| This program is that the Jilin Science and Technology Office of 2002 years imburseing the international cooperative research project: "Use the SPR immunity sensor detection research of typhoid Salmonella bacterium", project serial number: 20,020,802, undertaken from the Biology and Agriculture Engineering College of Jilin University. Along with the raising of the economic development and people's living standard of our country, the kind and the quantity of food are increasingly rich, and how to raise the food quality and safety stick out increasingly. After having solved the basic need, the customers of town and country ask to eat more safety, this is the social tendency. During the food processing, store, transportation, sell , till to be eaten , the food has possibility to get biological pollution and endangers the health of human body. According to domestic and international statistics, in various bromatoxisms, the germ bromatoxism is the most. E.Coli is the most common pathogenic bacteria in germ bromatoxism, and endanger for human body greatly. The Centers for Disease Control and Prevention (CDC) approximates that 6,500,000 people in the United States are annually affected by pathogen in food, including 9,000 deaths. E.Coli is especially harmful to human health. With highly contagion, E.Coli is the main pathogen of infant diarrhea and seriously can lead to death. Especially E.coli O157:H7 can lead to hemorrhagic colitis (HC). E.coliO157:H7 is the most common in germ bromatoxism, take the 42.6% ～60% of germ bromatoxism approximately according to statistics. Bromatoxism leads to urgent disease, patient centralize, thus fast accurate diagnosis is particularly important for epidemic situation control. So food industry needs an rapid and simple detection assay for the pathogen in food, especially for E.ColiO157:H7. Now in the world, the SPR biosensor used in the research of microorganism detection is not much, and the detection limits of E.Coli is not high. General research can only reach the detection limit of 106 cfu/ml. Medina et al used SPR-immunochemical methods to detect E.Coli O157:H7.The direct detection of bacteria cells provided low responses. So sandwich immunoassays were utilized. This approach has produced an assay with detection limits of about 106cfu/ml for E.Coli O157:H7. Use avidin-biotin system and bind the antibody to the avidinated surface via avidin-biotin interaction for signal amplification of SPR biosensor. Thus the detection respond signal increase substantially, at the same time have raised detection limit notably. The Neutravidin we use can guarantee the accuracy of detection. To raise the sensitivity of detection, Sandwich assay has been used to amplify the detection respond .In order to increase assay sensitivity, we use sandwich assay to detect. At first the secondary antibody is made by avidin and biotin. Later use antibody labeled by colloid gold as second antibody to signal amplification. Use all these methods to detect E.Coli and Salmonella pullorum extend the detection limit and realized fast detection. This paper draws conclusions as below through experiments: 1. Avidin and biotin system applies to SPR biosensor detection. Avidin joints of the golden membrane surface, and to make detection microorganism become possible. The very strong affinity between avidin and biotin, also combine the antibody labeled by biotin to golden membrane surface. Finally the combination between antibody and antigen has realized the combination of microorganism and sensor, such the multilayer membrane structure forms. Thus the respond signal has been amplified, and is helpful for microorganism detection. 2.After using the avidin-biotin system ,the SPR biosensor can detect the E.Coli O157:H7,whose concentration is 106cfu/ml. 3. Adopt the method of joining secondary antibody, that is to combine secondary antibody after the combination of bacteria and antibody, so make detection respond signal increase. The secondary antibody is compound antibody made by avidin and biotin. This method extends the detection limits from 106cfu/ml to101cfu/ml, and the detection time is less than 1hour. 4. When use antibody labeled by biotin and avidin to make compound antibody, the proportion of avidin and biotin is 1:0.8. 5. Sandwich assay is used to detect E.Coli O157:H7 & Salmonella pullorum and immunity colloid gold as secondary antibody to amplify the response signal obviously, the detection limits have been extended from 106cfu/ml to101cfu/ml. 6. Mey tests determine the most suitable quantity of E.Coli O157:H7 antibody is 8 μg/ml. 7. When we use SPR sensor to detect the bacteria concentration, the test value is uncertain because it is influenced by test time, the zero point position, random error, whether rinsed by LB and other factors, which bring many problems on the actual application. 8. The slope of curve with immunity colloid gold of combination as the standard of detection bacterium concentration has much more accuracy and can detect the E.coli O157:H7 of 101cfu/ml.. It avoids the error caused by the zero point, test time, random error, whether it is rinsed by LB and other factors. Thus the assay detects bacteria more reliable and practical. 9. Rinse with LB after the combination with secondary antibody can make the value of index of refraction not only rises overall, and makes the test value per point increase. This explains that rinse with LB after the combination with secondary antibody is feasible, and it is helpful for the detection for microorganism.10. The slope of curve with immunity colloid gold of combination as the standard of detection bacterium concentration has much more accuracy and can detect the E.coliO157:H7 of 101cfu/ml.. It avoids the error caused by the zero point, test time, random error, whether it is rinsed by LB and other factors. Thus the assay detects bacteria more reliable and practical.
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