The Detection Of Food Borne Pathogen’s DNA Based On Logic Gate Technique

Molecular logic gate was proposed by De Silva and his team for the first time in1993, they constructed a molecular switch based on photoinduced electron transfer, the work did great contributes to the development of molecular logic gates and published in Nature. In this thesis, we detected three kinds of food borne pathogens character DNA by using DNA molecular logic gate technique combined with electrochemical methods."We successfully detected Escherichia coli DNA, Shigella DNA and Salmonella DNA by construction of redox-tag labeled hairpin DNA molecular logic gate, redox-tag labeled hairpin stem three helix structure DNA molecular logic gates and label free triplex structure of DNA molecular logic gates. The main research contents of this thesis are as follows:(l)As the detection of foodbome pathogenic bacteria do great contribute to ensure food safety, we mainly discussed the detection methods of foodborne pathogenic bacteria in introduction part. By given the contrast of modern methods and traditional methods, introduced the detection method of this thesis. We combined the electrochemical methods with molecular logic gate technology for food borne pathogen detection in this thesis. The second part of introduction introduces the concept of molecular logic gates and research progress of DNA molecular logic gates closely related and the research method of the thesis.(2)An "OR" logic gate based on ferrocene labeled harpin probe which can detect coli-DNA and Shi DNA simultaneously is constucted. According to literature and the related database, simulation calculation of Escherichia coli, Shigella feature gene sequence with the help of professional software. The features of DNA as a target molecule, and then, designed the ferrocene labeled hairpin structure DNA probe. We use Escherichia coli, Shigella DNA as inputs, the current intensity signal as outputs in order to construct OR logic gate. When any of the input or both of the inputs occur will result in the current reduction rate>54%, when no target DNA appears, current reducing rate<54%. Results of E. coli-DNA detection:the regression equation:y=-0.0027x+4.575, correlation coefficient R=09962, the linear range was:1.0×10-7-7.0×10-7mol/L, detection limit:1.0×107mol/L. Detection of Shi-DNA results:the regression equation is y=-0.0037x+4.54, correlation coefficient R=0.9973, the linear range is:1.0×10-7-7.0×10-7mol/L, detection limit:1.0×10-7mol/L.(3)We constuced an "OR" logic gate which can detect Sal-DNA and Shi-DNA simultaneously which based on ferrocene labeled stem-triplex hairpin structure probe.With the help of literature and the related database, simulation calculation of Shigella, Salmonella characteristic gene sequence were made according to professional software. Select the appropriate Shigella, Salmonella characteristic gene sequence as target. Improved in the last experiment principle, we designed ferrocene labeled stem triplex hairpin stucture DNA probe. We use Shigella DNA and Salmonella DNA as inputs, the current intensity signal as outputs in order to construct OR logic gate. When any of the input or both of the inputs occur resulted in the current reducing rate>83%. when no target DNA appears, current reducing rate<83%. Results of Sal-DNA detection:the regression equation:y=-1.291og (Csal/mol)-6.819, the correlation coefficient R=0.9974, linear range:3.0×10-7-2.5×10-11mol/L, detection limit:2.5×10-11nmol/L. The detection of Shi-DNA regression equation y=-1.261og(Cshi/mol)-6.496, the correlation coefficient R=0.0995, the linear range is:3.0×10-7-2.5×10-11mol/L, detection limit:2.0×10-11mol/L.(4)An "AND" logic gate based on triplex DNA which can detect Sal-DNA and Shi DNA simultaneously is constucted. Based on the previous work, select the appropriate probe of Shigella, Salmonella characteristic gene sequence as target molecular. Using methylene blue as the indicator of electrochemical activity, using the interaction between methylene blue and DNA hybridization signal of single stranded DNA, double stranded DNA and triplex DNA response signal as outputs. The construction of the AND logic gate can realize rapid simultaneous detection of Shigella, Salmonella DNA. Detection of Sal-DNA results:the regression equation:y=-1.2071og (Csal/mol) x+14.172, the correlation coefficient R=0.9986, the linear range is:2.0×10-7-3.5×10-11mol/L, detection limit:3.5×10-11mol/L. The detection of Sal-DNA regression equation y=-3.1031og (Cshi/mol)-7.34, the correlation coefficient R=0.9967, the linear range is:2.0×10-7-3.5×10-11mol/L, detection limit:3.5×10-11mol/L.