The Study On The Construction Of Sensors For The Detection Of Escherichia Coli O157:H7 In Food Based On Aptamer Technology

The World Health Organization lists Escherichia coli O157:H7 as one of the new infectious diseases.More than 2 million food safety incidents worldwide were caused by Escherichia coli O157:H7.Escherichia coli O157:H7 can contaminate soil and grain by contaminating irrigation water or organic fertilizers during cultivation,and are commonly found in uncooked foods,such as uncooked meat,raw eggs,salads,or unwashed fruits and vegetables.When Escherichia coli O157:H7 enters human body,it will produce shiga toxin,causing nausea,vomiting,diarrhea,hemolytic uremic disease,thrombocytopenia,etc.,which threaten human health seriously.Aptamers are ss DNA or RNA that can bind to the target specifically,which is obtained by repeated screening and exponential enrichment in the artificial nucleic acid library.Because of its high specificity to target,it has been widely used in biological,medical,food detection and other fields in recent years.Sensor is mainly composed of two parts:the identification element and the transducer.The working principle of the sensor is to transform the signal sent by the recognition element through the transducer in real time and amplify it into other forms of signal output,so as to realize the quantitative analysis of the target substance.Among them,UV absorption sensor and fluorescence sensor are widely used in food detection because of the advantages of economical and simple-operate.In this study,three sensors were constructed to detect Escherichia coli O157:H7 in food based on the specific recognition ability of aptamers to target substances.A ultraviolet absorption sensor sensor of Escherichia coli O157:H7 was constructed based on the specific recognition function of aptamer and the adsorption performance of graphene oxide.Aptamer and temperature were optimized through single-factor optimization experiments.The best experimental conditions were determined:the concentration of aptamer of 70n M,the reaction temperature of 37℃;then,the 10-fold serial dilution gradient concentration of Escherichia coli O157:H7 pure bacterial solutions were detected under the best experimental conditions,and the results revealed that the linear range was in the range of 3.5×10²-3.5×10⁶CFU mL^-1,the standard curve equation was y=-0.087x+0.889,R²=0.995,and the limit of detection was as low as 1×10² CFU mL^-1;the specificity experiment proved that the UV absorption sensor showed high specificity for Escherichia coli O157:H7.Finally,this study carried out spiked recovery experiments on apples,strawberries,cucumbers,and lettuce.The results showed that the spiked recovery rates were in the range of 90.4%-101.3%,and the relative standard deviations were all below 5%,the accuracy and precision of this method are in the acceptable range.The Escherichia coli O157:H7 fluorescence sensor was constructed in combination with the signal amplification of the hybridization chain reaction based on the high specificity of ap-tamer,the adsorption and fluorescence quenching performance of graphene oxide,.This study proved the method through control experiments firstly,the results proved that the fluorescence intensity was accumulated in the presence of Escherichia coli O157:H7,H0,H1,and H2,in this case,the fluorescence intensity would not be quenched completely by graphene oxide;then the best experimental conditions was obtained through single-factor optimization experiments:the concentration of graphene oxide of 25μg m L^-1,HCR reaction time of 90min,the concentration of H0 of 50n M,the concentration of H1 and H2 of 50n M;under the best experimental conditions,the gradient concentration of Escherichia coli O157:H7 pure bacteria solutions were tested,and the results showed that the detection range was in the range of 2.6×10²-2.6×10⁸ CFU m L^-1,the standard curve was y=591.1x-943.9,R²=0.995,the detection limit was as low as 5×10¹CFU m L^-1;the specificity test results showed that the method had good specificity for the detection of Escherichia coli O157:H7;through the recovery experiments on apples,strawberries,cucumbers,and lettuce,the results showed that the recovery rates of addition were in the range of 85.1%-101.3%,and the relative standard deviations were less than 5%,the accuracy and precision of this method are in the acceptable range.The Escherichia coli O157:H7 fluorescence sensor was constructed based on the specific recognition ability of the aptamer,the adsorption and the fluorescence quenching performance of the metal-organic framework.Control experimental group was used to prove the feasibility of the fluorescence sensor firstly,the experimental results proved this method is feasible for the detection of Escherichia coli O157:H7;then we optimized the experimental conditions through single-factor experiments,and the results showed that the best experimental conditions were as belows:the concentration of aptamer of 100n M,the concentration of MOFs of 200.50×10³μg m L^-1,the reaction culture temperature of 37℃,and the reaction time of 60min.In order to evaluate the analytical performance of the method,the gradient concentration of Escherichia coli O157:H7 pure bacterial solutions were tested under the best experimental conditions,the results showed that the detection range of the method for Escherichia coli O157:H7 was 7.8×10¹-7.8×10⁸CFU m L^-1,the standard curve equation was y=23.75x+97.48,R²=0.975,and the detection limit was as low as 6×10¹ CFU m L^-1;through the control experiments on other 9 kinds of bacteria,the results proved the fluorescence sensor owned good specificity for Escherichia coli O157:H7;in order to verify whether the method can be applied to the detection of actual samples,we carried out spiked recovery experiments on four food samples of apple,strawberry,cucumber,and lettuce,the results showed that the recovery rate was in he range of 88.4%-103.6%,the relative standard deviations were less than 5%,and the accuracy and precision of this method are within the acceptable range.In this study,the aptamer was used as the sensor response element.Based on the specific recognition function of the aptamer,three sensor systems for the rapid detection of Escherichia coli O157:H7 in food were constructed successfully:The aptamer ultraviolet absorption sensor based on GO for Escherichia coli O157:H7 was constructed;The aptamer fluorescence sensor of Escherichia coli O157:H7 based on hybrid chain reaction amplification was constructed;The aptamer fluorescence sensor based on Escherichia coli O157:H7 based on metal organic framework was constructed.These three aptamer-based detection methods provide new reference for the detection of Escherichia coli O157:H7 and other pathogens from different perspectives.