Simultaneous Detection Of Staphylococcus Aureus And Escherichia Coli O157: H7 By Technology Of Combining Aptamer And Quantum Dot

According to surveillance and data reports of disease outbreaks in China,there were 3,170food-borne diseases caused by food-borne pathogens between 2009 and 2019.S.aureus is at the top of the morbidity rate.Although the lethality is not high,it can contaminate almost all foods,especially meat products,and cause serious enterotoxic diseases.The next most harmful bacteria is Escherichia coli O157:H7,The disease caused by Escherichia coli O157:H7 can cause nearly600 million people a year.Illness is the main fecal-derived disease that causes diarrhea in our residents,which seriously endangers people's health.At the same time,it is easy to cause pollution in food processing,production,storage and transportation,and there are huge hidden dangers.Therefore,there is a huge demand for the establishment of methodologies for the simultaneous detection of two foodborne pathogens.this study established a fast,efficient and sensitive detection method for two food-borne pathogens,Staphylococcus aureus and Escherichia coli O157:H7.1. Use fluorescence detection technology to analyze the binding affinity of 4 aptamers of the same species and their target bacteria,calculate the dissociation constant based on spectral data,etc.,and correspondingly select an aptamer sequence with the smallest dissociation constant;Specificity tests were performed on the two selected aptamer sequences.Combined with surface-enhanced Raman spectroscopy,it was confirmed that the aptamer has high specificity with its target conjugate,and the results are good in repeatable experiments.Successfully selected two aptamers Apt S4 and Apt E1 that bind to the target bacteria and have good affinity,which will lay the foundation for the next experiments;2. Select superparamagnetic nanobeads and aptamers to covalently bind to form markers,and specifically isolate and enrich the two target bacteria;Two kinds of quantum dots(QD525,QD605)with different luminescence principles were selected as nano-fluorescent labels,combined with quantum dot fluorescent labeling technology to build a"sandwich structure"of"magnetic beads+target bacteria+quantum dots",and different fluorescence was obtained through fluorescence detection technology The intensity further realizes the qualitative and quantitative determination of the object to be tested.The"sandwich structure"was optimized for the concentration of the two bacterial aptamers,the nanomagnetic bead capture time,and the magnetic separation time,and the minimum detection limit for simultaneous detection of the two target bacteria was determined.The results proved that the two aptamers Apt S4 and Apt E1obtained from the screening had high specificity with the target bacteria,and could be coupled with magnetic beads to complete the simultaneous isolation and capture of two target bacteria.Two fluorescent markers with different emission wavelengths,QD525 and QD605,were selected for detection to form a complete"sandwich structure".The optimization results showed that the optimal concentration of the aptamer of the two target bacteria was 400 nmol/L;The best capture conditions are 45 min capture time and 2 min magnetic separation time;Using this structure to simultaneously isolate and detect two types of bacteria:the minimum detection limit of S.aureus is 10~1CFU/g,and the linear equation is Y=28.51X+126.67(R~2=0.973);The minimum detection limit of E.coli O157:H7 was 10~2CFU/g,and the linear equation was Y=92.86X-64.67(R~2=0.987).Among them,Y is the value of fluorescence intensity,and X is the logarithm of the number of bacterial colonies,and the fit is good;3. Apply the method developed by this research to food samples for testing.Commercially available sterile milk,raw pork meat minced meat,raw beef minced meat,and bagged soy sauce were used as samples for spiked recovery experiments.The results were compared with traditional plate counting methods.The recoveries of Staphylococcus aureus and Escherichia coli O157:H7 in milk samples were 94.6%?102.8%and 93.4%?100.4%,respectively;For the detection of spiked pork and beef minced meat,the detection recovery rates of S.aureus in food samples were between 83.5%?90.6%and 87.4%?93.4%,and the recovery rates of E.coli O157:H7 were 81.7%?90.6%and 82.4%?91.3%.The recovery rate of Staphylococcus aureus in the bagged soybean paste was between 92%?96.7%,and the recovery rate of Escherichia coli O157:H7 was between 92.8%?98.9%.The results prove that the method established in this research can simultaneously detect two food-borne pathogens of Escherichia coli O157:H7 and Staphylococcus aureus.The method is easy to operate and has high sensitivity,and it is safe to detect in food processing It has good application prospects.