Rapid Detection Of E.coli O157: H7by Immunocapture And Real-Time Fluorescence LAMP

Enterohemorrhagic Escherichia coli (EHEC) O157:H7is found that it is a serious intestinalinfectious disease in recent years. Besides cause diarrhea, hemorrhagic enteritis, it can also happenhaemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), and otherserious complications. The later are ominous and have high case fatality rate. Now for the detection ofpathogenic microorganisms relies mainly on the pathogens isolated culture methods, immunologicalmethods and PCR methods. However, the above methods are time-consuming and low sensitivity andspecificity and high detection cost, which are not suitable for on-site rapid detection and not for basingto be popularized and applied. LAMP technology overcomes the drawbacks of conventionaltechnologies, which can specificly, effectively, and quickly amplify out109copies of the DNA targetsequence in constant temperature in1h. So it is more suitable for clinical pathogenic microorganismtesting requirements. But it often requires pre-enrichment step in actual sample testing,and the time forenrichment is generally in18~24h. So it has important practical significance in improving the methodfor sample processing and shortening the processing time.This study effectively combined the immunocapture and loop-mediated isothermal amplification(LAMP) technology. Firstly preparated the monoclonal antibody against E. coli O157: H7and itspolyclonal antibody, after identification and purification, carried out specific tests. The monoclonalantibody had good specificity, while the polyclonal antibody also achieved the same effect afterabsorpting by the bacterias which had cross reaction with serum. Make the two antibodies as captureantibody to be enveloped in ELISA plates to specificly capture the E. coli O157∶H7in the samples.Secondly using the LAMP method, through Primer Explorer V4to design LAMP primers, to detect thesurface antigen gene rfbE of E. coli O157: H7, and to discuss its specificity and sensitivity. Finallydetect the sensitivity of the artificial contaminative full-fat milk with E. coli O157: H7using theestablished method of immunocapture and Real-Time Fluorescence LAMP.This study screened one hybridoma cell which can stably secrete themonoclonal antibody against E.coli O157: H7, and named2BA12.At the same time, successfully developed the monoclonal antibodyand polyclonal antibody against E. coli O157: H7. After purification and identify the quality, the proteinconcentration of the anti-E. coli O157: H7McAb is15.125mg/mL, and the PcAb is12.463mg/mL.The titer of the McAb is1:80000by indirect ELISA and the titer of the PcAb is1:64000. The limit of E.coli O157∶H7detection byMcAb and PcAb immunocapture and Real-Time Fluorescence LAMPreaction are2.5CFU/mL and1.8CFU/mL, which consistent with the level of LAMP method detectionlimit. The preprocessing time for samples by immunocapture is generally about2.5h, so in the context that ensuring the LAMP method detection limit level, this method is significantly shortened thepreprocessing time and has advantages of double specificity. Finally using McAb as capture antibodyfor sensitivity test of the artificial contaminative full-fat milk, the detection limit is8.0CFU/mL.