Study On Real-time Fluorescent Recombinase-aided Amplification For Rapid Detection Of Escherichia Coli O157:H7 In Milk

Escherichia coli O157:H7(Escherichia coli O157:H7,E.coli O157:H7)is a pathogenic Escherichia coli that produces Shiga-like toxins,human is infected by E.coli O157:H7 from contaminating food,which cause hemolytic uremic syndrome,hemorrhagic colitis,thrombotic thrombocytopenic purpura,and even permanent organ failure.Therefore,establishing a rapid and accurate detection method to monitor E.coli O157:H7 is of great significance to protect public health.E.coli O157:H7 mainly parasitic in the intestinal tract of ruminants(mainly cattle),milk and beef are easy to be contaminated by E.coli O157:H7.In this study,the detection method based on RAA(recombinase-aided amplification,RAA)technology was established with milk as the detection matrix to realize the sensitive and rapid detection of E.coli O157:H7 in milk.The contents of each chapter were as follows:Chapter one: This chapter reviews the application and research progress of RAA(recombinase-aided amplification,RAA)technology and live bacteria detection technology to provide theoretical guidance for the development of rapid and sensitive DNA amplification-based live bacteria detection methods.Chapter two: r RAA method was established to realize the rapid and sensitive detection of E.coli O157:H7 in milk.A pair of specific primers and corresponding fluorescent probes were designed based on the flagellin gene(flagellin,fli C)sequence of E.coli O157:H7 to amplifiy and detection.The agarose gel electrophoresis and measurements of fluorescent intensity were used to verify the feasibility of the proposed methods.The sensitivity and specificity was evaluated,the limit of detection(LOD)of the proposed methods for E.coli O157:H7 in pure culture or in milk was less than 10 CFU/m L,the detection was completed within 20 min at a constant temperature of 39℃,and there was no cross-reaction to other non-target bacteria in the experiment.Therefore,the proposed method is simple,rapid,and has good specificity for E.coli O157:H7 detection.Chapter three: The PMAxx-r RAA method was established,and the selective detection of viable E.coli O157:H7 was realized based on the difference of cell membrane integrity between live and dead bacteria.PMAxx only penetrates the damaged membrane of dead bacteria and inhibit DNA amplification.However,viable bacteria with intact membrane keeps PMAxx out.The measurements of fluorescent intensity and LSCM images were used to prove that PMAxx can permeate into dead bacteria.Some critical parameters have been optimized such as PMAxx concentration and exposure time,and also evaluated the LOD and specificity of the proposed methods.Under the optimized condition,the LOD of the proposed methods for E.coli O157:H7 is less than 10 CFU/m L in pure culture or in skimmed milk.And the PMAxx-r RAA can apply in skimmed milk in the presence of dead bacteria or nontarget bacteria,which indicated the proposed method can resist interference dead bacteria or nontarget bacteria.The proposed PMAxx-r RAA method was performed with simple,rapid,and sensitivity in E.coli O157:H7detection.,and has good specificity and capacity of resisting interference..Chapter four: We developed TOMA-r RAA to detect viable bacteria based on metabolic activity of cell.Thiazole orange monoazide(TOMA)is activity-labile nucleic acid-modifying compounds(ALCs),can penetrate bacterial cell membranes.and inhibit DNA amplification from dead bacteria.But the ester linkage of TOMA is cleaved by the active esterase that are found in metabolically active cells,DNA amplification is not suppressed.The feasibility of the proposed method has been verified via agarose gel electrophoresis,measurements of fluorescent intensity and LSCM images.Some critical parameters have been optimized such as TOMA concentration and exposure time,and also evaluated the LOD and specificity of the proposed methods.Under the optimized condition,the LOD of the proposed methods for E.coli O157:H7 is less than 10 CFU/m L in pure culture or in milk.And the TOMA-r RAA can apply in skimmed milk in the presence of dead bacteria or nontarget bacteria,which indicated the proposed method can resist interference from dead bacteria or nontarget bacteria.The proposed TOMA-r RAA method was performed with simple,rapid,and sensitivity in E.coli O157:H7 detection.,and has good specificity and capacity of resisting interference.In summary,the r RAA method was established in this study to achieve sensitive and rapid detection of E.coli O157:H7 in milk.In further studies,PMAxx and TOMA were respectively combined with r RAA,the selective detection of viable E.coli O157:H7 was realized.It provides a new reference scheme for rapid and sensitive detection of foodborne pathogenic bacteria and selective detection of foodborne pathogenic bacteria.