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| Fabrication of a Droplet Digital PCR Chip and Its Application in Pathogenic Bacteria Detection |
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| DOI： |
| KeyWord:digital PCR droplet microfluidic chip pathogenic bacteria Vibrio Parahemolyticus |
| Author | Institution |
| DENG Xue-lei,ZHANG Yuan-yi,YUAN Hao-jun,LIU Song-sheng，CHEN Ying-ying,GAO Wan-lei,ZHOU Hong-bo，JIA Chun-ping，ZHAO Jian-long,BIAN Xiao-jun |
1.上海海洋大学食品学院;2.传感技术国家重点实验室，中国科学院上海微系统与信息技术研究所;3.农业部水产品贮藏保鲜质量安全风险评估实验室上海;4.上海海洋大学食品学院食品热加工工程技术研究中心 |
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| Abstract: |
| 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 20μm could be produced within 30 min. 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. |
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