Microfluidic Chip for Direct Detection of E. coli O157:H7 in Ground Beef via Anti-Digoxigenin Immuno-PCR Assay

Microfluidic chip was successfully fabricated from polydimethylsiloxane (PDMS) and used for direct detection of E.coli O157:H7 in ground beef via anti-digoxigenin immuno-PCR. The PDMS channels were functionalized with 100 μg/ml streptavidin at room temperature for 6 min. A recovery of 60% ± 3.4 was obtained when bacterial cells were extracted from seeded ground beef using differential centrifugation. ProMag bindTM IT microspheres were effectively used to capture 66% ± 4.2 of bacterial cells from ground beef extract. TM Biotinylated PCR amplicons of E. coli O157:H7 intimin gene were detected using anti-digoxigenin labeled with FITC. This chip allowed sensitive and rapid detection of a 5 cells/g ground beef while agarose gel electrophoreses failed to detect 50 cells/g ground beef. These microchannels offer pathways to eliminate the time-consuming and messy slab gel process and generate much more reproducible and high quality data. Abbreviations:Intimin gene (eaeA) Shiga toxins (stx1 and stx2) Streptavidin (STA) Biotinylated PCR amplicons labeled with digoxigenin (dual-labeled amplicons) Polydimethylsiloxane (PDMS) Anti-digoxigenin immuno-PCR (anti-dig-IPCR) Mean grey Values (MGVs)