Study On Rapid Detection Of Salmonella And Escherichia Coli O157:H7 Based On Gold Nanoparticle And Dynamic Light Scattering Technique And Design Of Food Safety Inspection Vehicle

In recent years,food contamination caused by foodborne pathogens has become one of the major issues in food safety.Therefore,it is of great significance to establish simple,rapid and sensitive detection assays for foodborne pathogen detection.As a conventional nanoparticle analysis method,dynamic light scattering(DLS)technology has attracted more and more attention of researchers due to its advantages of sensitive detection,low cost,no separation,easy operation,and easy data analysis.The DNA from common foodborne pathogens of Salmonella and Escherichia coli O157:H7 were used for the synthesis of gold nanoparticles(AuNPs)probes to capture efficiently of the target DNA,and then combined with DLS technology to achieve rapid and sensitive detection of two target bacteria in milk.In order to further realize the detection of actual samples,a new food safety inspection vehicle program was designed on the basis of traditional food rapid inspection vehicles with serial reforms to provide an immediate,on-site inspection platform for food safety.Each chapter is described as follows:The first chapter summarizes the research progress of the rapid and sensitive detection of target by using AuNPs-DLS assay,and the application progress of the food safety inspection vehicle in the basic food safety inspection.In the second chapter,a rapid AuNPs-DLS method for the detection of Salmonella spp.in milk sample base on conventional base-pairing was developed.In this study,primers and probes from Salmonella specific invA were designed,and a large amount of single-stranded DNA(ssDNA)was prepared by asymmetric PCR(aPCR).The AuNPs probe captures the target ssDNA through conventional base-pairing and caused a obvious change of hydrodynamic diameter in the presence of target ssDNA.On the contrary,the AuNPs probe maintains a relatively dispersed state with a uniform hydrodynamic diameter in the absence of target ssDNA.The changes in hydrodynamic diameter of AuNPs were monitored for the detection of Salmonella.Under the optimum reaction conditions(NaCl of 0.6 mol/L and pH=6),the results showed that the limit of detection for Salmonella in milk was 2.6×10~1CFU/mL.The recovery rate with a range from 92.9%to 102.6%were carried out for detection the target bacteria with a range of concentrations of 10~1to 10~6CFU/mL.And showed good specificity for the detection of 7 common pathogenic bacteria in milk.In the third chapter,a rapid AuNPs-DLS method for the detection of E.coli O157:H7 in milk sample base on the special DNA structure of“triplex formation”was developed.In this method,a large amount double-stranded DNA(dsDNA)from E.coli O157:H7 was first prepared by PCR.Compared to ssDNA preparation by aPCR,the greatly improved preparation efficiency of dsDNA was obtained.The AuNPs probe aggregation based on binding with target dsDNA to forming a"triplex formation"by Hustin base pairing.On the contrary,the AuNPs probe maintains a relatively dispersed state in the absence of target dsDNA.The hydrodynamic diameter of AuNPs was determined by using DLS to detect E.coli O157:H7.Under the optimal reaction conditions,the limit of detection for E.coli O157:H7 was reached to 10~1 CFU/mL.A good specificity of this developed assay was proved by the cross-reactivity of the non-target bacteria.In Chapter four,a product design program of new food safety inspection vehicle was completed and make a successful application for this established rapid detection method.The design idea is:based on laboratory standardization requirements,completed the whole processing including grass-roots food sampling,testing,data processing with a good mobility,rapidity,and applicability in basic level detection.This food safety inspection vehicles design focuses on discussion the main functional units,such as vehicle infrastructure,experimental operating areas,sample and waste storage area,Water and electrical facilities,and data processing facilities,and to meet the requirements of rapid,accurate and economical testing,and to provide a favorable detection platform for basic level food safety protection.