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设计与制作了一种基于聚二甲基硅氧烷-玻璃(PDMS-Glass)的多功能集成式液滴数字聚合酶链式反应(ddPCR)芯片,该芯片由产生液滴的PDMS模块和收集液滴的玻璃腔体模块组成。PDMS模块采用双通道的T形结构设计,液滴产生速度快且通量高,在30 min内可生成2×10~6个直径约为20μm的微液滴。玻璃腔体模块中存储的液滴在整个实验过程中无需转移,可直接在原位PCR仪上进行扩增,每个液滴均是一个微反应器,经过多次热循环后,液滴仍能保持良好的稳定性。选用副溶血性弧菌(VP)作为食源性致病菌的研究模型,考察了ddPCR芯片对其基因组DNA的绝对定量能力,结果表明,该ddPCR芯片对VP基因组DNA绝对定量的线性范围宽,可跨越5个数量级(10~1~10~6 copies/μL),定量结果与DNA理论参考浓度间有很好的相关性。
A multifunctional integrated PDMS-glass chip based on polydimethylsiloxane-glass (PDMS-Glass) was designed and fabricated. This chip consisted of PDMS module to generate droplets and collect Droplet glass cavity module. The PDMS module adopts a two-channel T-shaped structure design. The droplet generation speed is high and the flux is high. Within 30 min, 2 × 10 ~ 6 droplets with a diameter of about 20μm can be generated. Droplets stored in the glass cavity module do not need to be transferred throughout the experiment and can be directly amplified on an in-situ PCR machine. Each droplet is a microreactor. After several thermal cycles, the droplets remain Can maintain good stability. Vibrio parahaemolyticus (VP) was selected as the research model of food-borne pathogenic bacteria to investigate the absolute quantification ability of ddPCR on its genomic DNA. The results showed that the ddPCR chip has a wide linear range of absolute quantitative determination of VP genomic DNA, It can span 5 orders of magnitude (10 ~ 1 ~ 10 ~ 6 copies / μL). The quantitative results have a good correlation with the DNA reference concentration.