Study On The Conditions Of Detecting Pathogenic Bacteria Based On RAFT Preparation Of DHPLC Stationary Phase

In recent decades,food safety issues caused by outbreaks of food-borne diseases have always been a hot issue worldwide.Controlling and reducing the outbreak of food-borne diseases is one of the issues that must be resolved in the food sector.The main cause of food-borne disease outbreaks is the infection of pathogenic bacteria.The infection of pathogenic bacteria has been constantly changing from traditional plate count detection methods to immunological technology detection,to molecular biology technology detection and combined detection technology methods.The improvement,from time-consuming and laborious to fast and efficient improvement.Although the current detection methods can meet the purpose of rapid detection and provide more effective methods for the rapid detection of pathogenic microorganisms in food in the food industry,this topic applies the thermal stability of non-imprinted materials to the stationary phase of denaturing high performance liquid chromatography.In this paper,a method combining polymerase chain reaction technology and denaturing high performance liquid chromatography was established to be used for rapid detection of food-borne pathogenic bacteria.In the research of this subject,in order to achieve rapid detection of pathogenic bacteria,this experiment proposes a method based on RAFT polymerization with porous silica as the carrier,and styrene and 2-VP reagents as functional monomers.Azobisisobutyronitrile（AIBN）is the initiator,divinylbenzene（DVB）is the crosslinking agent,1-butyl-3-methylimidazole tetrafluoroborate（BF₄）is the porogen and chain transfer reagent（RAFT reagent）and other non-imprinted materials prepared by polymerization reaction as the stationary phase of denaturing high performance liquid chromatograph（DHPLC）,established PCR-DHPLC method for rapid detection of pathogenic bacteria,this method can achieve rapid,efficient,and specific effects.The main contents are as follows:（1）In Chapter 2,the experiment is to prepare non-imprinted materials by the RAFT method.Two functional monomers,namely styrene and 2-VP,are combined with the carrier in the initiator,crosslinking agent and chain transfer reagent.Polymerization takes place at temperature and time.Analyze the non-imprinted material polymerized by RAFT by infrared characterization and analyze whether there are main groups of functional monomers on the carrier,perform electron microscopic characterization to analyze whether the pores of the material are uniform,and perform thermogravimetric analysis of the thermal stability of the non-imprinted material.The polymerization effect and stability of the polymer material polymerized by this method.（2）In Chapter 3,the experiment content is divided into two parts.The first part is to establish a multiplex PCR method to detect Salmonella typhimurium.In order to save testing time and achieve rapid detection of multiple virulence genes of Salmonella typhimurium,the experiment was conducted through primer design,primer screening and synthesis of primers for virulence genes of Salmonella typhimurium,and established multiple PCR technology based on conventional PCR technology for detection of Salmonella typhimurium Detection of virulence genes of Salmonella typhimurium virulence island.Through the experiment,the mgt C,mog A,ara B genes were screened out from mgt C,mog A,bcf A,inva,and ara B genes for multiplex PCR reaction.The response surface experiment was used to optimize the amount of primers,the amount of template added,and the annealing temperature to determine the multiplex PCR technique to detect Salmonella typhimurium.The detection limit of this method for Salmonella typhimurium is0.0165 ng/μL.The second part is the combination of multiple polymerase-linked reaction technology and denaturing high performance liquid chromatography technology,using the non-imprinted polymer material synthesized in Chapter 2 as the stationary phase to establish a denaturing high performance liquid chromatography method for rapid detection of Salmonella typhimurium Method,and improve the detection effect of the method by optimizing the ratio of mobile phase,and finally obtain a new detection method that is fast,efficient and specific.（3）In Chapter 4,an experimental summary of the whole research.In this study,it was determined that using Styrene as a functional monomer and porous silica support to conduct RAFT polymerization,a non-imprinted material with good thermal stability was obtained.In order to save time and achieve rapid detection results,a multiplex PCR technique was established based on the single PCR method.The method for detecting multiple virulence genes of Salmonella typhimurium has strong specificity and good sensitivity.Combine multiplex PCR technology with denaturing high performance liquid chromatography,and establish a PCR-DHPLC technology for rapid detection of food-borne pathogenic bacteria,which can achieve rapid,efficient,and specific effects.