| In recent years,a number of infectious diseases emerging increasingly as the pathogenic zoonosis with the rapid development of economyand the changing of peoples life style.Zoonoses have several characteristics with fast epidemic,diversity of disease transmission and spread widely.They not only caused huge economic losses to agriculture and animal husbandry,but also will constitute a serious threat to crowd and animal health.Most Zoonoses were not caused by a single pathogen.They were usually caused by a variety of at least two pathogens.The lack of specific clinical manifestations leads to great difficulty for clinical diagnosis and treatment.It is extremely important to strengthen monitoring on animal disease to reduce the harm of Zoonoses.Current detection methods of pathogen caused zoonosis mainly include traditional culture methods,immunological methods and molecular biological methods.However,these methods exist some disadvantages such as long detection time,operation time and tedious steps,high laboratory or operator requiremen,especially cannot be synchronous detection of various pathogenic bacteria detection,etc..Therefore,it is of great significance to develop new strategies which is simple,rapid and accurate for the simultaneous detection of multiple pathogens,which is simple,rapid and accurate for the prevention and control of zoonotic diseases,to reduce the economic losses and medical burden.Escherichia coli O157:H7(E.coli O157:H7),Listeria monocytogens(LM)and Brucella,as common and high incidence of zoonosis pathogen were selected as the target pathogen in this study.First,the trivalent egg yolk antibody(Ig Y)which could identify the three kinds of pathogenic bacteria was sprepared by Ig Y technique.By coupling carboxylated magnetic nanoparticles(MNPs)with trivalent Ig Y,the immune magnetic beads(IMB)were obtained for isolation and purification three kinds of pathogenic bacteria from samples.Then,anti-E.coli O157:H7 polyclonal antibody and anti-LM polyclonal antibody was prepared by polyclonal antibody preparation technology.Combined with the anti-Brucella polyclonal antibody prepared by our laboratory before,three kinds of quantum dots(QDs)biological fluorescence probes were produced by coupling three kinds of Cd Se/Zn S QDs with different emission wavelengths and the three kinds of polyclonal antibody,respectively.Eventually,a new synchronous,fast and accurate detection method of the three kinds of pathogenic bacteria was established based on immune-magnetic separation technique and quantum dots fluorescence technology.The detailed research works include the following four parts.Part 1 Preparation and identification of trivalent Ig Y(1)The trivalent inactivated vaccine was prepared according to the concentration ratio of 1:1:1 of E.coli O157: H7,LM and Brucella.Then healthy laying chickens were immured and the eggs were gathered.The trivalent Ig Y was extracted by PEG 6000 precipitation methods.(2)The indirect ELISA reaction conditions were optimized and the titer of chicken serum and trivalent Ig Y was detected by the optimal indirect ELISA method.The results demonstrate that with the prolonged time of immunization,the titer of serum and trivalent Ig Y was significantly increased.The serum antibody titer reached to the highest in sixth weeks after the first immunization.The antibody titer of anti-E.coli O157:H7 Ig Y and anti-Brucella Ig Y reached the highest in the tenth weeks after the first immunization and that of anti-LM Ig Y reached the highest in the twelfth weeks.(3)The protein content of trivalent Ig Y was determined by BCA protein assay kit.The results show that the range of protein content ranged from 5.73 to 21.03 mg·m L-1.On the tenth weeks later,the protein content reached the peak value with 21.03 mg·m L-1.(4)The specificity of trivalent Ig Y was identified by indirect ELISA method and the results indicate that trivalent Ig Y on the tenth weeks had cross reaction only with Vibrio parahaemolyticus and no cross reactivity with other nine strains.Purity of trivalent Ig Y was identified by SDS-PAGE electrophoresis and obviously heavy chain and light chain was observed which indicated the trivalent Ig Y had high purity.Part 2 Preparation and identification of the rabbit polyclonal antibody(1)E.coli O157: H7 and LM inactivated vaccine were prepared and used to immunize healthy New Zealand rabbits.The anti-serum was collected and the two kinds of polyclonal antibody extracted by saturated ammonium sulfate precipitation method,respectively.The indirect ELISA method was optimized and used to detect the antibody titer.The results showed that serum antibody titer of anti-E.coli O157: H7 serum was reached to 1:256000 in 10 days after the fourth immunization with the extension of the time of immunization.The serum antibody titer of anti-LM serum was reached to 1:256000 in the fourth immunization.On the tenth day after the fourth immunization,antiserum was collected and anti-E.coli O157: H7 polyclonal antibody and anti-LM polyclonal antibody was extracted.The antibody titer of anti-E.coli O157: H7 polyclonal antibody was 1:128000 and antibody titer of anti-LM polyclonal antibody was 1:512000.(2)The specificity of two rabbit polyclonal antibodies was identified by indirect ELISA method.The results indicated that anti-E.coli O157:H7 polyclonal antibody had cross reaction with Staphylococcus aureus,Shigella boydii and Serratia marcescens,but no cross reaction with other bacteria.The anti-LM polyclonal antibody existed cross reaction with Staphylococcus aureus,Streptococcus faecalis and Serratia marcescens,and did not exist cross reaction with other bacteria.