Study On The Detection Of Pathogenic Bacteria Based On Nucleic Acid Amplification Assisted Microchip Electrophoresis

Microchip electrophoresis(MCE),also known as microchip capillary electrophoresis,is a method of capillary electrophoresis separation on microchips.It has the advantages of fast analysis,high separation efficiency,less reagent/sample amount and miniaturization.And microchip gel electrophoresis has been used in the analysis and detection of gene mutations and specific DNA fragments because of its efficient separation and analysis of DNA.There are many kinds of pathogenic bacteria,some of which can cause infection at very low concentrations.In addition,rapid diagnosis of bacterial infection is also important so that treatment can be quickly determined to stop the spread of the disease and reduce the development of drug-resistant bacteria.At present,bacterial detection by microchip electrophoresis is usually through detection of specific DNA fragments of bacteria,or through aptamer specific recognition for signal conversion,the detection of bacteria into a DNA fragment detection.However,even if the laser-induced fluorescence detector with high sensitivity is used in the detection,its detection sensitivity will be limited due to the small sample consumption of MCE,which cannot fully meet the detection requirements of high sensitivity.In recent years,nucleic acid signal amplification technology has been widely used in the field of biomarker sensing and detection,which realized a significant improvement of detection sensitivity.Based on the advance of nucleic acid signal amplification technology,we combined nucleic acid signal amplification technology with MCE,and developed a series of sensitive pathogenic bacteria analysis methods.This paper consists of six chapters:Chapter 1 IntroductionThe characteristics,classification,detection method,principle of DNA separation and signal amplification method of MCE are briefly introduced.The introduce of some commonly used nucleic acid signal amplification methods,the research background and the significance of this paper.Chapter 2 Simultaneous Detection of Different Bacteria by Microchip Electrophoresis Combined with Universal Primer-Duplex Polymerase Chain Reaction.The specific and sensitive detection of multiple pathogens is critical for the prevention and identification of health-and safety-related problems.A microchip electrophoresis/LED-induced fluorescence(MCE-LIF)method,combining an aptamer-based probe and a novel universal primer-duplex polymerase chain reaction(PCR)process(UP-DPCR),was designed to simultaneously detect two kinds of bacteria.The probe consists of a recognition unit(aptamer)for specifically capturing bacterial cells and eventually releasing complementary DNAs(C1 and C2).The two released DNA strands(C1 and C2)can be simultaneously amplified by a pair of universal primers,because of the identical sequences designed at both ends of the two DNA strands.The UP-DPCR products of C1 and C2 can be separated and detected by MCE-LIF,and the heights of the two peaks are correlated with the concentrations of the corresponding bacteria.Here,Salmonella enterica serovar Typhimurium(S.Typhimurium)and Pseudomonas aeruginosa(P.aeruginosa)were detected as a proof of concept.Under optimal conditions,the limits of detection(S/N=3)were 15 CFU mL-1 for S.Typhimurium and 5 CFU mL-1 for P.aeruginosa.This approach was also applied for detecting these two types of bacteria in defatted milk,indicating its potential application in the analysis of real samples.This method can not only simultaneously detect two kinds of bacteria without lysing the bacterial cells,but also simplify duplex PCR with the use of universal primers.Chapter 3 Ultrasensitive Biosensing Pathogenic Bacteria by Combining Aptamer-Induced Catalysed Hairpin Assembly Circle Amplification withMicrochip Electrophoresis.Because foodborne pathogenic bacteria are in a great variety and may cause many infectious diseases even at low concentrations,a highly sensitive and selective method has long-been desired for bacteria detection.In this study,a microchip electrophoretic method for biosensing E.coli O157:H7 was developed by using E.coli O157:H7 aptamer(apt-E)for specific bacteria recognition together with aptamer-induced catalysed hairpin assembly(CHA)for significantly improving the sensitivity of bacteria detection.Briefly,three nucleic acid strands(apt-E,hairpin H1,and H2)were used in the CHA amplification.Because different quantities of H1/H2 