Methodological Study On Electrochemical Sensing Detection Of Mcr-1 Resistance Gene Based On DNA Self-assembly Amplification System

Polymyxin is a polypeptide antibiotic isolated from the culture medium of polymyxin and has a killing effect on most Gram-negative aerobic bacteria,but it is rarely used in the past because of its significant nephrotoxicity and neurotoxicity In clinical.However,with the recent years,super bacteria and other multi-drug resistant bacteria continue to emerge,most antimicrobial drugs helpless,and polymyxin drug susceptibility test results are "sensitive",it is back to medical,scientific research personnel vision.However,in November 2015,Liu Jianhua,who was on the lancet on South China Agricultural University,reported for the first time that there was a plasmid-mediated,level-resistant antimicrobial resistance gene mcr-1(mobile colistin resistance).Since then,the United States,France,the United Kingdom found and reported mcr-1 gene,and the isolated mcr-1 gene not only in pigs,chickens and other animals,from food,water,vegetables,etc.also separated Mcr-1 gene.Because of its breakthrough in the last line of defense of antibiotics,mcr-1 resistance gene global spread gradually attracted people’s attention.Electrochemical DNA biosensing technology is a single-stranded DNA or double-stranded DNA as a sensitive element,the nucleic acid molecules generated by the interaction of biological signals can be detected into electrical signals,in order to achieve qualitative or quantitative detection of DNA.Based on the widespread spread of mcr-1,and the severity of clinical treatment,we first explored the use of electrochemical DNA sensing technology to detect mcr-1,a plasmid-mediated bacteriocin-resistant gene Detection lay the theory and the basis of preliminary practice,but also for the clinical detection of other common drug resistance to provide a common technology platform.First,the design of the 5 ’end of the modified mercapto(HS-)ssDNA,through the gold sulfur bond to be fixed on the surface of the gold electrode,with mercaptohexanol closed blank site to reduce non-specific adsorption.HS-ssDNA is self-assembled into a stable monomolecular layer on the surface of the gold electrode,and the digested product of the plasmid DNA is specifically identified under suitable conditions(such as temperature,pH,ionic strength,etc.).The electrochemical workstation is used to detect the electrical signal,And the mcr-1 resistance gene fragment was successfully detected.The results showed that the expression of mcr-1 gene was detected by electrochemical biosensor technology.The results showed that the plasmid-mediated Escherichia coli Feasibility of Clostridium mcr-1 Resistance Gene.Secondly,in order to solve the problems of complex operation,time-consuming and labor-saving and insufficient detection sensitivity,we constructed a non-enzyme-free liquid phase biogas based on teohold-mediated hybrid chain reaction(HCR)and screen printing gold electrode Sensing technology platform.The toehold probe containing the sticky end is designed and the stem of the stem-loop DNA probe is opened by the toehold-mediated hybrid chain reaction when the target gene mcr-1 gene fragment is present in the solution,Exposing the triggering chain of the hybrid chain reaction(HCR),triggering the chain to initiate the toehold-mediated HCR reaction,and ultimately form a long double-stranded structure.The self-assembly and amplification system without enzyme-labeled DNA was constructed by combining the methylene blue(MB),which could be inserted into the dsDNA double helix structure.Compared with free MB,the diffusion rate of MB in MB/dsDNA complex was significantly decreased,so the binding of MB and dsDNA could significantly reduce the reaction current of MB in homogeneous solution,and the current was weakened on the screen printing gold electrode Change and degree.The experimental results show that the concentration of △ ip and mcr-1 gene in the detection system is linearly positive in the range of 10nM to 1μM,and through the investigation of the base mismatch ability of the detection system,the system With a good specificity,in order to achieve mcr-1 resistance gene more convenient and fast detection.