A quantitative gene detection technique targeting the pathogenic E. coli O157:H7 eaeA gene was developed using magnetic bead (MB)-quantum dots (QDs) nanoparticle complexes. MBs allowed for the separation of DNA-conjugated QD nanoparticles via magnetic field manipulation. QDs provided internal fluorescence calibration to account for the intrinsically different numbers of nanoparticles interrogated in each assay. Based on the measurement of normalized fluorescence (Cy3/QD655), the linear quantification ranges of ssDNA and dsDNA targets were determined to be 10 through 103 fM (R 2 = 0.992) and 2 × 102 through 6 × 10 7 gene copies (R2 = 0.972), with detection limits of 9.72 fM and 104 gene copies, respectively. The kinetic results indicate that adjustment of hybridization temperature in accordance to the amount of target DNA was required to maximize the efficiency of DNA hybridization. We were able to discriminate perfectly matched target DNA, 1-, 2-, and 41-base pair mismatched target DNAs in our approach and therefore demonstrated excellent selectivity. Our technique was also used on pure bacterial culture to showcase its ability to analyze environmental samples.
Bibliographical noteFunding Information:
This work was supported by a National Research Foundation of Korea Grant funded by the Korean Government (NRF-2009-52-D00169) and Auburn University start-up funds.
- eaeA gene
- Escherichia coli O157:H7
- magnetic bead, quantum dot