Research And Application Of Novel Methods For The Pathogenic Microorganisms Detection And Prevention System

Objective:As a most important indicators of detection in clinical examination and food safety, pathogenic microorganisms has been concerned by worldwide microbiologist. Developing a quick and simple microbial detection technology and prevention system applied during whole entire food production process have become one of the important means to control the pathogenic microorganism infection and pollution. Proteins and nucleic acids are the most important types of biological molecules, so combined with different fluorescent dyes or sensing technology have become the frontier research in the field of new method for pathogenic microorganisms detection. The purpose of this study is that we want to develop a series of new methods to detect pathogen and prevention system on the basis of new technology, and reduce the infection of pathogenic microorganisms and contamination of food.Methods:(1) With the characteristics of gold nanoparticles quenching the fluorescent dye, depending on the aptamer affinity for hemolytic Streptococcus target specific molecular and bacterial protein. Fluorescence signal was excited in the real reaction system. Hemolytic streptococcus was detected by the fluorescence signal intensity.(2) With the unique design of molecular beacons and complementary DNA deletion to the DNA locus, the clever use of the characteristics for DNA stem loop structure to hybrid complementary Staphylococcus aureus specific DNA sequence, the DNA structure phase changed, forcing ATMND fluorescencen re-excited. The fluorescence signal intensity could determine the presence of S.aureus.(3) Through immuned Escherichia coli0157:H7antigen, we have established hybridoma cell lines. Depending on the serum agglutination test and ELISA antibody test, we prepared monoclonal antibody of Escherichia coli O157:H7.(4)Using a similar ELISA reaction principle, a highly sensitive electrochemical immunosensor for detection of Escherichia coli O157: H7was constructed. Depending on the double antibody sandwich response system, combined with the catalytic silver characteristics of alkaline phosphatase, with glass as a micro gap chip substrate interdigitated electrode array sensor, Escherichia coli O157:H7would be detedcted through the electrochemical signal change.(5) With the aid of food microbiological hazard analysis and critical control point principle, we warned large enterprises for microbial contamination, getting microbial hazard assessment and finded out the key control points of process, using the conventional method and label free molecular beacon probe technology to reduce microbial contamination for food Food processing.Results:(1) With the aid of aptamers for affinity Hemolytic streptococcus, the method for detecting the bacteria in the linear range is4.9102-4.9107CFU/mL, the lowest value to calculate the detection limit was33CFU/mL.(2) With the label free dye binding design of beacon sequences probe and complementary DNA probe, its fluorescence intensity enhanced after the specific sequence of Staphylococcus aureus had been added. When the the target sequence without presence, the fluorescence intensity induced little. The changes of fluorescence intensity could directly reflect the presence of Staphylococcus aureus.(3) We obtained excellent monoclonal antibodies of Escherichia coli O157: H7, after bacteria antigen had been immuned. The ascites titer reached1:104.(4) Silver particle appeared on the surface of microarray gap interdigitated electrodes, so the conductive properties changed. The result showed that the Escherichia coli0157:H7concentration of1103CFU/mL,1105CFU/mL,1108CFU/mL have good linear relationship, and the method for other control gram positive and gram negative bacteria has apparently nonspecific. The device could reach the national standards for detection of microbial "detected or not detected".(5) After the application of control system and the microbial prevention, statistical differences in microbial test pass rate was significant, and Staphylococcus aureus detection time from72hours shortened to24-48hours.Conclusion:With the help of trace analysis of nucleic acid and protein,The detection methods to pathogenic microorganisms will have great application prospect in clinical diagnosis, health inspection and quality supervision. Because the gene probe methods to detect pathogenic microorganisms just needed fluorescence spectrophotometry, the test reagent was also cheap and easy to obtain, and aptamer sequence easy for synthesis too, the personnel skill requirements was not high, the detection system has strong versatility to microbial test. At the same time, electrochemical detection technology depended on simple equipment, low detection cost, response sensitivity, easy to miniaturize overall inspection system, so its application market is extremely broad. The price of glass substrate microarray interdigitated electrodes chip compared with traditional silicon chip was lower, and the supporting reagent and the experimental operation skills were not high, so the detection device could completely replace all detection methods which are similar to the ELISA principle in clinical examinations and food safety field in the near future. Moreover, the Escherichia coli0157:H7antibody which we had prepared has high titer and stable quality, which was good for the research of correlation detection method of immunology. Finally, we studied the microbial contamination assessment for food processing enterprises, combined with the methods for rapid detection of pathogen.With warning and controlling microorganism pollution during food processing, the system could try to apply in meat product enterprise.