Establishment And Evaluation Of Immune Detection Method For Foodborne Pathogens Based On Fluorescence Microspheres

In recent years,food safety problems have received more and more attention.To prevent people from infecting by foodborne pathogens,and reducing the risk of food poisoning,it is necessary for pathogen detection from the source of food,food production and processing.Therefore,it is of great significance to develop rapid methods for foodborne pathogens detection and explore the standardized detection methods.Immunochromatography assay has the characteristic of simplicity,rapidity,high sensitivity and good stability.Fluorescent microspheres have stable morphology and good luminescent behavior,which has little affected by environment than those of pure fluorescent compounds,and the number of bacteria was easily quantified by fluorescence intensity.So it has been widely used as biomarkers in detection in recent years.In this study,we combined fluorescent microspheres with immunochromatography assay for establishment of immune detection model based on fluorescence microsphere(FM-ICA).The immunomagnetic separation methods were used to concentrate the foodborne pathogens from foods.We also studied the influencing factors in the chromatography process,and evaluated the FM-ICA detection method.In this study,several polyclonal antibodies of anti-foodborne pathogens were prepared by immune male rabbit,and then they were used to prepare antibody pairs which can be used in sandwich format using ELISA method.The specificity of antibody pairs was investigated.The results showed that the antibody selected had good specificity.The magnetic microspheres were coupled with polyclonal antibodies by EDC/NHS coupling method.Four magnetic microspheres with good properties were screened.The average particle sizes of PM3,xMag-C,xMag-N and EM1 were 190.9 nm,1150 nm,1545 nm and 469.7 nm,respectively.The four magnetic beads had uniform particle sizes and dispersed wel.The amount of antibody conjugated to magnetic microspheres was different due to different particle sizes and different carboxyl content on the surface of magnetic microspheres.The amount of antibodies coupled to EM1,xMag-C,xMag-N and PM3 were 120?g/mg,160?g/mg,160?g/mg,and 240?g-280?g/mg separately.The immunomagnetic beads(IMB)of three foodborne pathogens were prepared using xMag-C and antibody.With 0.25-0.5 mg IMB,the capture rate was higher than 80%for 1×10~4 CFU/mL bacteria with a capture time of 30-45 min.The dilution fold of food matrics had a significant effect on the capturing rate,when solid samples were diluted for 20 times,the recovery rate of bacteria could reach 85%or even more.An immunochromatographic detection platform based on fluorescent microspheres was established,and the main factors affecting the chromatography process were studied.The application performance of the platform was evaluated.The results showed that the sensitivity of the established FM-ICA method for Vibrio parahaemolyticus(VP)detection was 4×10~5CFU/mL,the recoveries rate ranged from 81.75%to 110.50%,and the fluorescence was maintained stable at 37?for 28 days.The coefficient of variation(CV)between different batches was below 10%.There was no cross reaction with 23 non-target strains.FM-ICA detection had high sensitivity,precision,specificity and stability.The whole detection process could be completed within 30 minutes and it has good application value.It can be used in rapid food screening of VP outbreak in some emergencies,and also can be used in VP detection in seawater,or rapid diagnosis of VP infection in diarrhea patients.The fluorescence immunochromatography platform(FM-ICA)was combined with immunomagnetic separation(IMS)for Salmonella typhimurium(ST)detection in food.The effects of different elution methods and dilution fold of matrics on the detection were studied.The results showed that,the elution rates of ST reached about 65%when IMB was heated at70?for 15 min and this elution method had higher elution rate than the other elution methods.For the artificial contaminated eggs detection,the sensitivity of ST detection by FM-ICA+IMS was 5×10~4 CFU/mL,which was 20 times higher than detection by single FM-ICA method.There was no cross-reaction with 25 non-target bacteria,and the specificity was good.The detection was completed within 2 hours.It can be used for rapid screening of food during ST outbreak.A FM-ICA+IMS detection model for E.coli O157:H7 was established.The minimum detection limit was 3×10~3 CFU/mL.The recoveries of FM-ICA using the same PVC board ranged from 101.64%to 107.03%,different batches of PVC ranged from 95.62%to 110.2%,and with CV below 10%.It showed strong positive signals for 6 E.coli O157:H7 strains,no cross reaction with the other 15 strains.The established FM-ICA+IMS method had good specificity,stability and precision.The sensitivity of FM-ICA+IMS was 33 times higher than single FM-ICA method.This method can be used for rapid screening of E.coli O157:H7 during the outbreak of foodborne pathogens.Three fluorescence CdSe quantum dots were synthesized by using Se and Cd,the surface of CdSe/ZnS quantum dots were coated with ZnS,then the green fluorescence quantum dots were sweled into polystyrene microspheres for synthesis of quantum dots-polystyrene fluorescence microsphere(QD-PS).The QD-PS microspheres were conjugated to VP antibody and used as fluorescence probes in establishment of QD-PS-ICA.This detection method had the sensitivity of 1×10~4 CFU/mL,the quantitative limit was 4×10~4 CFU/mL,the linear detection ranged from 4×10~4 to 4.1×10~7 CFU/mL.The sensitivity of this method was 20 times higher than that of FM-ICA.The recovery,stability and precision were good.