Study On The Application Of Hybridization Chain Reaction-mediated Signal Amplification Strategy In Detection Of E.coli O157:H7

Escherichia coli O157:H7 is an important foodborne pathogen,which can cause fever,nausea,vomiting,abdominal colic,diarrhea,and other symptoms after infecting humans,and even lead to hemorrhagic diarrhea or hemolytic uremic syndrome in severe cases.According to the research report of the World Health Organization,the outbreak of the epidemic is caused by eating meat products,dairy products,fruit and vegetable products and other foods contaminated by E.coli O157:H7.Therefore,the establishment of accurate and effective detection methods is of great significance for the monitoring and prevention of food poisoning caused by E.coli O157:H7.Hybridization chain reaction is a nucleic acid isothermal signal amplification technology,which alternately hybridizes the target chain with two hairpin probes to amplify the nucleic acid signal without enzyme catalysis.HCR was widely used in the field of detection and diagnosis because of its simple operation,flexible design,and combining different signal output strategies.In this study,based on HCR nucleic acid signal amplification technology,combined with a novel type of blocker primers for the detection of E.coli O157:H7 in milk,the contents of each chapter are as follows:The first chapter summarizes the research progress of the application of hybridization chain reaction-mediated signal output strategies in the field of detection.In the second chapter,a PCR-HCR dual-signal amplification strategy was developed for the detection of E.coli O157:H7 in milk.The method uses E.coli O157:H7 specific gene fliCh7 design primers with forward blocker primers modified with oxyethylene glycol and single strand DNA fragments and reverse primers modified with biotin at the 5' end.Amplification of the target gene using modified primers enables the generation of ss-dsDNA-Bio products,followed by the capture of PCR products using magnetic beads modified with streptavidin on the surface,resulting in the formation of ss-dsDNA-Bio+SA?MBs compounds.Finally,ssDNA was used to open two hairpin probes(H1/H2)to initiate HCR,thereby obtaining many long dsDNA,by adding DNA double-stranded binding dye to produce fluorescence signals to achieve detection.In this chapter,the feasibility of the method was verified by agarose gel electrophoresis and laser confocal microscope imaging,the reaction conditions were optimized,and the sensitivity and specificity of the method were evaluated.Under optimal conditions,the minimum limit of detection for E.coli O157:H7 in pure bacterial solution was 7.2 ×101 CFU/mL,which was 100 times more sensitive than that without HCR amplification,and the minimum LOD for E.coli O157:H7 in milk spiked samples was 7.2 ×102 CFU/mL,and the specificity of the method was good.In the third chapter,a homogeneous detection system of HCR combined with polydopamine was proposed for sensitive detection of E.coli O157:H7.In this method,a hairpin blocker primer is designed based on chapter 2 blocker primers,which can be amplified by PCR to form ss-dsDNA products,and the hairpin probes designed according to the ssDNA sequence for HCR signal amplification.Using the difference in the adsorption force of PDA on ssDNA and dsDNA and the effect of quenching fluorescence,excess primers and hairpins are adsorbed,thereby establishing a homogeneous fluorescence detection method for E.coli O157:H7.In this chapter,PDA was first obtained by self-polymerization of dopamine under alkaline conditions and compared with graphene oxide and zinc oxide to verify the strong anti-interference properties of PDA,to optimize important parameters such as PDA reaction concentration and HCR reaction time,and to evaluate the sensitivity and specificity of the method.Under the optimal conditions,the minimum LOD of E.coli O157:H7 in pure bacterial solution was 7.2 × 102 CFU/mL,and the minimum LOD of E.coli O157:H7 in milk spiked samples was 7.2 × 103 CFU/mL,and the specificity of the method was good.