleneck of real-time PCR throughput, a new detection format based on fluorescence combination was proposed to enlarge the covering range of objects in a single reaction. The so-called Multicolor Combinational Probe Coding (MCPC) technology uses probes labeled with mixed fluorophores to detect the matched templates, generating characteristic fluorescence profiles correspondingly. This innovation allows identifying any of 15 templates in a single tube on a four-color fluorometric thermolcycler.In chapter two, multiplex real-time PCR with modified molecular beacons was adopted as a solution for broad-spectrum detection of foodborne pathogens. The established multicolor real-time PCR could detect seven kinds of pathogenic vibrios through two PCR tubes. The first four-color real-time PCR system could detect Vibrio cholerae O1, Vibrio cholerae O139, total and pathogenic V. parahaemolyticus simultaneously. The detection limit ranged from 29.6 to 630 CFU per reaction for single target vibrios. The second four-color real-time PCR system could detect V. vulnficus, V. mimicus, V. alginolyticus and V. fluvialis simultaneously. The detection limit ranged from 29.5 to 295 CFU per reaction for single target vibrios. This method provided a rapid and accurate approach to the detection of pathogenic vibrios. In chapter three, broad-range PCR coupled with MCPC technology was adopted as a solution for broad-spectrum detection and identification of foodborne pathogens. Displacing probes were chosen in the application of MCPC technology in the study. Three segments were amplified from 16S rRNA and 23S rRNA gene by universal primers. Amplified products were differentiated by the displacing probes according to their signature sequences. The assay could identify any of nine categories of foodborne bacterial pathogens, namely pathogenic E. coli & Shigella spp., Salmonella spp., Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Vibrio cholerae, V. parahaemolyticus, Proteus spp. and Streptococcus pyogenes in a single reaction. The specificity of the assay was validated by 129 bacterial isolates.In chapter four, HAND (Homo-Tag Assisted Non-Dimer) system coupled with MCPC technology was adopted as a solution for broad-spectrum detection and identification of foodborne pathogens. Modified molecular beacons were chosen in the application of MCPC technology in this study. The assay had the capability of detecting any of seven pathogenic vibrios in a single tube. The list of target vibrios were Vibrio cholerae O1, Vibrio cholerae O139, total and pathogenic V. parahaemolyticus, V. vulnficus, V. mimicus, V. alginolyticus and V. fluvialis. The detection limits for these vibrios ranged from 2 copies to 100 copies of genomic DNA per reaction. The assay showed good quantitative ability within a wide range (1pg to 100ng DNA per reaction for V. fluvialis, 100fg to 100ng DNA per reaction for the rest). The specificity assay with 145 strains demonstrated 100% accuracy. The assay showed great potential in identifying a pathogen from a number of suspected objects.The three strategies described in this dissertation should facilitate the broad-spectrum detection and identification of foodborne pathogens in food and environmental samples.