Ultrasensitive Sensing Strategy For Methicillin-resistant Staphylococcus Aureus Based On Isothermal Amplification

Bacteria can cause the infection of 900 million people every year.Bacterial infection has a significant mortality rate worldwide and has become a huge threat to global public health.Bacterial infections are mainly treated with antibiotics.However,the overuse of antibiotics has led to a dramatic increase in the number of important antibiotic-resistant bacteria,especially multidrug-resistant bacteria.At present,the World Health Organization has listed multidrug-resistant bacteria as a serious threat to human beings.The common methods for multi-drug resistant bacteria detection are mainly divided into three categories:culture,immune detection,drug resistance gene detection.The plate culture method is currently the gold standard method for detection of multi-drug resistant bacteria.The type of drug resistance can be determined by observing the size of the inhibition zone.Immunoassay is a method to detect the specific antigens on the surface of multi-drug resistant bacteria by antigen-antibody reaction.Drug resistant gene detection is to detect the specific gene loci of multiple drug resistant bacteria by a series of amplification techniques such as polymerase chain reaction（PCR）.These methods are both time-consuming,lack of sensitivity,and need professional equipment and technical support,which is difficult to achieve early and rapid detection of infection,causing serious medical burden for patients.In recent years,isothermal amplification technology has developed rapidly,and it is widely used in the detection of pathogenic microorganisms because of its advantages such as rapid,high amplification efficiency,no need of temperature change conditions,and professional equipment support.In this study,two isothermal amplification techniques,Exponential Amplification Reaction（EXPAR）and Palindromic-Powered Transcription Amplification（PPTA）,are used to construct biosensors to detect the specific PBP2a protein and specific mec A gene of MRSA.The following studies are carried out:（1）The signal conversion module based on aptamer functionalized magnetic beads（AFMBs）,the signal amplification module based on EXPAR and the signal readout module based on high performance Mo S₂ nanosheets are effectively integrated to construct the background free MRSA identification technology.AFMBs is formed by connecting amino modified aptamer-blocker double strain DNA with the EDC activating carboxyl modified magnetic beads,and is characterized by Scanning Electron Microscopy,Dynamic Light Scattering,Vibrating Sample Magnetometer and UV/VIS.In the presence of MRSA,it will bind closely with AFMBs and change the conformation of the aptamer,thus releasing the single strain blocker which realizes the conversion from protein signal to nucleic acid signal.The released blocker was combined with the amplification template to trigger the EXPAR achieving signal amplification,and we add tetramethyl ammonium chloride to inhibit its non-specific amplification,which is characterized by polyacrylamide gel electrophoresis.Subsequently,high-performance Mo S₂ nanosheets with adsorption single-strand DNA and fluorescence quenching properties are synthesized by hydrothermal method.Transmission Electron Microscopy,X-ray Photoelectron Spectroscopy and X-ray Diffraction are used to characterize them.Then,a Cy5-labeled single-strand DNA complementary to the blocker was designed and adsorbed on the surface of the Mo S₂nanosheets to form a signal readout module.When the EXPAR amplified block is added,it hybridizes with the Cy5 fluorescent probe to form a double strand and falls off from the surface of the Mo S₂ nanosheets,restoring the fluorescence of Cy5.Under optimized experimental conditions,this strategy shows a good linear relationship in the range of10-10⁶ CFU/m L,and the detection limit can reach 6.39 CFU/m L,and it could effectively distinguish MRSA from multiple cocci,including Staphylococcus aureus.Repeated experiments show that this method has good repeatability,with a relative standard deviation of 2.5%.Water and milk were selected as the actual samples for detection,and the results proved that the MRSA detection method had high reliability.（2）Combined with PPTA,an efficient and practical mec A gene electrochemical sensor is established.A hairpin structure probe（HP）was designed concluding palindrome sequence,mec A gene binding region,T7 promoter sequence and transcription template.When mec A gene closes to the HP probe,it hybridizes with the recognition region and opens the stem loop,exposing the T7 promoter sequence.After recognition by T7 RNA polymerase,the tail of the palindrome sequence is extended as a primer to form a double-stranded T7 promoter,which triggers in vitro transcription and produces a large number of RNA products.These RNA products can be hybridized with the capture probe on the electrode to effectively bind to the interface and perform an electrochemical signal readout using methylene blue（MB）.The feasibility of PPTA was evaluated by polyacrylamide gel electrophoresis.Differential pulsed voltammetry（DPV）was used to verify the feasibility of transcription product capture and electrochemical sensing for mec A gene.Under the optimized experimental conditions,the method has a good linear relationship in the range of 1 f M to 100 n M,the detection limit can reach0.81 f M,and has good repeatability.The two new biosensors constructed in this paper are used to identify MRSA from the two levels:MRSA specific protein and nucleic acid.Both methods showed a short detection period,high sensitivity,good repeatability,and no need of the support of large equipment.It provides a simple and practical platform for the detection of multi-drug resistant bacteria and is expected to become a potential tool for clinical identification of pathogenic microorganisms.