Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens
Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens
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用于高通量快速检测食源性细菌病原体的多通道振荡流多重 PCR 微流体
DOI：
10.1007/s10544-011-9558-y
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发表时间：
2011-10-01
影响因子：
2.8
通讯作者：
Xing, Da
中科院分区：
文献类型：
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作者：
Zhang, Chunsun;Wang, Haiying;Xing, Da
在连续流动PCR领域，单一反应溶液中的扩增通量低，并且通常使用单重PCR。在这项工作中，我们报告了一种基于流动的多重PCR微流控系统，能够进行高通量和快速的DNA扩增检测食源性细菌病原体。作为示范，与三种不同食源性病原体相关的DNA靶标的混合物被包括在单个PCR溶液中。然后，溶液以振荡方式流过并入三个温度区的微通道。实验结果表明，聚合酶浓度、循环次数、循环次数和模板浓度对多重PCR反应的影响较大。实验结果表明，在100 μl/min（70 μl/min）的流量下，仅需5 μl的流动式多重PCR，经过35个循环（25个循环）后，约13 min即可完成，约为常规PCR所需时间（70 min）的1/6。利用目前设计的DNA样品模型，在30 μl/min时可以检测到的最小目标浓度为9.8 × 10−2ng/μl（278-bp，S. enterica），11.2 × 10 - 2ng/μl（168 bp，E. coliO157：H7）和2.88 × 10−2ng/μl（106-bp，L.单核细胞增多症），分别对应于约3.72 × 104 copies/μl、3.58 × 104 copies/μl和1.79 × 104 copies/μl。这种水平的速度和灵敏度与大多数其他连续流PCR系统中可实现的速度和灵敏度相当。此外，四个独立的通道用于并行地实现来自不同食物来源的三个不同DNA样品的多靶点PCR分析，从而实现另一个层次的多路复用。
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 × 10−2ng/μl (278-bp,S. enterica), 11.2 × 10−2ng/μl (168-bp,E. coliO157: H7), and 2.88 × 10−2ng/μl (106-bp,L. monocytogenes), which corresponds to approximately 3.72 × 104copies/μl, 3.58 × 104copies/μl, and 1.79 × 104copies/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.