Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne pathogen detection

•A high-throughput and rapid microfluidic method for pathogen detection is proposed.•The method offers a simple and convenient way toward high-throughput DNA analysis.•Important parameters of segmented continuous-flow multiplex PCR were investigated.•The proposed method is suitable for high-throughput biomedical monitoring.

High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m × n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19 min, and the limit of detection was low, down to 102 copies μL−1. As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4 × 3 with no detectable cross-contamination.

Graphical abstractOn a spiral-channel microfluidic platform, the high-throughput and rapid amplification for multiple foodborne bacterial pathogens was developed via the segmented continuous-flow multiplex PCR. Fig. 1 showed the design principle of spiral-channel segmented continuous-flow multiplex PCR amplification.Figure optionsDownload full-size imageDownload as PowerPoint slide