Biotin Exposure Based Magnetic Separation Method Establishment And Research On Viable Listeria Monocytogenes Detection

L.monocytogenes has been identified as one of the severe foodborne pathogens,which is a serious threat to human's health.The WHO Food Safety Work Program has included L.monocytogenes as key foodborne pathogens which should be closely monitored.L.monocytogenes can survive at wide pH range,high salt concentrations and grow at refrigeration temperatures.Thus,making control of L.monocytogenes in foods processing is challenging.Therefore,developing a rapid and sensitive method for L.monocytogenes detection is very essential to ensure food safety and protect health of consumers.Immunomagnetic separation(IMS)is a simple preenrichment method for foodborne pathogens rapid enrichment.However,traditional immunonanoparticles based IMS for L.monocytogenes enrichment usually use large amount of immunonanoparticles;therefore,the cost of the IMS is high.For the purpose of reducing the cost of IMS,we developed a biotin exposed and amplified magnetic separation method in this study.Combined with PMA-PCR and nucleic acid chromatography or flow cytometry,we could detect viable L.monocytogenes in lettuce samples.The main contents of this thesis are described as follows:In the first chapter,the research progress of application of magnetic nanoparticles in foodborne pathogen detection was reviewed.In the second chapter,biotin exposure and polyamidoamine(PAMAM)dendrimers mediated biotin amplified magnetic separation methods were developed.In biotin exposed magnetic separation method,biotinylated antibodies(biotin-mAb)were premixed with L.monocytogenes and anchored on the L.monocytogenes makes biotin exposed on the bacteria surface.Then streptavidin coated magnetic nanoprticles(MNP-SA)were added for capture of biotinylated antibodies labeled L.monocytogenes.Under this optimum conditions,the capture efficiency of biotin exposed magnetic separation method was more than 91.8%±1.3% when the concentration of L.monocytogenes was lower than 104 CFU/mL both in PBS buffer and lettuce samples.Compared with traditional immunonanoparticles based IMS method,the amount of antibody used in this method was reduced 10 times.In PAMAM dendrimers mediated biotin amplified magnetic separation methods,biotin amplified strategy can further reduce dosage of antibody,shorten incubation time between MNP-SA and biotin-PAMAM-mAb labeled L.monocytogenes and reduce the usage amount of MNP-SA.Under the optimum conditions,more than 89.15% ± 1.75% L.monocytogenes were captured based on the PAMAM mediated biotin amplified magnetic separation method when the concentration of L.monocytogenes was lower than 104 CFU/m L both in PBS buffer and lettuce samples.Compared with traditional immunonanoparticles based IMS method,the amount of antibodies,and MNP-SA were reduced 20 times and 40%,respectively,and the whole incubation time shorten by half.In the third chapter,PMA-PCR based viable L.monocytogenes detection methods were built combined with flow cytometry or nucleic acid chromatography strip method.When the amount of PMA was 10 ?g/mL and exposed to bright light for 5 min,PMA could restrict the PCR amplification of dead L.monocytogenes and there was negligible influence for viable L.monocytogenes PCR amplification.Based on PAMAM dendrimer-mediated biotin amplified magnetic separation method,combined PMA-a PCR and nucleic acid chromatography strip for viable L.monocytogenes detection,the limit of detection was 3.5×104 CFU/g in lettuce samples.Based on PAMAM dendrimer-mediated biotin amplified magnetic separation method,combined PMA-PCR with flow cytometry for viable L.monocytogenes detection,the limit of detection was 3.5×10~2 CFU/g in lettuce samples.