| Enterotoxigenic Escherichia coli is a common pathogens causing acute diarrhea in infants, tourists and pups in developing countries, E. coli K88 can be infected through drinking contaminated water or eating under-cooked food, then causing food-borne illness. With the food safety issues caused by the pathogenic microorganisms highlighted, as an important technical support to guarantee for food safety, the detecting technology, especially rapid detecting technology for pathogenic bacteria play an increasingly important role in food production, circulation process control and supervision as well as import and export trade. Therefore, the research and development of accurate, sensitive, simple and rapid detection methods for E. coli K88 and other pathogenic microorganisms is extremely important.Firstly this study prepared Au NPs using sodium citrate reduction, and optimized the preparation conditions of Au NPs and examined its characteristic, the results were as followed, the Au NPs of 16.0±0.70 nm with the concentration of 3.57×10-9 mol/L was prepared in conditions of being heated by hot type constant temperature heating magnetic stirrer with the stirring speed of 200 r/min, and continuing to heat 20 min after adding reductant. Furthermore, the storage stability of the Au NPs was evaluated, it was concluded that the Au NPs solution can stored for 50 d under 4 ℃ and avoiding light, while almost did’t occur the phenomenon such as aggregation and sediment. Finally the Au NPs was modified through the Au-S key to connect to the thiolation E. coli K88 aptamer, which lays a foundation for the next step in establishing the visual detection technology for E. coli K88 based on Au NPs and aptamer.Secondly, this study established a visual rapid detection method for E. coli K88 based on aptamer technology combined nanogold labeling with silver staining, through optimizing detecting conditions of the rapid detection method, determinated the optimal concentration of streptavidin coating microwell was 0.1 mg/m L; the best working concentration of biotinylated aptamer 1 and thiolation aptamer 2 was both 1.0×10-6 mol/L; the optimal combination condition of aptamer 1, Au NPs-aptamer 2 compounds and E. coli K88 strain was under 40 ℃ for reacting 50 min; the optimal silver staining time was 150 s, and the coloration result can be observed by the naked eye. Under the optimal experimental conditions, a series of concentrations of E. coli K88 were investigated quantitatively, there was a strong linear correlation(R2=0.9903) between the intensity of the signals and the concentration of E. coli K88 within the range of 1.0×101~1.0×105 cfu/microwell, with a detection limit of 10 cfu/microwell, detecting time was about 2 h; and detecting results were negative for other common bacteria, indicated that this method had high affinity and specificity.Lastly, the method was applied to three categories of food: milk, pork(lean pork and streaky pork) and raw vegetables(purple cabbage and lettuce) samples polluted E. coli K88 by artificial simulation. The results showed that the method in three categories of food samples detection sensitivity can reached 102 cfu/m L(or cfu/g), the whole detection process took 2 h, at the same time there was a strong linear correlation between the intensity of the signals and the contaminated concentration of E. coli K88 within the range of 1.0×101~1.0×105 cfu/m L(or cfu/g), and in three categories of food, R2 were all more than 0.97. These preliminary showed that the detection method can be used in three categories of food for the visual and quantitative detection of E. coli K88, and with high specificity and sensitivity. |
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