Research On Application Of Allosteric Transcription Factors In Antibiotic Detection

Antibiotics are used and even abused worldwide,resulting in the accumulation of antibiotics in the animal organism and its living environment,which can cause hypersensitivity reactions,antibiotic resistance,teratogenicity and cancer in humans after long-term intake.In recent years,in order to limit the development of antibiotic resistance,the European Union(EU),the Food and Agriculture Organization of the United Nations(FAO)and the Food and Drug Administration(FAO)have developed specific regulations to monitor antibiotic residues in animal origin foods.As national organizations continue to regulate antibiotic abuse,standards and regulations for Maximum Residue Limits in animal origin foods are gradually being improved.Rapid and accurate detection of antibiotics is a key aspect of effective antibiotic abuse control.Traditional detection methods have the advantages of high accuracy and sensitivity,but these methods usually require complex,time-consuming and expensive sample pretreatment procedures,and they also require specialized personnel and expensive instrumentation.Therefore they can only be applied under special laboratory conditions.New techniques for rapid on-site detection of antibiotics are needed to meet realistic requirements.Allosteric transcription factors(aTFs)are regulatory elements of gene expression widely found in prokaryotes and contain an effector binding domain(EBD)and a DNA binding domain(DBD).Under natural conditions,aTFs bind specifically to effectors,resulting in conformational changes that alter their binding ability to DNA.In this work,aTFs were used to identify tetracyclines and macrolides,and rapid,convenient and sensitive field detection methods were innovatively developed.The specific implementation method and content are as follows.(1)A novel ELISA technique for the detection of tetracycline based on aTFs.In this chapter,a novel aTFs-based ELISA detection technique has been developed for semi-quantitative and quantitative on-site detection of tetracycline in seawater,sewage,milk and honey.Firstly,QD-TetR,a coupling between Quantum Dot(QD)and TetR,was prepared by chemical coupling,and QD-TetR was characterized by fluorescence spectrophotometry,zeta potential,infrared spectroscopy and transmission electron microscopy;Then,biotin-modified double-stranded DNA(ds DNA)was immobilized in the bottom of a 96-well plate coated with streptavidin,and QD-TetR was added to the 96-well plate,and the fluorescence was determined after washing with PBST;Next tetracycline was added and the fluorescence decreased after washing with PBST,and the ELISA technique was successfully established.Different concentrations of QD-TetR were added into the 96-well plate fixed with ds DNA,and the fluorescence of different QD-TetR was completely disappeared with different concentrations of tetracycline,and the field semi-quantitative assay of ELISA technique was successfully established.Finally,the practicality of the method was verified by detecting seawater,sewage,milk and honey samples.Under the optimal conditions of 1:1 molar ratio of COOH functionalized QD to TetR,the fluorescence value after detection of tetracycline by ELISA technique has a good linear relationship with the logarithm of tetracycline concentration,and the linear equation is Y=-211.9*X+729.9(R~2=0.9825),where Y is the fluorescence value and X is the logarithm of tetracycline concentration,and its detection limit is 0.05μM.In addition,it can be detected by semi-quantitative on-site detection of 0.5-500μM tetracycline can be completed by the naked eye in 5 min.(2)The research on detection technology of antibiotics based on aTFs.In this study,an in vitro transcription(IVT)biosensor based on aTFs for the detection of tetracyclines and macrolides was developed using Nuclear acid sequence-based amplification(NASBA).Firstly,the in vitro transcription system was constructed with ds DNA containing T7 promoter,aTFs binding site,3WJd B expressed gene and T7 terminator as the reaction template;Then,the binding and dissociation processes between aTFs and DNA were described by BIAcore analysis and electrophoretic mobility shift assay(EMSA);Next,appropriate concentrations of aTFs were added to the in vitro transcription system to construct the IVT biosensor and to complete the quantitative detection of four tetracycline antibiotics and four macrolide antibiotics;Finally,NASBA was used to amplify the RNA produced by IVT to achieve signal amplification.Innovative use of BIAcore to directly observe the binding and dissociation of aTFs with DNA in real time.Classical EMSA was used to characterize the binding and dissociation of aTFs with DNA in an IVT buffer system.Under the optimal conditions,the fluorescence value generated by this detection system had a good linear relationship with the logarithm of the concentration of antibiotics.Four tetracyclines(anhydrotetracycline,oxytetracycline,doxycycline and chlortetracycline)and two macrolides(roxithromycin and azithromycin)were quantified in the range of 0.5-15μM,while erythromycin and clarithromycin were quantified in the range of 0.1-15μM.The use of NASBA resulted in a 27-fold increase in fluorescence signal and a reduction in the limit of detection to 0.01μM for both anhydrotetracycline and erythromycin,meeting international MRL requirements.The biosensor was used in milk samples to prove its field detection performance.In this work,based on the binding and dissociation properties of aTFs and DNA.Firstly,QD were selected as the signal output to directly monitor the binding and dissociation of aTFs with DNA to establish a novel ELISA assay technique for the rapid detection of tetracyclines.And then,RNA was selected as the signal output so that aTFs act as a switch to initiate the transcription process,indirectly using its binding and dissociation properties with DNA,and with the recognition and binding of aTFs to the target to initiate the transcription of downstream genes,the corresponding RNA signal was generated to establish the IVT biosensor.This research demonstrates the feasibility of the two detection techniques,providing a new means of antibiotic detection.And then,RNA was selected as the signal output so that aTFs act as a switch to initiate the transcription process,indirectly using its binding and dissociation properties with DNA,and with the recognition and binding of aTFs to the target to initiate the transcription of downstream genes,the corresponding RNA signal was generated to establish the IVT biosensing assay.This research demonstrates the feasibility of the two detection techniques,providing a new means of antibiotic detection.And it provides a new idea for the development of detection methods based on aTFs to detect contaminants.