Research On Rapid Detection Of Foodborne Pathogen Based On Immunomagnetic Separation And Aptamer Technology

Foodborne pathogens cause outbreaks of foodborne diseases,which pose a threat to human health.It is urgent to set up monitoring system and develop rapid,specific and sensitive detection methods for the prevention of their cross contamination in foods and.Traditional microbiological method is time-consuming and laborious,which can not fulfill requirement of the modern food industry for real-time monitoring of microorganism.Rapid detection methods could be devided into two categories:(1)molecular biology-based detection method,(2)specific recognition-based detection method.The goal of this dissertation was to develop rapid methods for detection of foodborne pathogens(Escherichia coli O157:H7,Staphylococcus aureus,Salmonella Typhimurium and Shigella sonnei),which is follows:(1)Development of an immunomagnetic separation-propidium monoazide-polymerase chain reaction assay with internal amplification control for rapid and sensitive detection of viable E.coli O157:H7 in milkImmunomagnetic separation(IMS)was used to capture E.coli O157:H7.Then propidium monoazide(PMA)was used to treat E.coli O157:H7,and inhibit the PCR amplification of DNA from dead cells,of which cell membrane was disrupted,and IAC was incorporated into the system to eliminate false negative results from the PCR inhibitors in the food matrix.When the concentration of cells increased from 102 to 107 CFU/m L,the capture efficiency was reduced from 100% to 87%.PMA could effectively inhibit the PCR amplification of DNA from dead cell,of which cell membrane was disrupted.The detection limit of this assay was 2.1×102 CFU/m L for pure culture.The IMS-PMA-PCR with IAC showed a detection limit of 5×103 CFU/m L in spiked milk.(2)Rapid and accurate detection of viable E.coli O157:H7 in milk using a combined IMS,sodium deoxycholate,PMA and real-time quantitative PCR processTo eliminate the PCR amplification of DNA from dead cells(intact cell membrane and disputed cell membrane),q PCR method combined with sodium deoxycholate(SD)-PMA treatment was developed to rapid and sensitive detect E.coli O157:H7.The optimal SD concentration and the optimal incubation time with SD were recorded at 0.1% and 20 min,respectively.The number of bacteria survivors using plate counts was close to that using SDPMA-q PCR assays.However,q PCR or PMA-q PCR method was used to enumerate the number of bacteria survivors,which showed high values.The detection limit of this IMS-SD-PMA-q PCR assay was 101 CFU/ m L for pure culture.The IMS-SD-PMA-q PCR showed a detection limit of 102CFU/m L in spiked milk.(3)Development of an SD-PMA-m PCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp.,Shigella spp.and S.aureus in food productsTo eliminate the PCR amplification of DNA from dead cells(intact out cell membrane and disputed out cell membrane),m PCR method combined with SD-PMA treatment was developed to rapidly and sensitively detect the target bacteria S.Typhimurium,S.sonnei.and S.aureus.After optimization,the detection limits of SD-PMA-m PCR assay were 6.8×104,7.9×103 and 5.8×104 CFU/m L for S.Typhimurium,S.sonnei.and S.aureus in pure culture,respectively.The detection limits of this assay for S.Typhimurium,S.sonnei,and S.aureus in spiked milk or ground beef were 101 CFU/m L or 101 CFU/g after 15 h enrichment.(4)QCM-based aptamer selection and detection of S.TyphimuriumQCM sensor is a highly sensitive instrument for mass detection,which make it possible to select aptamers and track the affinity of ss DNA pool in selection process.After eight rounds of selection and two rounds of counter-selection.Three aptamer candidates A24,B5 and B30 were obtained.Aptamer-based QCM method was developed to detect S.Typhimurium using the best aptamer B5.This assay could detect 103 CFU/m L of S.Typhimurium within 1 h.(5)An aptamer-based PCR method coupled with immunomagnetic separation for sensitive detection of S.Typhimurium in ground turkeyA rapid,sensitive method was developed to detect S.Typhimurium.IMS was used to capture S.Typhimurium from food samples.Aptamer B5 was added and incubated with S.Typhimurium.The aptamer B5 binding on the surface of S.Typhimurium was recycled for PCR.After optimization,the capture efficiency of immunomagnetic beads almost reached90%.This assay was able to detect 102 CFU/m L of S.Typhimurium in pure culture,and 103CFU/m L of S.Typhimurium in ground turkey.