| In view of the development trend of portable,miniaturized and integrated analytical instruments in the future,a micro-total chemical analysis system was established by using micromachining technology,among which the development focus was microchip electrophoresis(MCE).This technology takes the chip as the core,and efficiently integrates the preparation,enrichment,derivation,separation and detection of chemical or biological samples on the micrometer scale chip operating platform.It is widely used in the fields of chemistry,biology and molecular science as a powerful nucleic acid analysis technology.However,due to the short optical path of microfluidic chip and the small amount of sample injection,the detection sensitivity is not satisfying.The nucleic acid amplification strategy can take effect by the amplification ability of nucleic acid,amplifing the signal and improving the sensitivity through certain methods.The analysis of pathogenic bacteria is of great significance for food safety and clinical diagnosis.In this paper,the microfluidic chip analysis and nucleic acid signal amplification strategy were combined to efficiently and sensitively detect the pathogenic bacteria(methicillin-resistant S.aureus,S.typhimurium)in the actual samples.Through the optimization of various experimental conditions,the detection sensitivity was improved.The research content of this paper mainly includes the following three parts:Chapter 1,a brief introduction of microchip electrophoresis,basic characteristics,online enrichment technology,detection methods,application fields and various DNA signal amplification strategies,and the specific content and significance of the research were introduced in this paper.Chapter 2,a method based on isothermal strand-displacement polymerase reaction(ISDPR)with MCE was proposed to detect mecA gene in methicillin-resistant Staphylococcus aureus(MRSA).In this work,the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe(HP),and then the primer hybridized with the probe and released the target sequence in the presence of Klenow Fragment exo~-(KF exo~-)polymerase.The released target sequence hybridized with the next HP and then was displaced by the primer again,consequently achieving target recycling and amplification.The amplified products of the HP-c DNA duplex were separated rapidly from other DNAs by MCE.Under optimal conditions,the LOD of the target DNA was as low as 12.3 p M(S/N=3).The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S.aureus,and the experimental results showed that it had some advantages such as being label free,ultrasensitive,rapid and well separated.Chapter 3,An aptamer-mediated two-step strand displacement amplification combined with microchip electrophoresis was proposed for sensitive detection of Salmonella typhimurium.In this work,the specific binding between the aptamer and bacteria was used to achieve signal transformation,and the aptamer-mediated two-step strand displacement amplification combined with microchip electrophoresis was used to sensitively detect Salmonella typhimurium.The trigger DNA sequence was released by the specific binding of bacteria to the aptamer,initiating the two-step cyclic amplification to produce a large number of target single strands DNA.The concentration of aptamer-trigger duplexes,the concentration of template chain C1 and C2,the amount of KF exo~-polymerase and Nb.bpu10I nicking endonuclease and incubation time were optimized.The sensitivity was significantly improved,and the detection limit was as low as 6 CFU/m L.This method was successfully used to detect Salmonella typhimurium in defatted milk samples.And satisfactory results were obtained,which reflected the reliability,accuracy and development potential of this method. |
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