Study On A Rapid Detection Chip For Foodborne Pathogens

Foodborne pathogenic bacteria refer to pathogens that can cause human diseases through food,cereals or other media.In recent years,food safety accidents caused by foodborne pathogenic bacteria are common in the world and China.The national standard method for pathogen detection has the disadvantages of tedious operation,long detection time and low sensitivity.Loop-mediated isothermal amplification technology(LAMP)is favored by researchers because of its mild reaction conditions.Although LAMP has advantages in all aspects,it still cannot do without nucleic acid extraction,amplification and product analysis.How to simplify and integrate these cumbersome steps has become the key to rapid on-site detection of pathogenic microorganism nucleic acids.In order to solve this problem,this paper combined a microfluidic chip technology to build a paper-based microchip that integrated DNA extraction and LAMP amplification.It has been successfully applied to the detection of pathogenic bacteria in real samples,greatly improving the detection efficiency.Firstly,this study outlines the characteristics and hazards of four important pathogenic bacteria in response to food safety problems caused by foodborne pathogens.By comprehensively discussing the research progress of rapid detection techniques for food-borne pathogens,this study analyzes the future development trend of detection techniques for food-borne pathogens.The theoretical basis of LAMP technology is expounded systematically.Then,based on theoretical research,this paper built a paper-based microchip that is simple and inexpensive to manufacture.In this study,the material selection,structural design,temperature control system,and detection system of the microchip were designed,and the working conditions of the microchip were optimized.Taking E.coli O157:H7 as the target strain,this study tested the performance of the microchip.The results showed that the logarithm of the DNA concentration and the fluorescence intensity value showed a good linear relationship(R~2=0.9889).The sensitivity of the microchip was 0.0134 ng/u L and could simultaneously detect four pathogens(E.coli O157:H7,Salmonella spp.,S.aureus and V.parahaemolyticus).The microchip has been proven to have high sensitivity,specificity and high-throughput detection performance.Finally,the microchip was transformed into a paper-based microchip integrating DNA extraction and LAMP amplification.Salmonella was used as the target strain.We tested the simulated real sample(milk added with Salmonella spp.).The results showed that the logarithm of the bacterial concentration and the fluorescence intensity value had a linear relationship(R~2=0.9896).The minimum detection concentration of the microchip was 12 CFU/m L.It taked about 68 minutes from bacterial lysis to fluorescence detection.Finally,the national standard method and the microchip method were used to detect the same batch of blind samples.The detection results of the two methods were consistent.The theoretical value of the blind sample concentration was 5.1 10~3 CFU/m L.In short,this paper build a paper-based microchip that integrates DNA extraction and LAMP amplification based on LAMP technology.It has the advantages of simple fabrication,low cost,fast,strong specificity,high sensitivity and high throughput detection.In the future,it is expected to form a high-performance and high-efficiency professional instrument for the immediate detection of microorganisms.