液滴数字聚合酶链式反应芯片及其在致病菌检测中的应用

摘要

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2×106个直径约为20 μm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(101 ～ 106 copies/μL),定量结果与DNA理论参考浓度间有很好的相关性.%A polydimethylsiloxane-glass(PDMS-Glass) based droplet digital polymerase chain reaction (ddPCR) chip was designed and fabricated,which was integrated with multiple functions.The PDMS-Glass chip was composed of two modules,of which one was a PDMS module for droplet generation,and the other was a multi-functional glass chamber for droplet collection,in situ ddPCR reaction and on-chip fluorescence readout.The PDMS module has a T-shaped structure with two channels,where the aqueous phase with sample was cut into water-in-oil droplets by the oil phase.The droplets generated by the PDMS-Glass chip have the advantages of high-throughput and high-frequency.About two million uniform droplets with an average diameter of 20 μm could be produced within 30 minutes.The glass chamber was employed for droplets collection.No transfer of the droplets were required during the whole experiment.The droplets filled in the glass chamber were directly injected into the in situ PCR instrument for amplification reaction,where each droplet was a micro-reactor.The glass chamber provided the ddPCR with a good environment for ddPCR amplification,where the droplets kept stable after many times of thermal cycling.As one of the common pathogenic bacteria,Vibrio Parahemolyticus(VP) was selected to investigate the property of the PDMS-Glass based ddPCR chip on the aspect of absolute quantificaiton.The results showed that the ddPCR chip has a wide linear range of five-order-magnitude towards the genomic DNA of VP from 101 to 106 copies/μL.Meanwhile,the detected results by ddPCR approach have a good relativity with the theoretical expected DNA concentration.