Study On Electrochemical Sensing Of Pathogenic Bacteria Nucleic Acid Based On Gene Editing Technology

Although disease outbreaks caused by pathogenic bacteria have declined with ongoing efforts to improve public health,they remain a pressing global problem.In addition,growing antimicrobial resistance in p athogenic bacteria threatens the effective prevention and treatment of infections,reminding humanity of the urgency and importance of increased surveillance and prevention of the growing risk of these pathogenic bacteria.In Chapter 1,various pathogenic bacteria detection and identification methods that are widely used at this stage are summarized.The pathogenic bacteria can be systematically detected by traditional culture identification methods and gene sequencing methods,both of which have some limit ations,such as the long identification time of the traditional culture method;gene sequencing,although capable of accurate detection,requires expensive detection instruments.Immunologyand aptamer-based assays have high specificity,but the difficult y of obtaining the corresponding antibodies and aptamers limits the widespread use of these two methods.Various other nucleic acid or gene-based detection methods,which have greatly improved in terms of detection sensitivity and detection time,are based on the principle of nucleic acid complementary pairing,and the nature of nucleic acid complementary pairing allowing the presence of base mismatches dictates that the method cannot achieve accurate detection.Therefore,there is an urgent need to explore new easy-to-operate and low-cost techniques for the systematic detection of pathogenic bacteria,not only to avoid the expensive instruments currently used in molecular biology and to find new means of detection,but also to be able to distinguish single base mismatches at a level that enables systematic and sensitive on-site systematic rapid detection.Transcription activator-like effector nucleases(TALEs)are secreted by the plant pathogen Xanthomonas campestris upon infection of various host species an d promote bacterial infection or trigger defense by binding to promoter regions to activate effector-specific genes or R genes in the host plant.TALEs recognize specific DNA sequences through a DNA-binding structural domain consisting of almost identical 34 amino acid repeat units and can bind to different DNA sequences by changing repeat variable residues(RVD).And when the target DNA length is between 17 and 19 bp,TALEs have the highest specificity and exhibit strong recognition of single-base mismatches.In Chapter 2,the RVDs sequence of TALEs was designed by combining the universal target sequence of pathogenic bacteria.With the "Golden Gate" assembly method of TALEs,the purity of 95% and 38 mg/m L.Finally,the binding rate of purified TALEs and DNA was 86% by electrophoretic mobility shift assay(EMSA).In Chapter 3,we designed a pathogenic pathogen SPQC sensor with selected 16 S r RNA base sequence fragments available as a pathogenic pathogen marker as the detection target and TALEs proteins that specifically recognize the nucleic acid target duplex as the recognition molecule.In the presence of pathogenic bacterial nucleic acid targets,the target strands hybridized to the capture strand to form duplexes,which were specifically recognized and bound by the TALEs nucleases,so that the subsequent HCR(hybridization chain reaction)couldn’t occur.TALEs nucleases couldn’t recognize capture probes and duplexes containing mismatched bases,so they couldn’t bind to the electrode surface.However,they could trigger HCR amplification reaction after adding the initiator chains and hairpin probes.Changes in the electrode surfaces under different conditions were further amplified by the silver staining technique.SPQC sensors could make sensitive responses to this difference,and it is possible to achieve accurate detection of pathogenic bacteria represented by E.coli at25 CFU/m L within 3 hours and pathogenic bacteria could be detected by recording the response of SPQC sensor.Isc B,a nuclease encoded by a subset of the IS200/IS605 transposon family,is most likely the ancestor of the RNA-guided endonuclease Cas 9.Isc B-ωRNA has the same function as CRISPR/Cas9,and the currently proven Isc B-ωRNA can not only recognize and cleave 15-35 bp DNA target duplexes,but also distinguish 16-bp single base mismatches.In Chapter 4,we purified the Isc B nuclease by prokaryotic expression and used it as a recognition molecule to construct a sensor for rapid detection of nucleic acid targets.The capture chains modified with nanoparticles in the sensor were attached to the electrode surface,and the target DNA sequences hybridized with the capture probes to form duplexes.Isc B-ωRNA were able to recognize and cleave the double-stranded DNA,and then the gold nanoparticles were moved away from the electrode surface in the presence of proteinase K.If the target DNA sequence hybridized with the capture strands to form double strands containing mismatched bases,the Isc B-ωRNA couldn’t cleave the target DNA,so the capture probes with gold particles remained attached to the electrode surface through the double strand s.These changes could be sensitively detected by SPQC,thus achievin g the goal of detecting target sequences and single-base mismatches.This method was capable of accurate detection of E.coli within 1.5 hours and the limit of detection was 90CFU/mL.Clusters of regularly spaced short palindromic repeats and associated n uclease(CRISPR/Cas)systems are present in prokaryotes to mediate adaptive immune defense of bacteria against viruses or invading nucleic acids.In this system cr RNA-tracr RNA,also known as guide RNA(sg RNA),recognizes a 20-bp target sequence on the genome.The Cas9 nuclease can specifically recognize and cleave target sequences and precisely identify single base mismatches 10-bp away from the PAM region.In Chapter 5,we proposed a new MSPQC sensing method based on the CRISPR/Cas9-specific recognition capability for the rapid detection of Mycobacterium tuberculosis(M.TB).First,the capture probes and hairpin probes were utilized to generate DNA lines spanning the electrodes between the electrodes.The capture probes formed DNA duplexes after capturing the M.TB signature nucleic acid target sequences,which could be specifically recognized and cle aved by CRISPR/Cas9 system,resulting in the inability to form silver lines between the electrodes during subsequent silver staining.In the absence of the M.TB signature nucleic acid sequence target,silver lines spanning the electrode interval were forme d after silver staining.This phenomenon could be detected by the MSPQC M.TB sensor,which enables the detection of Mycobacterium tuberculosis.The detection time was down to 2.3 h,and the limit of detection was 30 CFU/mL.