The Screening Of Cefquinome Specific Nucleic Acid Aptamer And Its Application In Food Detection

Cefquinome(CFQ)is the fourth generation of cephalosporin antibiotics,which has been approved for animal use in China.Usually,it is specially used for treatment of bovine mastitis induced by Escherichia coli,swine respiratory disease and other diseases caused by various bacterials.Normally,there are antibiotics residues when antibiotics are overused and the period of milk abandoning is not strictly executed,which will bring extremely serious consequences to human health.The European Union and China both stipulate that the residue limits of cefquinazone in milk should not beyond 20?g/kg.However,current methodologies are always time and labour-consuming,and have low sensitivity,which can not meet the requirement of spot rapid detection.Therefore,establishing novel highly sensitive method for fast detection of cefquinoxime residues in milk is particularly important.Aptamers,which are derived form SELEX(systematic evolution of ligands by exponential enrichment),are short oligonucleotides(ssDNA or ssRNA)bind to their targets with high affinity and specificity.Owing to their obvious features such as wider range of target molecules,better stability and easier modification,aptamer-based detection strategies have been widely applied in analytical chemistry,food safety and clinic diagnosis and treatment.Here,four cefquinoxime-specific DNA aptamers were screened by magnetic beads-based SELEX technology(Mag-SELEX).And then,a highly sensitive and specific fluorescence aptasensor was established for the detection of cefquinoxime residues in milk samples.These works are listed as follows:(1)Four CFQ-specific ssDNA aptamers(W1,W2,W3 and W4)were screened via 12 rounds of selection by Mag-SELEX in vitro.These 4 aptamers all have good specificity and binding affinity against CFQ.Among them,aptamer W1 showed better specificity and binding affnity than that of the other three aptamers,whose Kd value is 40.13±22.11 nM.Therefore,aptamer W1 is selected for the establishment of subsequent fluorescence aptasensor.(2)A fluorescence aptasensor for the detection of CFQ in milk samples was successfully established using aptamer W1.Then,the detection conditions of the aptasensor was' systematically studied.The results showed that the length of fixed probe was 17 bp;the optimal concentration of aptamer utilization was 2?M;the optimal incubation time double stranded DNA and streptavidin beads was 1 h;the optimal detection time was 30 min.This newly established aptasensor showed good specificity and highly sensitivety with a linear detection range of 0.5-150 ng/mL and a limit of detection limit of 0.09 ng/mL.Finally,the recovery rate of CFQ in milk samples was about 96.6%-102%,indicating the aptasensor an ideal approach for fast and highly sensitive detection of CFQ residues in animal-sourced food.