Establishment And Evaluation Of E.coli O157:H7 Detection Method Based On Magnetic Separation And Fluorescence Resonance Energy Transfer System

Objective:Escherichia coli is a common food-borne pathogen,gram-negative,which belongs to the Enterobacteriaceae family.Escherichia coli O157:H7（E.coli O157:H7）is a special serotype of Escherichia coli,which can be transmitted through food and drinking water,cause diarrhea in the human body.In severe cases,it can also cause hemorrhagic colitis or hemolytic uremic disease,which not only constitutes human health.but causes a serious economic burden.E.coli O157:H7 easily contaminates a variety of foods such as beef,milk and pasta,and is highly adaptable to temperature,p H and dry environments.Therefore,the establishment of a rapid and effective pathogen detection method is of great significance for the timely detection of pathogens,the control of foodborne disease outbreaks,and the treatment of foodborne diseases.Therefore,the establishment of rapid and effective pathogen detection methods in food safety testing is important to its detection,control and treatment.This research aims to apply nanomaterials that have emerged in recent years to establish a method that can overcome the shortcomings of traditional detection methods and detect E.coli O157:H7 in food quickly.Method:In this study,immunomagnetic separation technology and fluorescence resonance energy transfer（FRET）was used to establish a method to detect E.coli O157:H7 in food.The immunomagnetic nanoprobes（IMBs）that specifically capture E.coli O157:H7 are prepared by coupling streptavidin-modified magnetic beads（MBs）and biotin-modified antibodies to achieve the enrichment and separation of target bacteria.The prepared immunomagnetic nanoprobes were characterized by transmission electron microscopy and Fourier infrared transform spectroscopy.Gold nanoparticles（Au NPs）and sulfhydryl-modified E.coli O157:H7 nucleic acid aptamers were coupled via Au-S bonds to prepare nucleic acid aptamer-gold nanoparticle probes.The nucleic acid aptamer-gold nanoparticle probe was characterized by transmission electron microscopy（TEM）and Ultraviolet-visible spectrophotometer（UV-Vis Spectrophotometer）.IMBs are added to the sample to be tested to capture the target bacteria.After magnetic separation,gold nanoprobes（Au NPs）are added to form an IMBs-bacteria-gold nanoprobe sandwich structure.Rhodamine B was then added to the complex,and the fluorescence intensity of the negative and positive samples was measured with Fluorescence-Visible Spectrophotometer（FL-Vis Spectrophotometer）.Atλ=576 nm,calculate the fluorescence signal difference（F₀-F）,which can detect the content of the target bacteria in the sample through the difference of the fluorescence signal indirectly.In addition,we optimized the dosage of IMBs and the binding time with target bacteria,the dosage of gold nanoparticle-nucleic acid aptamer and the binding time with target bacteria,as well as the dosage and reaction time of rhodamine B,under the optimal experimental conditions.The experimental method was evaluated by specificity and the limit of detection（LOD）in the simulated sample.Result:The average particle size of the streptavidin-modified magnetic nanoparticles is about 73±6 nm,which is spherical with good dispersion.Compared with individual magnetic nanoparticles,the magnetic nanoparticles’size that coated by the E.coli O157:H7 antibody successfully have increased to about 128±7 nm,and the dispersion state has changed with a slight agglomeration.Observed by transmission electron microscope（TEM）,compared with a single magnetic nanoparticle,the surface of the immunomagnetic nanoprobe has a transparent cloud-like film,indicating that the surface of the magnetic bead is covered by an antibody.Fourier infrared transform spectroscopy（FT-IR）showed that the magnetic nanoparticles modified with E.coli O157:H7 antibody compared with the magnetic nanoparticles alone,the peak（1618.65）representing the amide bond（CO-NH）had a stretching vibration change.This change is related to the specific recognition of biotin by streptavidin.Integrating the characterization results of TEM and FT-IR,it can be determined that the surface of the magnetic nanoparticles has been successfully modified with the E.coli O157:H7 antibody,and the IMBs probe has been successfully prepared.The particle size of individual gold nanoparticles is about 30±7 nm,which is spherical with good dispersibility.While the size of gold nanoparticles coupled with nucleic acid aptamer was increased to about 44±4 nm with a slight dispersed change.Under the TEM,we can see bright white areas around the Au NPs coupled with nucleic acid aptamers.The peak of the UV-Vis absorption spectrum of individual Au NPs is at520 nm.After the nucleic acid aptamer is combined,the absorption peak is red-shifted from 520 nm to 522 nm,and the absorbance has some decrease.Combining the changes in particle size,the morphology under the TEM,and the shift of the peak value in the UV absorption spectrum,it can be indicated that the nucleic acid aptamer is successfully combined with the Au NP,and the gold nanoprobe has been successfully prepared.The two nanoprobes were used to establish a rapid detection method for E.coli O157:H7,and the experimental conditions involved in the detection method were optimized.The optimized experimental conditions are:the optimal amount of immunomagnetic probe is 90μL（0.45 mg/m L）,and the optimal incubation time is 50minutes.The optimal molar ratio of gold nanoparticles to aptamer is 200:1,and the optimal amount of nucleic acid aptamer-gold nanoprobe is 250μL（0.1 mg/m L）,the optimal incubation time of gold nanoprobe is 60 minutes.The optimal dosage of rhodamine B is 25μL（0.025 mg/m L）,and the optimal reaction time is 10 minutes.In TBST buffer（p H=7.4）,when the linear range of E.coli O157:H7 concentration is 10¹～10⁷ CFU/m L,there is a good linear relationship between fluorescence difference（F₀-F）and E.coli concentration.The linear regression equation is Y=36.965X-4.9429,R²=0.9918.The detection limit is 0.35 CFU/m L.In milk simulation samples,the detection linear range of this method is 10¹～10⁷ CFU/m L,the detection limit of E.coli O157:H7 is 1.03 CFU/m L,the regression equation is Y=12.697X-5.9771,R²=0.9423.The specific experimental results show that the established method can specifically recognize E.coli O157:H7 without interference from non-target bacteria,the experimental method has good specificity.In summary,this method does not require high operation requirements,and the time consumed is about 3 hours,and it has a higher sensitivity and a lower detection limit.Conclusion:In this study,a rapid detection method of E.coli O157:H7 was established based on the IMS and FRET.This method does not require pre-enrichment,and the required reagents and instruments are relatively simple.Under the best experimental conditions,this method has a wider detection range,lower detection limit and better specificity.