Study On Application Of Fluorescent Signal Amplification Methods Based On Hybridization Chain Reaction In The Detection Of Salmonella

Salmonella,one kind of foodborne pathogen widely existed in meat,milk,eggs and vegetables,was listed as the second common causes of food poisoning in the world by European Center of Disease Prevention and Control.Salmonella has been reported to have 2500 serotypes,which are characterized by nausea and vomiting,abdominal pain,diarrhea,headache and fever,etc.Hence,it is of great importance to establish rapid and sensitive detection methods for achieving effective monitoring of Salmonella.Beacause of simple operation,prominent specificity and high sensitivity,fluorescence amplification methods based on hybridization chain reaction?HCR?has been followed with interests of investigators.In this study,fluorescent biosensors based on HCR were established in order to realize the rapid and sensitive detection of Salmonella in food,and to provide new technical means for the monitoring system of foodborne pathogens in China.The contents of each chapter are described as follows:The first chapter reviews the research progress of nucleic acid hybridization mediated fluorescence signal amplification methods in biological detection.The second chapter established two kinds of novel methods based on HCR and magnetic beads for the detection of Salmonella.These methods used biotin labeled DNA probe?bio-probe?to fix target single-stranded DNA?ssDNA?from asymmetry polymerase chain reaction on the surface of streptavidin-magnetic beads?SA-MBs?,and ssDNA triggered two DNA hairpin probes?H1/H2?to generate SA-MBs^ssDNA^H1H2n polymer by HCR,moreover,fluorophore modification and fluorescent dye labeling were used to achieve the fluorescence signals output respectively.This chapter chose inverted fluorescence microscope to verify the feasibility of this methods,and the important parameters of the two methods were optimized.Under the optimal conditions,the detection limit of Salmonella reached10¹ CFU/mL in PBS,and the detection limit of Salmonella reached 10² CFU/g or 10²CFU/mL in food matrix,and good specificity has been observed.The third chapter established homogeneous fluorescence detection method based on HCR combined with graphene oxide?GO?for rapid and sensitive detection of Salmonella.In this study,the ssDNA was obtained by polymerase chain reaction?PCR?,PCR product purification and high temperature denaturation,and a pair of6-carboxyfluorescein?FAM?modified DNA hairpin probes which could be triggered with ssDNA were designed based on ssDNA sequence.A homogeneous fluorescence detection method was successfully constructed based on the difference between ssDNA and dsDNA in affinity and the fluorescence quenching properties of GO.This chapter optimized the important parameters of the method,such as the concentration of GO,the incubation time of GO and HCR time.Under the optimal conditions,this method was successfully used for the detection of Salmonella,and the detection limit reached 4.2×10¹ CFU/mL in PBS,and the detection limit of Salmonella reached 4.2×10² CFU/mL in milk,and good specificity has been observed.