| Foodborne illness is defined as any illness caused by eating food contaminated with pathogenic bacteria,viruses,parasites,or chemicals,which is a huge threat to human health,even life-threatening.According to the Chinese Center for Disease Control and Prevention,food poisoning caused by microbial accounts is about 45 to 55percent of foodborne diseases every year.The U.S.Food and Drug Administration(FDA)claims that there are about 48 million cases of foodborne illness in the United States each year.There are major 31 species of foodborne pathogens that can induce illness in our daily life,and Salmonella,Listeria,Staphylococcus,Escherichia coli,and Campylobacter are the most common bacteria.Usually,foodborne diseases are related to the mixing of several pathogenic bacteria.Therefore,simultaneous and accurate detection of various foodborne pathogens is one of the essential ways to effectively prevent and control the happening of foodborne diseases.Therefore,in this topic,a multiplex microsensor with small-hole resistance microsphere counting was constructed for detecting three common foodborne pathogens.The polystyrene microspheres(PM)which are cheap and stable were used as signal probes and read by the microsphere counting sensor to realize rapid qualitative and quantitative analysis of foodborne pathogens.Combined with superparamagnetic nanoparticles,DNA hybridization and streptavidin-biotin bio-amplifying signal system,this project is expected to achieve simultaneous,sensitive and efficient quantitative analysis of three foodborne pathogens.The specific research contents are as follows:1.Research of detecting Listeria monocytogenes based on resistance microsphere counting biosensorIn this chapter,the DNA molecular hybridization technology,oligonucleotide probes,and a resistance microsphere counting sensor were combined to construct a biosensor that can detect targets sensitively.The designed signal probe was decorated on 3μm polystyrene microspheres surface,and the capture probe was fixed on the surface of 1μm magnetic microspheres.When the target molecule presenting,a hybridization reaction could occur in the system,where the sandwich complexes were formed.After that,the magnetic separation was carried out by the magnetic microspheres,this step could divorce the PM-Target-MNP sandwich complexes from the unbound polystyrene microspheres.Thus,the number of PM-Target-MNP sandwich complexes was monitored by the resistance method,where the quantitative relationship with L.monocytogenes can be obtained.The results showed that the limit of detection for L.monocytogenes based on ERMC was lower to 17 CFU/m L,and the linear range was 10~3 CFU/m L-10~7 CFU/m L.Moreover,the method exhibited excellent specificity and a recovery rate between 83%and 109.1%.As an one-step method for the detection of foodborne pathogens,this method showed many advantages over traditional methods,such as simple operation,low price,time-saving,fast detection speed,good specificity,and the potential for other small molecule substances detection.This method can be applied in food safety detection,in vitro diagnosis,environmental monitoring,and other fields based on these advantages.2.Research of detecting three foodborne pathogens based on resistive microsphere counting biosensorBased on the effect of different impedance generated by the different particle sizes,the resistive microsphere counting system can accurately distinguish and sensitively detect different particle sizes of microspheres.In this chapter,polystyrene microspheres with 3μm,4μm,and 6μm diameters were used as signal probes to construct a biosensor that was capable of detecting multiple targets simultaneously for the detection of S.aureus,L.monocytogenes,and S.typhimurium,respectively.The polystyrene microspheres used as signal probes were covalently coupled with oligonucleotide probes on their surfaces through DNA hybridization reactions,and the remaining polystyrene microspheres in the supernatant after magnetic separation were counted by a resistive microsphere counting system to indirectly quantify S.aureus,L.monocytogenes,and S.typhimurium.The results showed that the limits of detection for S.aureus,L.monocytogenes,and S.typhimurium were 94 CFU/m L,20 CFU/m L,and89 CFU/m L,respectively,and the linear range of 10~3 CFU/m L-10~7 CFU/m L.The recoveries ranged from 89%to 117%with a coefficient of variation lower than 20%.The method was in good agreement with q PCR for the actual detection of egg samples.3.Research of highly sensitive detection to three foodborne pathogens by small-hole resistance microsphere biosensor based on Streptavidin-Biotin amplification systemIn the fields of food safety,medical diagnosis and environmental monitoring,some test samples often need to be combined with signal amplification technology to meet the needs of ultra-sensitive detection due to the complex matrix and small amounts of pathogenic microorganisms.Based on these merits,the capture probe was modified with biotin on one end,magnetic beads were modified with streptavidin,and three different particle sizes of polystyrene microspheres of 3μm,4μm,and 6μm were used as signal reporters,were utilized to develop a multiplexed detection sensor by combining the DNA hybridization reaction and bio-signal amplification system.So that the number of 3μm,4μm,and 6μm polystyrene microspheres which represented the concentration of S.aureus,L.monocytogenes,and S.typhimurium in the final system can be detected.The limits of detection were 30 CFU/m L,10 CFU/m L,and 28 CFU/m L for S.aureus,L.monocytogenes,and S.typhimurium,respectively,with a linear range of 10~2 CFU/m L-10~7 CFU/m L.And the LOD is 2 to 3 times higher than before.The method also showed good agreement with q PCR and the detection process could be controlled within 2 hours.This method can realize highly sensitive and rapid detection of three foodborne pathogens without nucleic acid pre-amplification,avoiding the false positive and false negative caused by nucleic acid cross-contamination. |
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