Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

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 mu l, could be completed in about 13 min after 35 cycles (25 cycles) at 100 mu l/min (70 mu l/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 mu l/min was 9.8 x 10(-2) ng/mu l (278-bp, S. enterica), 11.2 x 10(-2) ng/mu l (168-bp, E. coli O157: H7), and 2.88 x 10(-2) ng/mu l (106-bp, L. monocytogenes), which corresponds to approximately 3.72 x 10(4) copies/mu l, 3.58 x 10(4) copies/mu l, and 1.79 x 10(4) copies/mu l, 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.