Establishment And Application Of Rapid Detection Method Of Escherichia Coli O157:H7 By Paper-based Sandwich ELISA

Enterohaemorrhagic escherichia coli(EHEC)plays an important role in foodborne pathogenic microorganisms.EHEC mainly includes O157:H7,O26:H11 and O111 serotypes,among which O157:H7 is the typical strain.O157:H7 infection can induce hemorrhagic colitis(HC),appendicitis,esophageal stenosis and severe gastrointestinal complications,colon perforation.It can also cause hemolytic uremic syndrome(HUS)and thrombotic thrombocytopenic purpura(TTP)and other serious complications in children and the elderly,and the serious can cause death.Each year,more than 2 million acute illnesses worldwide are attributed to E.coli O157:H7.Various techniques have been developed to detect this foodborne pathogen,such as bacterial culture,polymerase chain reaction,electrochemistry,and surface plasmon resonance.However,they are time-consuming,require professional equipment and complicated operation processes.Therefore,it is necessary to establish a sensitive,reliable and easy-to-detect method for the detection of O157:H7.O157:H7 is becoming more and more harmful.In order to establish a rapid and sensitive detection method,this study established a specific monoclonal antibody against O157:H7 serotype.A double antibody sandwich ELISA method was established.A paper-based sandwich ELISA(P-ELISA)method for rapid detection of O157:H7 was developed.Compared with conventional ELISA,P-ELISA has the advantages of low cost,fast detection,convenient portability,and can be used for on-site and timely detection.1.Establishment and application of a double-antibody sandwich ELISA method for the detection of E.coli O157:H7The two hybridoma cells 2E3 and 2G5 secreting anti-E.Coli O157:H7 monoclonal antibodies prepared in the previous stage were separately resuscitated to prepare ascites.After ascites purification,the titer of 2G5 antibody was 1:512000,with 2E3 antibody 1:256000.In order to establish a double-antibody sandwich ELISA method for the detection of E.coli O157:H7,2G5 was as the capture antibody,and enzyme-labeled monoclonal antibody 2E3(HRP-2E3)as the detection antibody.Optimized the reaction conditions.The results of square method showed that 5.75 ?g/ml was the optimum coating concentration of antibody 2G5,and 20.3 ?g/ml was the best detection concentration of HRP-2E3.Determining the optimal reaction conditions was that the capture antibody coated overnight at 4?,5%skimmed milk closed for 1.5h at 37?,antigen and capture antibody combined for 1.5 h at 37?,detection antibody and antigen combined at 37? for 45 min,and the optimal action time of the TMB chromogenic solution was 15 min.And this method had no cross-reaction with other serotypes of E.coli and non-E.Coli such as Salmonella,Listeria,which showed high specificity.The limit of detection(LOD)of E.coli O157:H7 reached 105 CFU/mL,with good sensitivity.Variable coefficient of intra-and inter-plate were all less than 7%showing high precision.The method was used for the detection of beef samples,which was compared with the national standard method and the conventional molecular biological method.LOD of E.coli O157:H7 in artificial contamination of beef samples was 1 CFU/25g after enrichment for 8h.It was determined that this method was superior to molecular biology method in detection sensitivity and was superior to national standard method in detection time and cost.2.Application of paper-based microfluidic chip and sandwich ELISA detection methodEscherichia coli O157:H7 is a severe foodborne pathogen that causes lots of lifethreatening diseases.To search for a rapid,sensitive,portable and low-cost method detecting this pathogen,we developed a wax-printed paper-based enzyme-linked immunosorbent assay(P-ELISA)based on microfluidic paper-based analytical device(?PADs),with the whole operation time less than 3 h and only needing 5 ?l samples to detect.P-ELISA combines sandwich ELISA with paper-based microfluidic chip technology,with the whole operation time less than 3 h and only needing 5 ?l samples to detect,which can be used for rapid clinical detection.The optimal P-ELISA detection system was that the optimal working concentrations 2G5 and HRP-2E3 were 22.6 ?g/mL and 20.3 ?g/mL,respectively,both of which are diluted 1:100;2G5 antibody was coated at 4? for 30 min;10%BSA was added 5ul to each well to block for 20min;antigen combined with 2G5 for 30 min at 4?;HRP-2E3 combined with antigen for 20 min at 4?;the chromogenic solution was allowed to react at room temperature for 3 min.The whole detection time did not exceed 3h,which greatly shortened the detection time compared with conventional ELISA(C-ELISA).The limit of detection(LOD)of E.coli O157:H7 reached 104 CFU/mL,which is an order of magnitude higher than C-ELISA.LOD in artificially contaminated beef samples is 1 CFU/25g after enriched culture for 8 h.This method is superior to molecular biology method in detection sensitivity and superior to C-ELISA and national standard method in detection time and cost.Thus,the established P-ELIS A method has good sensitivity,specificity and repeatability.It can be suitable for pointof-care testing without expensive and bulky instruments and can also provide a platform for detecting other pathogens,especially in areas that lack advanced clinical equipment.