| Food safety has always been a major issue affecting human health and have attracted the attention of governments and people all over the world.Among many factors affecting food safety,food-borne pathogens are one of the main factors leading to food-borne diseases.Monitoring of food-borne pathogens has always been the focus of food-borne diseases surveillance in various countries.Traditional food-borne pathogen detection methods such as plate culture and enzyme-linked immunosorbent assay have been unable to meet the requirements for rapid detection in food safety monitoring due to the shortcomings such as detection circumference,time-consuming and laborious,and low sensitivity.Therefore,it is urgent to develop easy-to-operate,rapid and sensitive detection methods to reduce the occurrence of food-borne diseases.Lectin is a kind of protein which has strong affinity with food-borne pathogens.Compared with the antibodies used in the field of separation and detection of food-borne pathogens,lectin has the advantages of low cost,easy preparation and preservation,so is widely used in separation and rapid detection of food-borne pathogens.In this paper,wheat germ agglutinin(WGA)was used as a recognition agent,and the"two-step"magnetic separation technique was used to enrich Staphylococcus aureus in food samples.S.aureus separated was detected by various methods,including AuNPs-based colorimetric,Selective culture,Polymerase chain reaction(PCR),and 3,3′,5,5′-Tetramethylbenzidine(TMB)-based colorimetric methods.In order to make the magnetic separation method established in this study more widely used,we designed a magnetic separation device specially used for magnetic separation of 10 mL system,which provides a powerful guarantee for the promotion and application of new methods for food microbial detection.The contents of each chapter are as follows:Chapter 1:The research progress of the application of lectin in the detection of food-borne pathogens was reviewed.Chapter 2:A“two-step”Lectin-magnetic separation(LMS)method based on WGA and an AuNPs-based colorimetric system were developed to detect S.aureus.In this study,biotinylated WGA was firstly anchored with S.aureus to carry biotin on the surface of S.aureus.Then streptavidin modified magnetic nanoparticle(MNP-SA)were added to bind biotin to achieve the enrichment and separation of S.aureus.Under the optimum conditions,the capture efficiency of S.aureus in PBST was higher than 90%at the concentration of 3.5×10~0-3.5×10~5 CFU/mL,and the capture efficiency of low concentration S.aureus in lettuce samples(3.5×10~0-3.5×10~3CFU/g)was also significant(higher than 80%).Compared with the traditional immunomagnetic separation method,the method uses WGA as the recognition agent of bacteria,and uses the“two-step”method to improve the reaction efficiency,which greatly reduces the cost of magnetic separation.Combined with the gold nanoparticles(AuNPs)aggregation based naked eye detection system,the method realizes the ultra-sensitive detection of S.aureus.The minimum detection limits in PBST and lettuce samples were 3.5×10~0 CFU/mL and 3.5×10~1 CFU/g,respectively.This method demonstrates the potential application value of lectin in bacterial magnetic separation,and highlights the obvious advantages of“two-step”method in improving reaction efficiency and reducing detection cost.Chapter 3:A“two-step”LMS method based on polyethylene glycol(PEG)-mediated streptavidin(SA)exposure was established.The rapid detection of S.aureus was realized by selective culture,PCR and TMB based colorimetric methods.The PEG-mediated streptavidin-modified magnetic nanoparticle(MNP-PEG-SA)effectively improved the binding efficiency of streptavidin and biotin in“two step”magnetic separation system,and the amount of MNP reduced by 37.5%.Moreover,the separation efficiency of the magnetic separation method for different concentrations of S.aureus is higher than 90%,indicating that the LMS method has superior separation effect.The S.aureus separated was specifically tested by selective culture,PCR and TMB based colorimetric methods.The results showed that the three rapid detection methods are easy to operate and are suitable for the detection of S.aureus under different sensitivity and detection time requirements:BP medium-based selective culture method can detect 3×10~0 CFU/mL S.aureus within 15 h.;the detection period of PCR method is 4 h,and the sensitivity reached 3×10~2 CFU/mL;TMB based colorimetric method only cost 2 h,and the limit of detection is 3×10~5CFU/mL.This study established a method for the detection of spiked juice samples.The results show that the three specific detection methods established showed great detection results in spiked juice samples,indicating that the method has broad application prospects in actual sample detection.Chapter 4:A 10 mL system magnetic separation device was designed.In order to make magnetic separation technology more widely used in food safety testing,a magnetic separation device for separation of food samples in 10 mL system was designed.The magnetic separation device has good separation effect,solid structure,convenient storage and use.The design idea of the magnetic separation device is:analyzing the stress of the MNP in the experiment,selecting the appropriate permanent magnet material according to the force situation,designing the appearance structure according to the actual use requirements,and finally verifying the separation effect of the designed magnetic separation device.After reasonable evaluation,the NdFeB permanent magnet material was selected as the magnetic field source of the magnetic separation device,and the centrifuge tube hole position was set on both sides of the permanent magnet material for magnetic separation.The magnetic separation device can realize rapid separation of MNP,and the capture efficiency reached 95%for the magnetic separation of pathogens in 10 mL system in 4 min.The device has the advantages of solid structure,convenient storage and use,and can meet the detection requirements of samples under different environmental conditions. |
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