| E. sakazakii (Enterobacter sakazakii), a kind of pathogenic bacteria found in dairy products recently, has been identified as an important opportunistic pathogen leading to the death of infants and the mortality is as high as 50%. A rapid method to enrich and detect E. sakazakii becomes the key to control and prevent its contamination in food. Based on current development of immune technology, the immune magnetic microspheres and immune fluorescent probe specific to E. sakazakii are prepared in this study. Moreover, the combination of these two technologies is used to detect the E. sakazakii fast and sensitively, which can overcome the weakness of traditional detection, molecular biotechnology detection and single immune detection. The study can provide the theoretical basis and reference to the research and application of immune technique in field of pathogen detection. The main results are as follows:(1) The study established a fast and efficient method of preparing polyclonal antibody of E. sakazakii. Specifically, the short-range immune method of subcutaneous multipoint injection combined with ear marginal vein injection was used to inject the E. sakazakii into New Zealand white rabbit. Antiserum could be obtained after injecting for 19 d and the purified titer was up to 1:20480, which fully satisfied the needs of immune detection. Specific research results showed that the preparation of polyclonal antibody of E. sakazakii had highly specific identification to E. sakazakii and no cross reaction with related strains of Escherichia coli, Enterobacter cloacae and Enterobacter aerogenes was observed. The study first revealed and indicated that two antigens in E. sakazakii were pyruvate dehydrogenase subunit El and molecular chaperone GroEL respectively. The method of preparation of polyclonal antibody is novel, simple and efficient.(2) The reverse suspension crosslinking method was used to prepare the magnetic chitosan microspheres with chitosan and nano-sized particles of Fe3O4 as raw materials. Results illustrated that the magnetic chitosan microspheres possessed an uniform size and some good properties including magnetic responsiveness, superparamagnetism and thermal stability. The surface, which was smooth and had no holes, could reduce nonspecific adsorption in the later using process.(3) Carboxyl-modification was performed in the surface of magnetic chitosan microsphere, which was reacted with chloroacetic acid. Then anti-E. sakazakii polyclonal antibodies were connected with carboxyl-modified magnetic chitosan microsphere by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HC1) and N-Hydroxy succinimide (NHS). The best coupling conditions were as follows:the dosage of the microsphere was 0.01 g, pH of activated buffer was 5.4, reaction time was 80 min, the polyclonal antibodies (12.6 mg/mL) coupled was 25 μL. Thus, the first immune magnetic microsphere was prepared. Enrichment effect of the immune magnetic microsphere on E. sakazakii was studied and the best enriching conditions were as follows:magnetic microspheres was 0.01 g, temperature was 37℃, pH of activation system was 7.4 and enrichment time was 60 min. The immune magnetic microsphere had a highly specificity to E. sakazakii and no cross reaction with related strains. The sensitivity was 6 cfu/mL. Reused results showed that the reusable frequency was more than 19 times when eluted for 7 min using glycine-hydrochloric acid buffer (0.1 M, pH 2.5).(4) Amino-modification was performed in the surface of magnetic chitosan microsphere, which was reacted with ethylenediamine under alkaline conditions. Then the anti-E. sakazakii polyclonal antibodies were connected with the amino-modified magnetic chitosan microsphere with glutaraldehyde. The best coupling conditions were as follows:the dosage of the microsphere was 0.01 g. pH of activated buffer was 7.4, reaction time was 20 min, the polyclonal antibodies (12.6 mg/mL) coupled was 50 μL. Thus, the scecond immune magnetic microsphere was prepared. Enrichment effect of immune magnetic microsphere on E. sakazakii was studied and the best enriching conditions were as follows:magnetic microspheres was 0.01 g, temperature was 37℃, pH of activation system was 7.4 and enrichment time was 45 min. Enrichment of the immune magnetic chitosan microsphere indicated that microsphere had a highly specificity to E. sakazakii and no cross reaction with related strains was observed. The sensitivity was 4 cfu/mL. Reused results showed that the reusable frequency was more than 27 times when eluted for 9 min using glycine-hydrochloric acid buffer (0.1 M, pH 2.5).(5) The synthesis of aqueous CdTe/CdS quantum dots (QDs) with core-shell structure was developed by salt water bath heating with the ultrasonic-assisted technique using thiol compounds as stabilizer. The pH value of reaction system was 10 and the reaction temperature was 105℃. The CdTe/CdS QDs had a core-shell structure with different colors under different emission wavelength. CdTe/CdS QDs also had high fluorescence intensity and a long fluorescent life. The synthesis method, which was simple, efficient and fast, pioneered a new field for the preparation of aqueous CdTe/CdS QDs. The CdTe/CdS QDs were regular microspheres about 4 nm and had good monodispersity. Fluorescence lifetime of CdTe/CdS QDs was more than 600 ms and the fluorescence intensity had not been weakened after placed in the dark for 24 months.(6) The immune fluorescent probe was prepared by connecting CdTe/CdS QDs and anti-E. sakazakii polyclonal antibodies using EDC and NHS. The best preparation conditions were as follows:the amounts of CdTe/CdS QDs and polyclonal antibodies were 125 μL and 75 μL, respectively. The pH value of reaction system was 8.2, reaction temperature was 35℃ and reaction time was 2 h. Fluorescence intensity of the immune fluorescent probe was enhanced compared with CdTe/CdS QDs and fluorescence lifetime of immune fluorescent probe had no significant change. The immune fluorescent probe has great potential in application of pathogen detection.(7) Rapid detection method was established by combing enrichment of immune magnetic microspheres with fluorescent mark of fluorescent probe. The effect of the method was studied through detecting the E. sakazakii in milk and the best detection conditions were as follows:0.05 g immune magnetic microspheres were added in 250 mL samples diluent, and the pH was adjusted to 7.4, then the samples were incubated in shaker at 37℃ for 60 min. After magnetic separation, the immune magnetic microspheres were washed thoroughly and reacted with 50 μL immune fluorescent probes in the shaker for 60 min. The whole testing time was 2 h. The results showed that the method had high specificity to E. sakazakii and the antibodies had no cross reaction with related strains. The results had a high accuracy and great temporal stability. The limit of detection was 15 cfu/mL.The pollution problem of E. sakazakii in infant food has aroused high attention to many people, however there has few mature rapid detection technology for detecting or monitoring the pathogen. Thus, this research has prepared immune magnetic microspheres and immune fluorescent probe specific to E. sakazakii and developed a rapid detection method for E. sakazakii by combing enrichment of immune magnetic microspheres with fluorescent probe labeling. The study provides theory reference for the development of rapid detection technology of pathogen. |
