On-site DNA extraction method for the detection of Escherichia coli (Migula)Castellani and Chalmers O157:H7 using a DNA-based nanobiosensor

Escherichia coli O157:H7 (ECO157:H7) is one of the serotypes belonging to the enterohemorrhagic E. coli (EHEC) group causing hemorrhagic colitis and hemolytic uremic syndrome which can be fatal. The minimum infectious dose is approximately 10 cells thus, on-site detection of this pathogenic bacteria such as ECO157:H7. However, DNA-based nanobiosensor requires rapid and simple DNA extraction method prior to detection. Hence, this study was aimed to developing a simple ECO157:H7 genomic DNA extraction method can be applied on-spite for nanobiosensor-based detection. A Philippine clinical isolate of ECO157:H7 BIOTECH 10307 was used in this study. Five different bacterial genomic DNA extraction protocols were designed, and were evaluated using different concentrations. These protocols included: (1)boiling method, (2)boiling coupled with centrifugation method, (3)use of magnetic microparticles, (4)use of electrochemically active magnetic nanoparticles (EAM-NP), and (5) use of magnetic naked nanoparticles FE2O3 NP). A commercially available DNA extraction kit (Zymo Research, CA) was used to control protocol. Analysis of the extracted DNAs using gel electrophoresis showed visisble band for the genomic DNAs using these methods, together with the ECO157:H7 PCR amplified stxl gene, were utilized in the nanobiosensor-based detection system. All the extracted and PCR-amplified DNAs were individually hybridized with the detector probe labelled EAM-NP-(DP) and biotinylated capture probes (BCP). The target DNA hybrids were detected on the streptavidin modified screen-printed carbon electrodes (SPCE) electrochemically using cyclic voltammetry. Cyclic voltammograms (current versus potential curves) were obained to determine the catholic (reduction) and anodic (oxidation) peak potentials of the target hybrids. Results showed that only the stxl PCR-amplified DNA and Fe2O3 NP extracted DNA were clearly detected as indicated by the distinct anodic and cathodic peak signals. However, the genomic DNA extracted using MP and EAM-NP methods did not produce distinct redox peak signals. Based on these results, the use of Fe2O3 NP extracting DNA was the best method for nanobiosensor-based detection. Therefore, the use of Fe2O3 NP method was evaluated using five different cell concentrations, which included 1 cell; 10 cells; 1000 cells; 100000 cells; and 10000000 cells. The extracted DNAs which were quantified using spectrophometry revealed that 11.6 ng/muL can be extracted from on cell. About 1.0 ng/muL from this extracted DNA was prepared and utilized for the nanobiosensor-based detection. Electrochemical detection of hybrids showed distinct anodic and cathodic peak signals indicating that dual hybridization occurred between the DNA and the probes. These results imply that the nanobiosensor-based ECO157:H7 detection system can detect one cell. Validation of the Fe2O3 NP extraction method was done using 50 mL tap water spike-inoculated with 2.6 x 10 sup 5 cells. About 500 muL of the inoculated water was used for genomic DNA extraction and approximately 12.8 ng/muL with A260/A280 ratio of 1.4 was obtained. Dilution was done to achieve approximately 1.0 ng/muL of DNA which was utilized for nanobiosensor detection. Cyclic voltammograms showed distinct redox peak signals comparable to a signals obtained using pure culture. Based on all results obtained in this study, the use of magnetic naked nanoparticles is efficient in extracting genomic DNA for DNA-based nanobiosensor detection which can be applied on-site.