Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate. / QC 20100824
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:kth-9187 |
| Date | January 2008 |
| Creators | Liu, Yanling |
| Publisher | KTH, Bioprocessteknik, Stockholm : KTH |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Trita-BIO-Report, 1654-2312 ; 2008:16 |
Page generated in 0.0028 seconds