(3)The protein content of two rabbit polyclonal antibody was detected by the BCA protein assay kit.The protein content of anti-E.coli O157:H7 polyclonal antibody and anti-LM polyclonal antibody was 29.06 mg·m L-1 and 38.27 mg·m L-1 respectively.The purity of two rabbit polyclonal antibodies was determined by SDS-PAGE electrophoresis.The results revealed clearly heavy chain and light chain,indicating a high purity of the two rabbit polyclonal antibodies.Part 3 Preparation and characterization of Quantum Dots-based biological fluorescent probes(1)Three kinds of Cd Se/Zn S QDs with different emission wavelengths were synthesized by organic phase synthesis.By using MPA as a stabilizer,Cd Se/Zn S QDs was shifted from the organic phase to the aqueous phase.The three kinds of Cd Se/Zn S QDs were characterized by TEM,UV Vis and fluorescence spectroscopy.The results showed that the fluorescence emission wavelength of the three kinds of Cd Se/Zn S QDs was 539 nm,583 nm and 634 nm respectively.The shape was approximately spherical with uniform particle size and good dispersion.(2)Three kinds of Cd Se/Zn S QDs with different emission wavelengths were coupled with anti-E.coli O157: H7 polyclonal antibody,anti-LM polyclonal antibody and anti-Brucella polyclonal antibody respectively by EDC and NHS agents.Three kinds of QDs fluorescent probes were obtained and then characterized by FTIR,zeta potential and fluorescence spectra.FTIR results showed the a group of peptide functional groups(-NH-CO-)absorption band appeared near 1550 cm-1 in the spectrum of QDs.Zeta potential results showed the surface potential of QDs displayed a positive increase when the QDs conjugated the polyclonal antibody.In the fluorescence spectra,the three fluorescence emission peaks were symmetrical and the position was separated from each other.All characterization results described the QDs was coupled with the polyclonal antibody and the the QDs-based biological fluorescent probes were successfully prepared.Part 4 Establishment and evaluation of simultaneous detection method of three kinds of bacteria of Zoonosis(1)IMB was prepared by coupling the magnetic beads and trivalent Ig Y through EDC and NHS agents and characterized by FTIR,zeta potential and DLS methods.The FTIR results showed that features of the absorption bands of(-NH-CO-)functional peptide appears on the surface of MNPs after coupling with trivalent Ig Y.The zeta potential results showed that the bead surface potential move to the positive direction after coupling with trivalent Ig Y.The DLS results showed that the particle size of MNPs increased after coupling with trivalent Ig Y.The above characterization results confirmed the trivalent yolk antibody was successfully conjugated to carboxylate MNPs surface.(2)IMB prepared above was used to capture and rapid separation the target bacteria include E.coli O157: H7,LM and Brucella in samples.The three QDs fluorescent probes with different emission wavelengths were used to label the target bacteria.Based on the immune-magnetic separation technique and quantum dots fluorescence technology,a synchronous detection method was established and the reaction conditions were optimized in this part.The results showed that the optimum dosage of IMB was 50 μL,the accumulation time was 60 min.The optimization experiment also displayed the optimal amounts of three QDs fluorescent probes was respectively 200 μL and the labeling time was 60 min.If ignoring the magnetic separation and washing time,the entire detection procedure requires 120 min.(3)The synchronous detection method of the three kinds of bacteria of Zoonosis was evaluated.The LOD of E.coli O157:H7,LM and Brucella in PBS buffer solution was 103 CFU·m L-1,103 CFU·m L-1 and 104 CFU·m L-1,respectively.The recovery rate of the three kinds of pathogenic bacteria was 90%~110%,indicating favorable accuracy.The results of repeatability experiments showed that the CVs of three bacteria detection methods were lower than 10%,which showed that the method had good stability.The specificity results showed that the established methods had a cross reaction with Vibrio parahaemolyticus and Serratia marcescens and no cross reaction with other strains.(4)Simulated samples of milk,lamb and cabbage were tested by our synchronous detection method.The LOD of E.coli O157:H7,LM and Brucella was all 103 CFU·m L-1 when the simulated samples were milk.The LOD of E.coli O157:H7,LM and Brucella was 104 CFU·m L-1、103 CFU·m L-1 and 104 CFU·m L-1,respectively when the simulated samples were lamb.The LOD of E.coli O157: H7,LM and Brucella was 103 CFU·m L-1、104 CFU·m L-1 and 105 CFU·m L-1,respectively.when the simulated samples were cabbage.In summary,a synchronous detection method was established and evaluated in this study,which can detect three kinds of bacteria caused Zoonosis(E.coli O157:H7,LM and Brucella)in one sample.This strategycombined the Ig Y technology,polyclonal antibody technology,immune-magnetic separation technique and QDs labeling technology.The detection method with short assay time showed good specificity,high sensitivity,excellent repeatability and stability,and can be used for rapid and quantitative detection three target pathogenic bacteria.This study provided an effective and reliable method which laid the foundation for the further development of a variety of fast and accurate synchronization detection kit for zoonotic pathogen. |
