Establishment And Evaluation Of Detection Methods For Vibrio Parahaemolyticus

Objective:Food poisoning caused by foodborne pathogenic bacteria has been serious public health concern and Vibrio parahaemolyticus is one of the foodborne pathogenic bacteria that are mainly monitored in China.At present,the detection methods of Vibrio parahaemolyticus are complicated and time-consuming.Due to the complicated composition of the food sample they were difficult to meet the sensitive detection requirements.Development of rapid,accurate and reliable methods for Vibrio parahaemolyticus detection in food samples is beneficial to timely find out the contamination of Vibrio parahaemolyticus and prevent,control foodborne diseases caused by Vibrio parahaemolyticus.This thesis aims to construct sensitive and accurate methods for Vibrio parahaemolyticus detection as well as evaluate the detection effect of the established methods.Eventually,the work of this thesis can provide theoretical basis and new detection method for the monitoring of foodborne pathogenic bacteria in food samples,and provide technical support for ensuring food safety in our country.Methods:Two novel methods were established for Vibrio parahaemolyticus detection.One is based on immunomagnetic separation technique combined with manganese dioxide nanoparticles to form magnetic beads-bacteria-manganese dioxide nanoparticles immune sandwich complexes.The complexes were separated by an external magnetic field,then the products were reduced by ascorbic acid and added to gold nanoparticles colorimetric system.If the sample contains Vibrio parahaemolyticus,the manganese dioxide nanoparticles attached to the target bacteria can be reduced by ascorbic acid to produce abundance of Mn²⁺,which were made the gold nanoparticles colorimetric system turned to blue;Otherwise,if there was no Vibrio parahaemolyticus,the manganese dioxide nanoparticles would be washed away,the gold nanoparticles colorimetric system was remained red.Above all,a novel visualization colorimetric method was established for Vibrio parahaemolyticus detection.The other was based on molecular imprinting technology for Vibrio parahaemolyticus detection.Firstly,Vibrio parahaemolyticus was used as bacteria imprinted template and polydimethylsiloxane（PDMS）was used as polymer monomer.Then the imprinted films were modified with the fluorosilicate layer on the surface by chemical vapor deposition method to enhance the selectivity of the imprinted films.The prepared imprinted polymer films were used as a device for identifying and capturing Vibrio parahaemolyticus.Then the captured Vibrio parahaemolyticus DNA were extracted by boiling method and polymerase chain reaction（PCR）was carried out to realize rapid and accurate detection of Vibrio parahaemolyticus in the sample.Results:Based on the immunomagnetic separation technique method and under the optimized detection conditions,the response signals linearly correlated with the concentration of Vibrio parahaemolyticus over the ranges of 10¹0⁶ CFU·mL^-1.As the concentration of Vibrio parahaemolyticus increased,the aggregation degree of the gold nanoparticles gradually increased,the color of the gold nanoparticles solution gradually changed from red to purple and finally to blue.A linear correlation existed between the absorbance of gold nanoparticles solution at 525 nm and the concentration of Vibrio parahaemolyticus from 10 to 10⁶ CFU·mL^-1.The equation of linear regression was A_norm=-0.128lgN+0.893,R²=0.997.The limit of detection was 10 CFU·mL^-1,the relative standard deviation and the recoveries were 6.48%⁹.65%（n=3）and 95.78%¹01.83%,respectively.The specificity evaluation results were satisfactory.The method was applied to detect fresh oyster samples,the range of Vibrio parahaemolyticus was10 to 10⁶ CFU·mL^-1.The proposed method can be completed within 45 min,allows direct analysis of the samples by the naked eyes without large instrument,which can meet the requirement for rapid detection of Vibrio parahaemolyticus.Based on the molecular imprinting technology of the established method,the surface of the prepared imprinted polymer films was consistent with the size and morphology of Vibrio parahaemolyticus by atomic force microscopy（AFM）characterization.When the imprinted polymer films were incubated with Vibrio parahaemolyticus for 2 h,the amount of Vibrio parahaemolyticus captured by the imprinted polymer film could reach 3.7×10⁷ CFU·mL^-1.The method had a detection range from 10⁴ to 10⁸ CFU·mL^-1 for Vibrio parahaemolyticus and the limit of detection was 10⁴ CFU·mL^-1.The established method showed good specificity.The method has been used for the detection of oyster samples,which imply that the setup method can be used for food matrices analysis.Conclusion:1.The method based on the immunomagnetic separation technique for Vibrio parahaemolyticus detection was combined the high enrichment efficiency of immunomagnetic beads and the sensitive signal amplification of gold nanoparticles,avoid the complicated pre-enrichment process.2.The method based on the molecular imprinting technology for Vibrio parahaemolyticus detection proposed a novel imprinted polymer film,by modifying the surface with fluorosilicate layer,the selectivity of molecular imprinted polymer films to Vibrio parahaemolyticus were improved.3.Two methods for Vibrio parahaemolyticus detection were successfully established,that is based on immunomagnetic separation technique and molecular imprinting technology for Vibrio parahaemolyticus detection respectively.Both of the methods showed good specificity,sensitivity,low cost,simple and time-saving.4.Both the two methods mutually complement,showed widely detection range,applicable to different detection conditions,convenient for popularization in the base layer detection work.