| Enterohemorrhagic Escherichia coli O157:H7(EHEC 0157:H7)is one of the important foodborne pathogens,which was found in recent years.It can spread to people through meat products with the result of infectious diarrhea.What's more,with the deterioration of severity,it can cause more serious diseases of hemorrhagic colitis(HC),hemolytic anemia(HA),thrombotic thrombocytopenic purpura(TTP)and hemolytic uremic syndrome(HUS).The death rate of these diseases are high and the dose of infection is extremely low.Human consumption of more than 10 to 100 CFU live bacteria can cause disease.Therefore,it is urgent to establish a rapid,accurate and highly sensitive detection method to prevent infection of EHEC O157:H7 from meat products.Recently,it was reported about the detection methods of EHEC O157:47 were mainly included selective medium separation identification,PCR detection,enzyme-linked immunosorbent assay(ELISA),serotyping detection,IMB separation,and so on at home and abroad.However,with the rapid development of food processing and transport,the traditional methods of incubating overnight to separate and enrich microbial pathogens have certain defects,such as spending long time,easy to disturbance,and false positives.Therefore,it is high time to combine the method of optimizing pretreatment of sample and the measurement technique of high sensitive,which to prevent the infection of meat products.The detection methods of EHEC O157:H7 were are prepared in this assay,such as preparation of IMB,detection methods of IMS-RT-PCR and preparation of LFM-IC strips.The detection methods with rapid speed,low cost,high sensitivity and high specificity were built to prevent diseases caused by EHEC O157:H7.Test ? Preparation and identification of McAb anti-EHEC O157:H7 O-SPthe o-spectific polysaccharides(O-SP)was prepared by acid hydrolysis method from phenol phase(smooth)of LPS by hot phenol-water method.The concentration of O-SP estimated by phenol-sulfuric acid method is 40 ?g/mL.The inactivated EHEC O157:H7 with concentration of 2×109 CFU/mL was used as whole-cell antigen.EHEC O157:H7(107 CFU/mL)and O-SP(2 ?g/mL)were used as the coating antigen respectively to screen positive putative hybrids by indirect-ELISA(i-ELISA)and E.coli 06 for the negative control(108 CFU/mL).The potency of McAbs was 1:106 in this assay and the concentration of McAbs anti-EHEC O157:H7 O-SP was 1.79 mg/mL after purification.The McAbs against EHEC O157:H7 O-SP in this assay had no cross-reaction with non-O157 E.coli strain or other bacterial strains which indicated that McAbs against EHEC O157:H7 O-SP was high specificity.These characteristics of McAbs was helpful to be applied in detecting technology.Test ? Rapid detection of EHEC O157:H7 in meat products by IMS-RT-PCR methodTo establish a method for rapid detection of Escherichia coli O157:H7(EHEC O157:H7)in meat products by immunomagnetic beads separation(IMS)and real-time fluorescence quantitative PCR(RT-PCR).The O-spectific polysaccharides(O-SP)of EHEC O157:H7 were extracted using the hot-phenol water method,and it was acted as the antigen to prepare monoclonal antibodies against EHEC O157:H7 O-SP.Monoclonal antibodies(McAbs)was coupled to magnetic nanoparticles(about 20 nm in diameter)which activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS),and the immunomagnetic beads(IMB)were prepared with anti-EHEC O157:H7 O-SP McAbs of 150 ?g per magnetic beads of lmg.The maximum of capturing EHEC O157:H7 of IMB(0.2 mg)was 13748.33 CFU,and the lowest detectable limit of IMB was 2.2 CFU.According to gene rfbE(Genbank login:S83460)that specific gene of serotype of EHEC O157:H7,the primers and fluorescence probes were designed.The method of combining IMS and RT-PCR was effective to enrich and detect EHEC O157:H7 in meat products.When the concentration of EHEC O157:H7 in meat products was higher than 1 CFU/g(meat of 2.5g),IMB could capture it,and RT-PCR could detect EHEC O157:H7 successfully after culturing 3h in EC broth culture-medium.The entire assay only took 6 to 7 hours.The detection method of IMS-RT-PCR has advantages of short time,high sensitivity and high specificity.It is of great significance to prevent the infection of EHEC O157:H7 from meat products.Test ? Preparation of Lanthanide-labeled fluorescent microspheres immunochroma--tographic stripsA simple and rapid detection method for enterohemorrhagic Escherichia coli O157:H7(EHEC O157:47)using lanthanide-labeled fluorescent microspheres immunochromatogra-phic strips(LFM-IC strips)was presented.Monoclonal antibody(McAb)against EHEC O157:H7 O-SP(McAb anti-EHEC O157:H7 O-SP)was coupled to lanthanide-labeled fluorescent microspheres which activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS).The immunochromatographic strips reader was based on a sandwich immunoreaction performed that basing on the binding between fluorescent McAb-bacteria and rabbit polyclonal antibody(PcAb)against EHEC O157:H7(PcAb anti-EHEC O157:H7)coated on the test line on nitrocellulose membrane(NC membrane)reaction zone.The strip reader was used as a quantitative system.In this study,and under optimal conditions,the visual limit of detection(LOD)of the strip was 104 CFU/mL and the detection limit was determined as 102 CFU/mL by using strip reader.The detection limit for EHEC O157:H7 was two orders of magnitude lower than those displayed by colloidal gold-labeled strips or ELISAs.No cross-reactions were observed with the other 11 non-EHEC O157:H7 strains that indicating the high specificity of LFM-IC strips.Good reactions with other 5 EHEC 0157 stains which isolated from the excrement of swine or pork that demonstated LFM-IC strips could be used for detecting these isolated strains of EHEC 0157 from different source.This study using the lanthanide-labeled fluorescent microspheres immunochromatographic strips(LFM-IC strips)were easy,rapid,sensitive and specific to perform.This method was a meaningful for the quantification of EHEC O157:H7. |
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