Study On The Bacterial Luciferase Bioluminescent System In Vitro And Its Application In Detection Of Pathogenic Bacteria In Fishery Products

Pathogenic bacteria which can cause a variety of food-borme diseases is the primary problem of food safety, and it does great harm to people's health. Therefore, there is an urgent need to develop rapid, sensitive, specific and reliable methods for the detection of pathogenic bacteria.According to the bioluminescence of NADH catalyzed by the luciferase:FMN-NADH oxidoreductase bioluminescent system in vitro (the coupled enzymes system) and the proportional relationship between the concentration of NADH and the luminescence intensity of the coupled enzymes system, a method of detecting live bacterial cells was developed. For purpose of detecting the aim bacteria specifically, bacteriophages which can infect their host bacteria and release NADH by specific lysis were used in the detection system. The coupled enzymes system was used to detect the pathogenic bacteria by detecting bacteria intercellular NADH. Then the detection system was used to the rapid detection of Klebsiella and E.coli in fishery products. The results are as follows:1. Based on the crude extract from Photobacterium leiognathi YL, the single bacterial luciferase bioluminescent system in vitro and the coupled bacterial luciferase:FMN-NADH oxidoreductase bioluminescent system in vitro were set up separately by optimizing the amount of different substrates. Furthermore, a method for quantitative detection of NADH was developed according to the bioluminescence of NADH catalyzed of bacterial luciferase:FMN-NADH oxidoreductase system in vitro. A good linear relationship of NADH concentration was in a range of 1.0×10-10 to 1.0×10-8 mol/L2. The bacterial luciferase:FMN-NADH oxidoreductase system is unable to identify intracellular NADH of different bacteria strains. To improve specificity of the detection system, a dauntless try to detect live bacterial cells by the coupled enzymes system combined with the bacteriophage lysis was established in this paper. Standard curves to detect Klebsiella and E.coli by combining the coupled enzymes system in vitro with the bacteriophage lysis were established respectively, and bacteria with the total bacterial count of above 107 cfu/mL in pure culture could be readily detected in an hour. The detection system also proved to be specific.3. The coupled enzymes system was applied to the rapid detection of Klebsiella and E.coli in fishery products. Bacteria with the total bacterial count of above 108 cfu/g could be directly detected from Spanish Mackerel. In order to improve the practicality of the detection system, the pre-enrichment of Klebsiella and E.coli was then studied.100 cfu/g of Klebsiella enriched in nutrient broth for 5 hour could be detected obviously and 100 cfu/g of E.coli enriched in nutrient broth for 6 hour could be detected significantly by the detection system. The coupled enzymes system combined with the bacteriophage lysis proved to be a advantageous tool for detecting live bacterial cells, and it laid a good foundation for detecting pathogenic bacteria in fishery products.4. Coimmobilization of bacterial luciferase and FMN:NADH oxidoreductase using sodium alginate as the carrier was also studied in this paper. The coupled enzymes system immobilized on sodium alginate was used to quantitative detection of NADH, and the limit of detection was 1.0×10-9 mol/L.In summary, the coupled enzymes system combined with the bacteriophage lysis is a simple, sensitive, rapid and specific method for the detection of pathogenic bacteria in fishery products.