Journal
ANALYTICA CHIMICA ACTA
Volume 826, Issue -, Pages 51-60Publisher
ELSEVIER
DOI: 10.1016/j.aca.2014.04.017
Keywords
Microfluidics; Segmented-flow; Continuous-flow; Multiplex polymerase chain reaction; High-throughput; Foodborne pathogens
Categories
Funding
- National Natural Science Foundation of China [61072030]
- National Basic Research Program of China [2010CB732602]
- Key Program of NSFC-Guangdong Joint Funds of China [U0931005]
- Scientific Research Foundation of Graduate School of South China Normal University [2013kyjj005]
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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 x 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 10(2) copies mu L-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 x 3 with no detectable cross-contamination. (c) 2014 Elsevier B. V. All rights reserved.
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