| Enterohemorrhagic Escherichia coli O157:H7(E.coli O157:H7)which was discovered in recent is a serious enteric pathogens.Infection with E.coli O157:H7 follows ingestion of contaminated food or water.E.coli O157:H7 infection can lead to severe symptoms or even cause death.E.coli O157:H7 infection which threatens human health has gradually become a major public health problem.Establishment of rapid,high sensitivity detection methods is the key of E.coli O157:H7 infection prevention and control.Quantum dot is a new type of nano-materials and has excellent fluorescence properties.Compared with other immunology analysis method,the immune analytical method based on the quantum dot has great advantages in specificity,the sensitivity and accuracy.It has a broad application prospect in the detection of the food microbiology.In this study,the whole cell of E.coli O157:H7 was used as immunogen.Polyclonal antibody of E.coli O157:H7 was prepared.On this basis,we do some researches on water-soluble core/shell quantum dots,quantum dots coupling with antibody and immune chromatography.A fluorescent immune chromatography strip was fabricated to detect E.coli O157:H7.The research content is as follows:Preparation of polyclonal antibody:E.coli O157:H7 which was heat inactivated was used as the immunogen.Polyclonal antibody of E.coli O157:H7 was obtained successfully by intradermal injection.After being purified by caprylic acid-ammonium sulfate,the titer of the polyclonal antibodiy was 1:25600 and the protein concentraton was 4.07 mg/mL.The polyclonal antibody had good purity tested by SDS-PAGE.the ralative molecular weight of the polyclonal antibodiy antibody was about 152 kD.Preparation of water-soluble core/shell quantum dots:CdTe/CdS core/shell QDs were prepared in Water Phase in the presence of mercaptopropionic acid(MPA)as a stabilizer.Preparation conditions were optimized by single factor experiment and the optimal level of various factors are as follows:the mole ratio of Cd2+and Te2-was 5:1,the mole ratio of MPA and Cd2+was 2.5:1,the pH of reaction system was 9,reaction temperature was 95℃,the reflux time of CdTe core was 1 h and the reflux time of CdS shell was 1 h.In this case,the fluorescence intensity of CdTe/CdS QDs was 726.41.The fluorescence intensity increases by 50.10%than in initial conditions.Compared with CdTe QDs,the fluorescence intensity and stability of CdTe/CdS core/shell QDs are greatly increased.Conjugation of QDs with antibodies:polyclonal antibody was conjugated to CdTe/CdS QDs by electrostatic conjugate and covalent conjugate respectively.The results of Agarose gel electrophoresis and fluoresence spectra show that QDs were successful coupled with the polyclonal antibody,and covalent conjugate mothod get a better effect.conjugate effect factors were optimized and the optimal conditions were as follows:pH of 7.4,0.01 mol/L PBS buffer system,37℃ for 1.5 h,100 μL CdTe/CdS QDs and 80 μL 1 mg/mL polyclonal antibody.The preparation of fluorescent immune chromatography strip for E.coli O157:H7:A fluorescent immune chromatography strip using QDs-antibody conjugates was fabricated.Results are determined by fluorescence banding.The test line and control lines of strip were coated with 1 mg/mL polyclonal antibody and 1 mg/mL goat anti-rabbit IgG.The test strip showed good specificity of inter-generic cross and did not react with other group.The detection limit was 104-105 CFU/mL.After 35 days in room temperature in dark,the test strip could use normally. |
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