The Construction Of Fluorescent Sensors For Unlabeled Nucleic Acid Fits And The Application Of Tetracycline Tetection In Milk

Tetracycline is widely used in animal husbandry and aquaculture to prevent bacterial infection and improve growth rate because of its broad-spectrum activity and low cost.However,the excessive and improper use of tetracycline leads to drug residues in meat,milk,honey,fish,eggs and shrimp,which seriously threatens human health.Therefore,strict regulations have been formulated on the residue of tetracycline in animal products.High performance liquid chromatography,capillary electrophoresis and gas chromatography-mass spectrometry all need to use some large instruments and organic solvents,which costs a high cost and does great harm to human health and the environment.In addition,the extraction process is very timeconsuming.Enzyme linked immunosorbent assay may obtain false positive results because it may cross react with substances with similar chemical structure.Thioflavin t(THT)can be embedded into special nucleic acid structure,so that the fluorescence intensity can be strongly excited to realize signal output.Based on this,the following studies are done in this study:1.The sequence of tetracycline aptamer was optimized to obtain the bivalent aptamer sequence with G-quadruplex formation potential.Taking the tht fluorescence change as the output signal,a non enzyme immunolabeling sensor based on conformational transformation was constructed for the accurate detection of tetracycline.The sensor is at 10 nm ? 1 ? The concentration range of M showed a good linear relationship,R2 was more than 0.99,and the detection limit was 96 PM.The sensor has strong specificity,short time-consuming and simple operation.It can realize the rapid detection of tetracycline in milk samples within 20 minutes and meet the maximum residue of tetracycline(100%)in the national food safety standard maximum residue limit of veterinary drugs in food ? G / kg),which has a good application prospect.2.The sequences needed to construct a double strand competitive cleavage visual nucleic acid sensor were optimized.Because the background fluorescence of tht binding between G-quadruplex and tetracycline aptamer is too high,the G-quadruplex is cut into g-triplex and tetracycline aptamer.The change of tht fluorescence intensity before and after tetracycline is used as signal output to reduce the detected background value.The g-triplex sequence was optimized,and finally the best nucleic acid sequence was selected and verified by g-triplex base mutation.Finally,the optimized aptamer apt4 was characterized by colloidal gold as having the ability to recognize tetracycline.3.Tetracycline aptamer can be combined with G-rich sequence to form double chain,but tetracycline is preferentially combined with aptamer in the presence of tetracycline.The unbound G-rich sequence can fold itself to form g-triplex structure,enhance the fluorescence intensity of THT,and realize the quantitative detection of tetracycline by measuring the fluorescence intensity of THT.The constructed sensor has a tetracycline concentration of 1 nm ? 1 ? It has good linearity in the range of M,R2 is up to 0.99,the detection limit is 0.07 nm,and the detection can be completed within 25 min.Therefore,a visualization method of double chain competitive cleavage based on g-triplex has been developed,and the detection of tetracycline is simple,rapid,sensitive,specific and low-cost.