Progress 05/01/05 to 12/31/06
Outputs
We designed and developed a sonic mixing flow cell, referred to hereafter as the sample preparation "flow cell". The flow cell used sonic mixing technology to disrupt laminar flow and improve the capture of E. coli by antibodies immobilized on the glass surface of the flow cell. We showed that mixing improved the capture efficiency, and the sonic mixing flow cell was able to capture 8% of the cells from a 10 ml sample in 45 min, in a flow cell volume of 900 ul. In an effort to reduce cost and complexity, we also developed another format of the sample preparation flow cell using capillary tubes and air bubbles to disrupt laminar flow and capture E. coli by antibodies immobilized on the surface of the capillary tube. Again, we showed that mixing improved the capture efficiency, and the air bubble format flow cell was able to capture 2% of the cells from a 5 ml sample in 1 hr, in a flow cell volume of 50 ul. Capture efficiency of 20% is predicted using a 500 ul flow cell.
Preliminary data of the capillary flow cell shows that capillary flow cells can capture more cells than sonic mixing flow cell and provide more DNA copies for PCR. We have developed two simple and rapid DNA extraction methods that are able to extract the genomic DNA from the antibody-captured bacteria in both flow cells. The extracted DNA is subject to real-time PCR assays. We have successfully developed two real-time PCR assays. One is a quantitative real-time PCR assay to determine the numbers of E. coli cells that have been immuno-captured on the sonic mixing or capillary flow cells. The second assay is the multiplex real-time PCR assay for the confirmation and identification of E. coli O157:H7 serotypes and virulence factors. For the quantitative real-time PCR assay, a standard curve was first constructed using a plasmid standard containing E. coli lacZ gene. There was a strong linear inverse relationship (R2 = 0.9997) between threshold cycle (CT) and the log10 number of lacZ
copies over 7 orders of magnitude. The equation describing the relationship is CT =−3.1998xlog10 (lacZ) + 39.54. A master mix of quantitative real-time PCR was prepared and optimized for the quantitative assays. For the multiplex real-time PCR assay, we have designed primers and probes to determine 5 genetic markers on the E. coli O157:H7 genome including the rfbEO157, fliCH7, stx1, stx2 and eaeA gene. The rfbEO157, and fliCH7 genes code for specific somatic and flagellar antigens associated with O157 and H7 serotypes, respectively. The stx1, stx2 and eaeA genes code for Shiga-Like Toxin I, Shiga-Like Toxin II, and Intimn outer membrane proteins responsible for bacterial pathogenesis. The primers and probes for these gene markers have been tested with a panel of 14 E. coli strains with different serotypes. The results obtained from the multiplex real-time PCR assays were consistent with that of serotyping, suggesting the primers and probes developed in this Phase I study were
highly specific. In addition to quantification of E. coli O157:H7 cell numbers, our PCR assays are capable of confirming and identifying bacterial serotypes and virulence factors.
Impacts
The sample preparation flow cells, in conjunction with multiplex real-time PCR, reduces potential interference from PCR inhibitors, minimizes sample splitting, and provides high sensitivity and specificity for rapid detection of water-borne pathogens.
Publications
- Zhu, P., Shelton, D. R., Karns, J. S., Sundaram, A., Li, S., Amstutz, P. and Tang, C.M.(2005) Detection of water-Borne E. coli O157 using the integrated waveguide biosensor, Biosens. Bioelectron. 21, 678-683.
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