title: Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens.
abstract: In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10(-2) ng/μl (278-bp, S. enterica), 11.2 × 10(-2) ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10(-2) ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10(4) copies/μl, 3.58 × 10(4) copies/μl, and 1.79 × 10(4) copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China. zhangcs@scnu.edu.cn
authors.forename: Chunsun
authors.initials: C
authors.lastname: Zhang
authors.forename: Haiying
authors.initials: H
authors.lastname: Wang
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis; D004867:Equipment Design; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis*; D005517:Foodborne Diseases / Q000382:microbiology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification; D008461:Meat Products / Q000382:microbiology; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D008892:Milk / Q000382:microbiology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D028521:Musa / Q000382:microbiology; D019779:Salmonella enterica / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial