Rapid And Sensitive Strategy For Salmonella Dedection Using An InvA Gene-Based Electrochemical DNA Sensor

Salmonella, as one of the most common pathogens of foodbornedisease worldwide, generally spreads through the consumption ofcontaminated food of animal origin （mainly meat, poultry, eggs and milk）.Pathogenic Salmonella strains cause food poisoning, gastrointestinalinflammation, typhoid fever, and septicemia in both humans and animals. Itconstitutes a major public health burden and represents a significant cost inmany countries. Therefore, sensitive and rapid detection of Salmonella is ofout-most importance to prevent and control the infectious diseases.Electrochemical DNA biosensor has been widely used due to its highsensitivity, simple, fast, good selectivity and low cost. Various methods havebeen used for the detection of Salmonella, including conventional culturemethods, enzyme-linked immunosorbant assay （ELISA） and PCR,but theyall have many disadvantages. In this work, in order to overcome the inherentdisadvantages of traditional methods, a simple strategy for detection ofSalmonella was developed by integrating rapid DNA extraction, specificPCR with an invA gene-based electrochemical DNA sensor. This strategy presented a simple, rapid and sensitive platform for Salmonella detectionand would become a powerful tool for pathogenic microorganisms screeningin clinical diagnostics, food safety and environmental monitoring. Thisdissertation includes the following three parts:Preparation of the invA gene-based electrochemical DNA sensorThe invA gene was used to design specific probes and target forSalmonella by exploring the Gene Bank database. The specificity of primersfor the PCR amplification of invA gene fragment and probes or DNAsensing had been positively verified via the BLAST（Basic Local Alignment Search Tool）. The high sensitivity of the DNAsensor was achieved due to low nonspecific absorption on the electrodesurface, the strong binding of streptavidin-biotin, and efficient signalamplification through catalytic generation of a large number of alkalinephosphatase. The fabrication and hybridization processes were characterizedwith electrochemical impedance spectroscopy, square wave voltammetryand surface plasmon resonance respectively. The linear calibration range fortarget DNA detection was from1pM to10nM with a detection limit of0.5pM and the correlation coefficient of0.9984. DPV responses of the DNAsensor were compared after hybridization with the three differentoligonucleotides, respectively. The reproducibility of the proposed sensorwas investigated by detecting synthetic target DNA at5nM and100pM.The low LOD of the DNA sensor was achieved due to low nonspecific absorption on the electrode surface, the strong binding of streptavidin-biotin,and efficient signal amplification through catalytic generation of a largenumber of alkaline phosphatase. Thus，the electrochemical sensor based onthe highly specific DNA probes for invA gene sequence recognition wassuccessfully developed， which exhibited high sensitivity, satisfactoryselectivity and good reproducibility.PCR amplification of nucleotide sequence within the invA gene ofSalmonellaSalmonella typhimurium strains were cultured under suitableconditions, a simple boiling method was used to extract Salmonella genomeDNA, and PCR was then performed using the genomic DNA extracted fromSalmonella. PCR products were determined by2%agarose gelelectrophoresis and observed under ultraviolet light. The resultsdemonstrated that the amplification of a284bp fragment of invA gene wassuccessfully achieved. PCR was then performed using the genomic DNAextracted from Salmonella with a series of concentrations. PCR product wasdenatured by heating in water bath, and immediately chilled in ice to obtaindenatured ssDNA before detection. The electrochemical DNA sensor wasthen applied to analyze the denatured PCR products. Under optimalconditions, the proposed strategy could quantitatively detect Salmonellafrom10to10⁵CFU mL^-1within3.5h. The sensitivity of the proposedstrategy was further compared with other methods reported previously for the detection of Salmonella including surface plasmon resonance （SPR）,field effect transistor （FET）, fluorescence, magnetoelastic biosensor,capacitive immunosensor, quartz crystal microbalance （QCM）, fiber-opticbiosensor, piezoelectric immunosensor. This proposed strategy possessedthe advantages of excellent sensitivity, rapid detection and low cost, whichwould provide a convenient platform for Salmonella screening inbiomedical diagnostics, food safety, biothreat detection and environmentalmonitoring.