complexes were formed due to the circle amplification induced with different amounts of apt-E and the correlation between the concentrations of apt-E and E.coli O157:H7,E.coli O157:H7 thus could be quantified by the detection of H1/H2 complexes with microchip electrophoresis(MCE).Under the optimal conditions,the limit of detection was 75 CFU mL-1.This method was also applied to detect E.coli O157:H7 in defatted milk with a satisfying recovery rate.The proposed strategy for E.coli O157:H7 detection is label-free,enzyme-free,ultra-sensitive,and cost-effective.It is also practical and could be applied to detect other bacteria in food samples.Chapter 4 Probe-lengthening Amplification-assisted Microchip Electrophoresis for Ultrasensitive Bacteria Screening.Probe-lengthening amplification(PLA)is a target-specific nucleic acid amplification method that realizes good selectivity by effectively avoiding nonspecific amplification.Here,we propose a probe-lengthening amplification-assisted microchip electrophoresis(MCE)strategy for sensitive analysis of 16S rRNA genes of five bacteria.In this assay,four specific short probes were designed for a target bacterium to recognize its bacterial 16S rRNA gene,integrated into longer DNA ligation duplexes using Ampligase,and subsequently separated and detected by MCE.Along with the rapid generation of ligation duplexes,this approach provides exponential amplification of nucleic acid signals that are useful for sensitive bacterial quantification.Through tactfully combining PLA and MCE,the detection sensitivity of bacterial genes was significantly improved,and a limit of detection(LOD)of 30 fM was realized for the artificial target DNA.This approach was also applied to detect actual bacterial genomic samples with excellent results,demonstrating the potential application of this methodology in infection diagnosis.Chapter 5 Study on defective T junction-mediated strand displacement amplification and its application in microchip electrophoretic detection of longer bacterial 16S rDNA.Current strand displacement amplification(SDA)-based nucleic acid sensing methods generally rely on a ssDNA template that involves complementary bases to the endonuclease recognition sequence,which has the limitation of detecting only short nucleic acids.Herein,a new SDA method in which the defective T junction structure is first used to support SDA(dT-SDA)was proposed and applied in longer DNA detection.In dT-SDA,an auxiliary probe and a primer were designed to specifically identify the target gene,following the formation of a stable defective T junction structure through proximity hybridization,and the formation of defective T junctions could further trigger cascade SDA cycling to produce numerous ssDNA products.The quantity of these ssDNA products was detected through microchip electrophoresis(MCE)and could be transformed to the concentration of the target gene.Moreover,the applicability of this developed strategy in detecting long genomic DNA was verified by detecting bacterial 16S rDNA.This proposed dT-SDA strategy consumes less time and has satisfactory sensitivity,which has great potential for effective bacterial screening and infection diagnosis.Chapter 6 Biosensing Bacterial 16S rDNA by Microchip Electrophoresis Combined with Transcription Amplification Derived Real-Time crRNA Generation CRISPR-Cas12a Method.Accurate and sensitive detection of bacterial infection at early-stage is highly valuable to prevent the widespread of disease.Recently,CRISPR-Cas12a enzyme derived nucleic acid detection methods are emerging along with the discovery of the indiscriminate single-stranded DNA(ssDNA)cleavage activity of Cas12a.These kinds of nucleic acid detection methods are effective and sensitive by combining with isothermal amplification.However,most of the proposed CRISPR-Cas12a strategies are Cas-crRNA complexes preassembled mode,which exist inevitable nonspecific backgrounds.Besides,the signal ssDNA used in these strategies were need tedious pre-label of signal molecules.Herein,a post-assembly CRISPR-Cas12a method was proposed based on target induced transcription amplification and real-time crRNA generation for bacterial 16S rDNA biosensing.This strategy is label-free through the combination of microchip electrophoresis(MCE)detection.In addition,this method eliminated the need of protospacer adjacent motif(PAM)on target sequences,and has the potential to be an effective and simple method for nucleic acid detection and infectious diseases diagnosis.