Detection Of Food-borne Pathogenic Bacteria By Amino Functionalized Magnetic Nanoparticles Enrichment Combined With Multiplex PCR

During food processing,it may be polluted by multiple pathogens,which poses a serious threat to the health of consumers.So it is of great importance for the rapid detection of the pathogenic bacteria in food to ensure food safety.However,pathogenic bacteria in food are usually in a low concentration,and there is the presence of disruptive substances in food components,all of which leads to a decrease in the sensitivity and specificity of the detection method.Therefore,it is very important to isolate or enrich pathogenic bacteria from foods.Magnetic nanoparticles?Magnetic Nanoparticles,MNPs?have been favored in the separation and detection of food-borne pathogens due to their good biological properties and easy to separate.The aim of this paper is to prepare amino-functionalized magnetic nanoparticles?AF-MNPs?with positive charge.Based on the electrostatic interaction between AF-MNPs with positive charge and bacteria with negative charge on the surface,the enrichment of bacteria by AF-MNPs can be realized.And then,the multiplex PCR method combined with the enrichment of bacteria by AF-MNPs was used to detect Staphylococcus aureus,Listeria monocytogenes and Salmonella enteritidis in food.The detailed contents and results of the study are as follows:1)Preparation and characterization of AF-MNPs.We applied the chemical coprecipitation method to prepare Fe₃O₄ MNPs Then the surface modification of 3-aminopropyl triethoxysilane?APTES?was carried out on the surface of Fe₃O₄ MNPs to synthesize the AF-MNPs with positive charge.The results showed that the prepared AF-MNPs were spherical or spherical,and the particle size was about 10 nm under electron microscope.The polydispersion index?PdI?showed good dispersion by ZetaSizer Nano-Particle size analyzer.And the isoelectric point of AF-MNPs is about 9.4,also when the pH is in the range of 2-9,the surface of AF-MNPs is in positive charge.2)Enrichment of food-borne pathogens by AF-MNPs.The capture of AF-MNPs on bacteria was mainly based on the electrostatic interaction between AF-MNPs with positive charge and bacteria withnegative charge.We use Staphylococcus aureus,Escherichia coli,Listeria monocytogenes and Salmonella enteritidis as representative strains to study the influencing factors of interaction between AF-MNPs and bacterium,including the amount of AF-MNPs,bacteria and buffer system.The results showed that the capture efficiency of AF-MNPs to S.aureus,E.coli and L.monocytogenes was higher than 97%when the bacteria with 10³CFU/mL was captured by 100?g AF-MNPs,however the capture efficiency for S.enteritidis was less than 40%.The capture efficiency of AF-MNPs to S.aureus and E.coli were higher than 80%when the 10 mmol/L PBS was in the range of pH 5-9,while the capture efficiency of AF-MNPs to L.monocytogenes was higher than 75%when the 10 mmol/L PBS was in the range of pH 4-8.When the concentration of PBS was 5-30 mmol/L,the capture efficiency for S.aureus,L.monocytogenes and E.coli were more than 75%,85% and 90% respectively.Among the four bacteria studied,the Zeta potential of S.enteritidis was the smallest and the capture efficiency was the lowest.The capture efficiency of AF-MNPs was higher than 85% when the concentration of bacteria was in the range of 10²-10⁶CFU/mL.When the system volume increased to 10 mL and the bacterial concentrations were at 4 CFU/mL,40CFU/mL and 400 CFU/mL,the capture efficiency was still more than 90%.The capture efficiency for bacteria in artificially contaminated milk samples by AF-MNPs decreased significantly due to the effect of composition in milk.For S.aureus,E.coli,L.monocytogenes and S.enteritidisthe capture efficiency was 67.14%±1.33%?62.89%±0.68%?57.92%±2.93%respectively.For S.aureus,E.coli,L.monocytogenes and S.enteritidis,the capture efficiency for bacteria in artificially contaminated watermelon samples by AF-MNPs was 85.38%±0.63%,69.84%±2.97%,79.28%±0.90%and 31.66%±3.30%respectively.In order to detect the applicability of AF-MNPs,8 kinds of pickle samples and 6 kinds of brine samples were randomly selected in this chapter.The capture efficiency of bacteria in 8 kinds of actual salted vegetable foods by AF-MNPs were higher than 75%,and the capture efficiency of 6 kinds of brine meat samples were higher than 65%.3)Detection of Food-borne pathogenic bacteria by AF-MNPs enrichment combined with multiple PCR.Using S.aureus,L.monocytogenes and S.enteritidis as target bacteria,a multiplex PCR was established.The three kinds of food-borne pathogens in artificially contaminated milk samples were detected by multiplex PCR after magnetic separation.After magnetic separation and PCR detection,the results showed that the sensitivity of S.aureus,L.monocytogenes and S.enteritidis were 2 CFU/mL,2 CFU/mL and 2×10²CFU/mL respectively.However,using the direct multiplex PCR method,the sensitivity of S.aureus,L.monocytogenes and S.enteritidis were 2×10² CFU/mL,2×10³ CFU/mL and2×10³ CFU/mL respectively.Three kinds of food-borne pathogens were mixed and contaminated with the same concentration.After detection by multiple PCR combined with capture and enrichment by AF-MNPs,the sensitivity of S.aureus,L.monocytogenes and S.enteritidis was 20 CFU/mL,2×10² CFU/mL and 2×10² CFU/mL respectively.Compared with the direct multiplex PCR method,the sensitivity of S.aureus,S.enteritidis and L.monocytogenes increased by 100 or 1000 times respectively.Therefore,AF-MNPs enrichment combined with multiplex PCR can effectively improve the sensitivity of multiple PCR detection.