Devlopment Of Magnetic Fluorscent Motheds For Rapid Detection Of E.Coli

Along with the development of the global industrialization process, the environment pollution and the food security problem have become more and more serious. Environmental issues and food security problem have become one of the most important problem which deeply influence human survival and development in this century. Enterohemorrhagic escherichia coli (EHEC) is a kind of human foodborne and waterborne pathogens. When eating the food contaminated with the bacteria, diarrhea and enteritis would attack our health. More seriously, it may lead to a hemorrhagic colitis and hemolytic uremic syndrome, and even go to a death. EHEC is represented by O157:H7, which showed atrend of outbreak and mutate constantly. It is of gteat significance for environment detection and clinical epidemiological diagnosis to establish a rapid detection mothod. In consequence, A novel sensitive assay is investigated for the highly sensitive and rapid detection of Escherichia coli O157:H7. It is based on magnetic Fe3O4/graphene oxide (MGO) and DNA probes which is modified by 4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide (AHA). The results suggest that the linear relationship is found between log (CFU/mL of E.coli) and the ratio of fluorescence intensities (F/Fo) from 150 to 1.5×106 CFU/mL. The detection limit is 100 CFU/mL. The highly sensitive, rapid and simple means is demonstrated to be a noble tool for the detection of pathogenic bacteria.In this paper, the mothod is based on magnetic Fe3O4/graphene oxide (MGO) and DNA probes which is modified by 4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide (AHA). First, the ssDNA labelled with AHA was employed as a capture probe and adsorbed on the surface of MGO. When the target ssDNA appeared, capture probe hybridized with it in part. Then target ssDNA was fished and enriched via external magnetization to remove the original solution. Afterwards, a solution of release probe was added to the magnetic particles to complete the hybridization which resulted in separating the AHA from MGO. Then, high sensitive detection of E.coli O157:H7 would achieved by measuring the fluorescence intensity. In this paper, the target DNA T synthesized by Shanghai biological engineering corporation, and E. coli O157:H7 liquid bacterial germ were carried on the test. The former was employed to explore the feasibility of the test preliminarily, and the latter acted as the application of this system for the further exploring of detecting E.coli.The main results of this present work were concluded as follows:1. In this test,4-N-aminoethyl-N-hydroxyethyl-1,8-naphthalimide was synthesized. This material show superior water solubility and it is employed as fluorescer agent.2. High sensitive detection of E.coli O157:H7 had achieved by measuring the fluorescence intensity. The target DNA T synthesized by Shanghai biological engineering corporation was employed to explore the feasible conditions of the experiment, including hybridization temperature, hybridization time, absorption time, concentrations of MGO, concentration of release probe R and times of enrichment. Under the optimal conditions, the linear relationship is researched between log(CFU/mL of E.coli) and the ratio of fluorescence intensities (F/Fo).3. Cultivation of E.coli O157:H7 was conducted. The bacterial quantity was determined by plate colony counting method. Meanwhile, the DNA was extracted through gene extraction box which acted as the determinand under test. A novel method for detection of E.coli is successfully established, including studying the sensitivity and practical applications of this method.