Research On Electrochemical Biosensing Method For Detection Of MecA Gene In Methicillin-resistant Staphylococcus Aureus

Objective:Methicillin-resistant Staphylococcus aureus(MRSA) is a major cause of human bacterial infections worldwide. Clinical evidence has shown that rapid diagnosis and appropriate early treatment of MRSA infections have positive influence on making infection control strategies. The mechanism underlying resistance to a family of antibiotics is reportedly based on the penicillin-binding protein(PBP2a), which was encoded by mecA gene which placed on Staphylococcus cassette chromosome mec(SCCmec).Hence, detection of mec A gene is tantamount to methicillin resistance detection. Based on recycling amplification strategy and cascade recognition method, respectively, two electrochemical biosensor for highly sensitive and specific detection of mecA gene in MRSA were proposed in this study.Methods:1. Construction of two electrochemical biosensors: an ultrasensitive electrochemical biosensors based on target recycling signal amplification method and(ISDPR) strategy is presented; Using spontaneous cascade DNA branch migration and dual-signaling sensing strategy, an electrochemical biosensor was developed. Every step of biosensor was characterized by methods of electrochemical impedance spectroscopy(EIS),cyclic voltammetry(CV), atomic force microscope(AFM), fluorescence spectrophotometry, and agarose gel electrophoresis.2. Feasibility analysis and experimental conditions optimization: we investigated the immobilization order, ratio, immobilization time,temperature, and concentration of probes, concentration and reaction temperature of polymerase enzyme and so on.3. Analytical performance and sample detection: analyze the performance of two electrochemical biosensors, such as detection linear range, detection limit, specificity, repeatability, stability and so on. Use simple signal transduction mechanism output electrochemical signal and detect it using square wave voltammetry(SWV) method.Results:1. The preparation process of two electrochemical biosensors was characterized by different methods, such as EIS, CV, AFM, fluorescence spectrophotometry, and agarose gel electrophoresis. Different approachesto prove the success of the electrode modification and preparation together.2. Performance of electrochemical biosensor based on ISDPR: detect mec A gene sensitively; linear range is 0.075-200 pM; detection limit is 63pM; good repeatability and stability.3. Performance of electrochemical biosensor based on spontaneous cascade DNA branch migration reaction: this method showed good analytical performance; detection limit is 85 pM; it has high single-base mutation recognition ability.Conclusions:This study presented two novel electrochemical biosensors for detection of mecA gene in MRSA based on target recycling signal amplification strategy and spontaneous cascade DNA branch migration reaction, respectively. Using ISDPR, the sensitivity biosensor was significantly improved with good analytical performance. With the help of dual recognition of cascade DNA branch migration, the detection specificity of biosensor was significantly improved and realized single-base mutation recognition. Simple signal transduction mechanism of both biosensors significantly simplified the preparation and detection of electrochemical biosensor. Importantly, these two electrochemical DNA biosensor could be conveniently applied to other nucleic acids simply by altering the corresponding DNA sequences. Therefore, the proposed twoDNA biosensor has a great potential for further applications in biomedical research, food safety, early clinical diagnosis, and environmental monitoring.