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 × 104 copies/μl, 3.58 × 104 copies/μl, and 1.79 × 104 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.
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Acknowledgements
This research is supported by the National Natural Science Foundation of China (61072030, 30700155), the Key Program of NSFC-Guangdong Joint Funds of China (U0931005), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0829).
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Fig. S1
Effects of Taq DNA polymerase concentration on conventional multiplex PCR. Lane M: the DNA marker; Lanes 1-7: the Taq DNA polymerase concentrations used in the conventional multiplex PCR were 0, 0.025, 0.05, 0.1, 0.2, 0.3, and 0.5 unit/μl, respectively. (DOC 119 kb)
Fig. S2
Effect of various concentrations of BSA on oscillatory-flow multiplex PCR. Lanes 1-5: the oscillatory-flow multiplex PCR reactions were performed on the microfluidic device with five different concentrations of BSA (0, 0.3, 0.5, 0.7, and 0.9 μg/μl , respectively); Lane M: the DNA marker. (DOC 91 kb)
Fig. S3
Influence of the cycle number on the oscillatory-flow multiplex PCR in the case of the flow rate of 50 μl/min. Lane 1: the positive-control multiplex PCR product; Lanes 2-5: the oscillatory-flow multiplex PCR was carried out with the cycle number of 35, 30, 25, and 20, respectively; Lane 6: the negative-control multiplex PCR; Lane M: the DNA marker. (DOC 113 kb)
Fig. S4
Oscillatory-flow multiplex PCR from different concentrations of relatively pure DNA samples extracted by using the bacterial DNA extraction kit with a preliminary lysozyme digestion. Lane M: the DNA marker; Lane 1: the positive-control PCR product; Lane 2: the concentration of each bacterial genomic DNA template was 1.0 ng/μl; Lanes 3-8: the concentrations of the used DNA template samples were the 101, 102, 103, 104, 105, and 106-fold dilutions of the DNA template concentration stated in Lane 2; Lane 9: the negative-control multiplex PCR solution without DNA template flowed at 30 μl/min. (DOC 173 kb)
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Zhang, C., Wang, H. & Xing, D. Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens. Biomed Microdevices 13, 885–897 (2011). https://doi.org/10.1007/s10544-011-9558-y
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DOI: https://doi.org/10.1007/s10544-011-9558-y