Detection Of Enterohemorrhagic Escherichia Coli O157: H7 By Immuno-PCR

E.coli O157:H7 is a pathogen that is transmitted through the food chain.It can result in haemorrhagic colitis,haemolytic uraemic syndrome. The mortality is very high. Although outbreaks have not occurred in our country,the bacteria have isolated throughout our country.So we must pay more attention to it.Traditional method for detecting O157:H7 are bacterial culture, serological test and PCR .But these methods need long time and have low sensitivity ,so it can't make rapid, accurate diagnosis and miss the opportunity of treatment. Therefore,it is critical whether the methods of detection is rapid,sensitive and specific.The purpose of this study was to set up a new technology–Immuno-PCR.This technology combined ELISA and PCR. DNA molecule which replaced the enzyme in ELISA was used as a marker of antibody.It formed the first antibody-antigen- second antibody-biotin IgG-biotin DNA complex through immune response.The bound biotinylated reporter DNA was detached from the plate by denaturing at 96℃in a hybridization oven.Then they were transferred to PCR tubes, amplified,electrophoresed.Finally, according to the presence and quantity of PCR products, antigen was detected qualitatively and quantitatively. The reaction process of the technology included the immune response and PCR amplification .In order to prepare the polyclonal antibody of E.coli O157:H7 as the detection antibody of immune-PCR,the bacterial antigen of E.coli O157:H7 was used to immunize the rabbit.As a result, the specific anti-serum was prepared by immunization for five times. The anti-serum dilution was detected using the chessboard titration,it had arrived to 1:25600 ,and had meet the experimental needs. Then the antibody was purified using saturated ammonium sulfate. The best concentration of O157:H7 monoclonal antibody and polyclonal antibody were also detected by chessboard titration,they were 1:1000 and 1:100.They provided a strong guarantee for the follow ELISA and immuno-PCR.To prepare the biotinylated DNA reporter,the biotin-labeled primers of the mouse genome were used to synthesize the GAPDH gene which was tagged biotin at one end. In order to link the DNA reporter to the antibody, streptavidin(STV) which has a specific affinity for biotin was used to link the biotinylated antibody and biotinylated DNA. In this study, the goat anti-rabbit biotinylated antibody IgG, STV and biotinylated DNA reporter were added step by step to exclude the possibility which biotin can saturate streptavidin binding sites.Biotinylated DNA and STV concentrations are the key factors which can affect the non-specific bind of immuno-PCR. In this study, the optimal concentrations of biotinylated DNA and STV were determined by the chessboard titration.They were 1g/ml and 100ng/ml.If the concentration was too high,it would lead to the non-specific binds and false positive,if the concentration was too low, the sensitivity of immuno-PCR would decline. In order to determine the sensitivity of immuno-PCR to detect E.coli O157:H7, O157:H7 were diluted serially,then sonicated on ice for 30s to break the cell.Finally they were added into the ELISA plate which were coated by O157:H7 monoclonal antibody. The results showed that the minimum detection limit of sandwich immuno-PCR was 10CFU/ml.It enhanced 1000 times than the sandwich ELISA .To define the specificity of the method,we used E.coliO26:H11, E.coliO5:NM, E.coliATCC23716, Staphylococcus aureus, Proteus, Streptococcus pneumoniae , Pseudomonas aeruginosa and beta-hemolytic streptococcus in place of E.coli O157:H7 and performed the same immuno-PCR assay. All of these showed negative results,indicating there was not cross reaction.It indicated that immuno-PCR had high specifity. Fifty one strains of O157:H7 which were isolated from the food, animal fecal and the stools of the diarrhea patients were used as samples,then they were detected by immuno-PCR , the results showed that they all produced the specific DNA bands.So this technique was viable for detecting O157:H7 which were isolated from samples.E.coli O157:H7 are transmitted by food chains. Beef and milk are the important source of infection. The contamination level of E.coli O157:H7 in ground beef is quite low, but low infectious dose can be pathogenic. In this study,we inoculated the ground beef and pasteurized milk with different concentrations of O157: H7,after eight hours culture, the detection limit can achieve 10-3 CFU/ml.It can meet the requirements of the food test.In summary,Immuno-PCR has high sensitivity and specificity. It doesn't need special equipments and reagents,it can operate in the routine laboratory. The establishment of this method for diagnosing Escherichia coli O157: H7 provides a new path.