A microfluidic droplet digital PCR for simultaneous detection of pathogenic Escherichia coli O157 and Listeria monocytogenes

Sensitive and rapid identification of pathogenic bacterial is extremely important due to the serious threat of pathogens to human health. In this study, we demonstrate the simultaneous and sensitive detection of pathogenic Escherichia coil O157 and Listeria monocytogenes using a novel duplex droplet digital PCR (ddPCR) platform. The ddPCR platform, which uses a mineral oil-saturated polydimethylsiloxane (OSP) chip to overcome the problem of droplet evaporation, integrates the functions of droplet generation, on-chip amplification and end-point fluorescence readout. Simultaneous detection of two kinds of bacterial is achieved by the design of differentially labeled TaqMan-MGB fluorescent probes. Compared with a quantitative real-time PCR approach, the OSP chip-based duplex ddPCR platform exhibits high sensitivity, which is at the level of single molecule resolution without significant cross-assay interference. Moreover, the applicability of the proposed method is also evaluated in artificially contaminated drinking water sample, which displays a low detection limit down to 10 CFU/mL for both pathogenic bacterial within 2 h. (C) 2015 Elsevier B.V. All rights reserved.