title: Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne pathogen detection.
abstract: 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 19min, and the limit of detection was low, down to 10(2) copiesμL(-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.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Bowen
authors.initials: B
authors.lastname: Shu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. Electronic address: zhangcs_scnu@126.com.
authors.forename: Chunsun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. Electronic address: xingda@scnu.edu.cn.
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D005516:Food Microbiology; D008461:Meat Products / Q000382:microbiology*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008892:Milk / Q000382:microbiology*; D028521:Musa / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: Continuous-flow; Foodborne pathogens; High-throughput; Microfluidics; Multiplex polymerase chain reaction; Segmented-flow