Visual Detection Of Vibrio Parahaemolyticus Based On Magnetic Separation And G-quadruplex DNAzyme

Vibrio parahaemolyticus is a common foodborne pathogen.Eating seafood contaminated with Vibrio parahaemolyticus can cause acute gastroenteritis,shock,septicemia,and even death.The existing methods for the detection of Vibrio parahaemolyticus have some shortcomings,such as complex sample pretreatment steps,poor sensitivity,long analysis time,high analysis cost and expensive instruments.Therefore,the development of rapid,accurate and low-cost detection methods for early detection and identification of Vibrio parahaemolyticus is essential for risk management and disease prevention and diagnosis.Objective:This project aims to establish and optimize a rapid detection method for Vibrio parahaemolyticus by using the specific recognition ability of aptamer and antibody,combined with immunomagnetic separation and G-quadruplex technology,so as to provide new ideas and technical support for food safety detection.Methods:1.Culture and morphological identification of Vibrio parahaemolyticusMonoclonal colonies were isolated by high salt solid medium and expanded by high salt liquid medium.Vibrio parahaemolyticus was identified by TCBS selective medium and Gram staining.2.Preparation and identification of chicken yolk antibodyPreparation of egg yolk antibody by immunizing laying hens with Freund’s complete adjuvant/Freund’s incomplete adjuvant vaccine of Vibrio parahaemolyticus.Antibody titers were determined by indirect ELISA,antibody content was determined by BCA protein quantitative kit,and antibody purity was determined by SDS-PAGE.3.Design and structural analysis of DNA sequenceUse NUPACK website to design and analyse sequences of ss DNA and Hairpin.4.Preparation and characterization of magnetic nanomaterialsFe₃O₄ NPs was prepared by hydrothermal method,and then Ag NPs was coated on the surface of Fe₃O₄ NPs using PVP as crosslinking agent to generate Fe₃O₄@Ag NPs.Then,the mercapto-modified aptamer was combined on the surface of Fe₃O₄@Ag NPs by Ag-S bond to obtain Fe₃O₄@Ag NBs-apt.The prepared magnetic nanoparticles were characterized by ultraviolet and visible spectrum,transmission electron microscope,X-ray diffraction,Energy Dispersive Spectrometer,vibrating sample magnetometer and Zeta potential.5.Preparation and characterization of gold nanomaterialsAu NPs-Ig Y was prepared by sodium citrate reduction method,and Au NPs-Ig Y was obtained by coupling chicken yolk antibody to the surface by electrostatic adsorption.The gold nanomaterials were characterized by ultraviolet-visible absorption spectrum,transmission electron microscopy and Zeta potential.6.stablishment and optimization of detection method for Vibrio parahaemolyticusAfter preliminary exploration of the experimental procedure,key conditions such as Fe₃O₄@Ag NPs-apt dosage and enrichment time,Au NPs solution p H,antibody coupling amount,gold nanoprobe dosage,substrate color development time,etc.were optimized to establish the detection method.7.Evaluation of detection methods for Vibrio parahaemolyticusThe sensitivity and specificity of the method were evaluated and simulated samples were tested by the method.Results:1.Morphological identification of Vibrio parahaemolyticusRound,raised,smooth opaque colonies were found on the common high salt plate,and the colony size was about 2 mm.On the TCBS plate,blue-green colonies were presented.Gram dyed red.2.Identification of chicken yolk antibodyThe titer of Vibrio parahemolyticus egg yolk antibody prepared in this study could reach 1:256000.The antibody purity was 88.63%,and the antibody protein content was38.09 mg/m L.3.Results of structural analysis of DNA sequenceThe results of structure analysis of designed ss DNA and Hairpin conducted on the NUPACK website are in line with expectations and can meet the requirements of the experiment.4.Characterization results of magnetic nanomaterialsTEM results showed that Fe₃O₄@Ag NPs and Fe₃O₄@Ag NPs-apt were spherical,with particle sizes of（99.34±20.55）nm and（107.83±25.43）nm,respectively.The results showed that the enrichment efficiency of magnetic nanoprobe for Vibrio parahemolyticus at 1.0×10⁷ CFU/m L and below concentration could reach 95%.5.Characterization results of gold nanomaterialsTEM results showed that Au NPs were spherical with a particle size of（15.55±1.48）nm.After antibody coupling,the characteristic absorption peak was redshifted by 2 nm and the particle size increased to（19.70±1.88）nm.6.Establishment and optimization of detection method for Vibrio parahaemolyticusThe optimum reaction conditions were established:The optimal dosage and enrichment time of Fe₃O₄@Ag NPs-apt were 40μL and 60 min,respectively.The optimal p H of Au NPs solution was 8.0.The optimal antibody coupling amount of Au NPs was 15μg.The optimal dosage of Au NPs-Igy was 40μL.And the optimal substrate color development time was 7 min.7.Evaluation results of Vibrio parahaemolyticus detection methodThe detection time of the established method was 6 h.There were two linear relationships in the range from 1.0×10¹ CFU/m L to 1.0×10⁷ CFU/m L:The linear regression equation from 1.0×10¹ CFU/m L to 1.0×10⁵ CFU/m L is Anorm=0.093lg C-0.053,R²=0.990.The linear regression equation from 1.0×10⁵ CFU/m L to 1.0×10⁷CFU/m L was Anorm=0.268lg C-0.927,R²=0.992.The detection limit was 3.02CFU/m L.In the detection of simulated samples,there are also two linear relationships in the range from 1.0×10¹ CFU/m L to 1.0×10⁷ CFU/m L:The linear regression equation from 1.0×10¹ CFU/m L to 1.0×10⁵ CFU/m L is Anorm=0.086lg C-0.029,R²=0.994.The linear regression equation from 1.0×10⁵ CFU/m L to 1.0×10⁷ CFU/m L was Anorm=0.236lg C-0.735,R²=0.995.The detection limit was 54.95 CFU/m L.The specificity experiment results showed that the method had good specificity and could distinguish Vibrio parahemolyticus from interfering bacteria.In conclusion,the detection system has high sensitivity,good specificity,wide linear range and low detection line,which can meet the needs of rapid and sensitive detection of Vibrio parahemolyticus in food samples.Conclusion:1.The chicken egg yolk antibody against Vibrio parahaemolyticus with high titer and good purity was prepared.2.Single-stranded DNA and hairpin reporter probe were designed.And the conversion from Vibrio parahaemolyticus concentrations to visualized signals was achieved by using the catalytic activity of the G-quadruplex DNAzyme.3.A rapid detection method for Vibrio parahaemolyticus was established and optimized,which had good linear range,low detection limit,and could be applied to the detection of real samples.