Rapid Detection Of Food-borne Pathogens Based On Aptamer Functionalized Scattering Probe And Circular Strand Displacement Polymerization Reaction

In this age of globalization,illnesses caused by contaminated food are increasing in numbers and degrees which brings unceasing threats to people's health and socio-economic development,and became a significant public health issue worldwide.Due to its diverse categories,large production and wide distribution,food could be easily contaminated in each critical step of food chain,which means it is critical to develop reliable and rapid pathogen-detection methods for food safety.Along with the development of nucleic acid researches,functional nucleic acids such as aptamer,DNAzyme and molecular beacon were found having high affinity for targets like metal ion,protein,and cell surface.Functional nucleic acids were also advantaged from high reliability,easy synthesis and easy modifying which is reasonably applicable to food-borne pathogen detections.Two rapid detection methods which respectively based on aptamer-mediated bacterial cell recognizing and molecular beacon mediated bacterial specific nucleic acid recognizing have been established in this thesis.(1)The converted dark-field microscope based on 36XC monocular microscope was fabricated by assembling the dark-field condenser with the microscope as well as adding a height indicator.The aptamer sequences which were modified on 60 nm gold nanoparticles were used as scattering probe.The syntheses and modifications were characterized with UV-VIS spectroscopy,nanoparticle and Zeta potential analyzer and transmission electron microscope(TEM).As the TEM pictures revealed,the scattering probe specifically combined with target S.aureus only if the target cells were alive.Dark-field microscopy in this method can be classified into direct dark-field microscopy and indirect dark-field microscopy,and both two methods were applied using a 10×object lens.In direct dark-field microscopy,the botryoidal light spots with the diameter of about 1?m can be observed which indicated the existing of S.aureus.The indirect dark-field microscopy was processed by washing off the unbound gold nanoparticle scattering probes then eluting the bound scattering probes for observation.S.aureus in different concentrations were qualitatively determined using indirect dark-field microscopy with the detection limits of 4.6×10~6 CFU/m L.The procedures in indirect dark-field microscopy were then improved by replacing syringe filters with nucleic acid purification column which attributed to an improvement of the current limits by three orders of magnitude.This dark-field microscopy detection method can be applied in milk samples with a detection limit of 5.3×10~3 CFU/m L.(2)Molecular beacon mediated detections for S.aureus,Salmonella,Enterohemorrhagic Escherichia coli(EHEC),and Shigella were developed based on the principle of circular strand displacement polymerization reaction(CSDPR).The specific molecular beacon probes and primer were designed using NCBI database,DNAMAN multiple sequence comparison and Oligo 7 duplex analyzing.The optimal length of stem in molecular beacon probes is 8 nt while the optimal concentrations of probe and primer are both 200 nmol/L.The specificity of these four types of probes is determined,which revealed that the S.aureus probe can identify the species of S.aureus,the EHEC probe can identify EHEC only,meanwhile the Salmonella and the Shigella probe can identify the genus of the target bacterium.Genome DNA of these four types of pathogens was extracted,diluted into different concentrations and quantitatively determined by CSDPR in optimal conditions.The quantitative linear range of S.aureus was from 2×10~5 CFU/m L to 2.5×10~6 CFU/m L with the detection limit of 9.5×10~4 CFU/m L.The quantitative linear ranges of Salmonella,EHEC and Shigella were from 1.0×10~4 CFU/m L to1.0×10~6 CFU/m L,and the detection limits of these three pathogens are respectively 4.8×10~4CFU/m L,3.4×10~4 CFU/m L and 3.0×10~4 CFU/m L.The quantitative CSDPR method was applied in detection of S.aureus in steak samples and milk samples.The real sample tests revealed that the concentration requirements in national standard can be detected by a pre-enrichment within at least six hours.The average absolute relative deviation between the CSDPR quantitative results and plate count results in steak samples and milk samples are respectively 14.5%and 11.4%.