Study On PCR And ELISA Methods For Detecting Pathogenic Microorganisms By Magnetic Nanoparticles

Food-borne pathogens is one of the most frequently factors in food safety throughout the world. To control the disease caused by pathogenic microorganism, it is important to make a rapid and accurate detection of pathogenic microorganism in food. The traditional test methods based on biochemical tests need complicated methods and get results slowly, which can't meet the demands in modern society. It is necessary to establish an accurate and rapid detection method for this pathogen. In our study, Polymerase Chain Reaction(PCR)and ELISA methods by magnetic nanoparticles were developed.In PCR methods, the genomic DNA was extracted by using different Fe3O4 magnetic nanoparticles with three functional groups: carboxyl group, hydroxyl group and amino group, and the results were comparised. The genomic DNA extracted from the bacteria was used as the template, a PCR and multiplex-PCR methods for Salmonella, Staphyloccocus aureus and Shigella were developed. The agarose gel electrophoresis and DHPLC was applied in the detection of the result of PCR. The multiplex-PCR-DHPLC method in milk sample was 102cfu/mL, and the process was finished in 2hours.In ELISA detection method, the monoclonal antibodies were prepared with a high titer of the specific antibody to the E. coli O157:H7 on the bacterial surface. The magnetic nanoparticles were modified with monoclonal antibodies, E. coli O157:H7 was captures by immunomagnetic beads and the efficiency was 90%-92%. It developed a monoclonal sandwich ELISA for the detection of E. coli O157:H7 with a good specificity. The limit of detection was 105cfu/mL in pure culture, and there was a linear range in the concentration range of 5×105 cfu/mL to 1×10~8 cfu/mL. After E. coli O157:H7 in tea sample was captured by immunomagnetic nanoparticles, it was detected by the monoclonal sandwich ELISA, 1cfu/mL E. coli O157:H7 can be detected in artificially contaminated green tea samples with 6.5h.