Study On The Rapid Detection System For The Common Foodborne Pathogenic Bacteria

Objective:To study the adsorption capacity of lab made magnetic beads to the common food-bome pathogenic bacteria; to establish a method of rapid detection of Vibrio parahemolyticus (VP) using TaqMan real-time PCR for the Vibrio parahemolyticus food poisoning diagnosis and the investigation of Vibrio parahemolyticus contamination.Methods:Part One Study on the adsorption capacity of lab made magnetic beads1. Study on the adsorption capacity of lab made magnetic beads to the low number of bacteria in samplesStudy on the adsorption capacity of lab made magnetic beads to the different bacteria, at different temperature and tothe bacteria in different samples.2. Study on the adsorption capacity of lab made magnetic beads to the high number of bacteria in samplesStudy on the different adsorption methods of lab made magnetic beads to the bacteria, on the adsorption capacity of dry and wet lab made magnetic beads to the bacteria, the adsorption capacity of different amount of lab made magnetic beads to the bacteria, the adsorption capacity to the different high number of bacteria, in different reaction time, and at the different temperature.Part Tow Establish a method of rapid detection of Vibrio parahemolyticus using TaqMan real-time PCR1. Primers and probe designDesign the primers and TaqMan probe based on the sequence of tdh gene published on GenBank, estimate their feasibilities with BLASTN.2. Optimization of the PCR conditionsEstablish a rapid quantitative detection method for Vibrio parahemolyticus using TaqMan real-time PCR, optimize the PCR conditions, and analyze its sensitivity and specificity.3. Detection of the target bacteriaPlot the log cell number versus the TaqMan real-time PCR cycle threshold for 10 fold dilutions of template, fix quantify of the target bacteria.Results:1. The adsorption capacity of lab made magnetic beads to the low number of bacteria in samples: The lab made magnetic beads had great adsorption capacity to bacteria at 37℃; the adsorption efficiency could reach up to 97％. The component of the tested samples could decrease the adsorption capacity of the magnetic beads, and the adsorption efficiency decreased to 58.42％.2. The adsorption capacity of lab made magnetic beads to the high number of bacteria in samples: The adsorption capacity was correlative with the amount of the magnetic beads. The adsorption capacity was correlative with the number of the bacteria in orders. The temperature was an influential factor of the adsorption capacity of the beads. The adsorption capacity increased as the incubate time extend until to 40 minutes. The preservation method for beads whould likely not an obvious factor to influence the adsorption.3. The TaqMan real-time PCR assay is rapid, sensitive (1.3×10~4cfu/ml,33cfu/PCR reaction) and specific. It could be applied to the rapid diagnosis of Vibrio parahemolyticus food poisoning and investigation of the contamination status of it in food.Conclusions:The magnetic separation technology can rapid adsorb and enrichment the bacteria. The adsorption capacity of lab made magnetic beads to the bacteria in different numbers is quite well. Comparison of other detection methods, the TaqMan real-time PCR assay is rapid, sensitive and specific. The combination of magnetic separation technology and the TaqMan real-time PCR assay could be applied to the rapid diagnosis of Vibrio parahemolyticus food poisoning and investigation of the contamination status of it in food...