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
发布时间：2018-09-05 14:04
【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2×10~6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。
[Abstract]:A multifunctional integrated liquid drop digital polymerase chain reaction (ddPCR) chip based on polydimethylsiloxane glass (PDMS-Glass) was designed and fabricated. The chip is composed of droplet generation PDMS module and glass cavity module. PDMS module is designed with a two-channel T-shaped structure. The droplet can produce 2 脳 10 ~ 6 microdroplets with a diameter of about 20 渭 m in 30 min. The droplets stored in the glass chamber module can be amplified directly on the in-situ PCR instrument without transfer during the whole experiment. Each droplet is a microreactor. After several thermal cycles, the droplet can still maintain good stability. Vibrio parahaemolyticus (VP) was selected as the research model of foodborne pathogenic bacteria. The absolute quantitative ability of ddPCR chip to its genomic DNA was investigated. The results showed that the linear range of absolute quantification of ddPCR chip to VP genome DNA was wide. There is a good correlation between the quantitative results of 5 orders of magnitude (10 ~ (-1) ~ 10 ~ (6) copies/ 渭 L), and the theoretical reference concentration of DNA.
【作者单位】： 上海海洋大学食品学院;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;农业部水产品贮藏保鲜质量安全风险评估实验室(上海);上海海洋大学食品学院食品热加工工程技术研究中心;
【基金】：国家自然科学基金(21405167,81472751,61271162) 上海晨光计划(15CG54) 国家科技重大专项(2017ZX10302201-005-006)
【分类号】：R155.3
，
本文编号：2224498
[Abstract]:A multifunctional integrated liquid drop digital polymerase chain reaction (ddPCR) chip based on polydimethylsiloxane glass (PDMS-Glass) was designed and fabricated. The chip is composed of droplet generation PDMS module and glass cavity module. PDMS module is designed with a two-channel T-shaped structure. The droplet can produce 2 脳 10 ~ 6 microdroplets with a diameter of about 20 渭 m in 30 min. The droplets stored in the glass chamber module can be amplified directly on the in-situ PCR instrument without transfer during the whole experiment. Each droplet is a microreactor. After several thermal cycles, the droplet can still maintain good stability. Vibrio parahaemolyticus (VP) was selected as the research model of foodborne pathogenic bacteria. The absolute quantitative ability of ddPCR chip to its genomic DNA was investigated. The results showed that the linear range of absolute quantification of ddPCR chip to VP genome DNA was wide. There is a good correlation between the quantitative results of 5 orders of magnitude (10 ~ (-1) ~ 10 ~ (6) copies/ 渭 L), and the theoretical reference concentration of DNA.
【作者单位】： 上海海洋大学食品学院;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;农业部水产品贮藏保鲜质量安全风险评估实验室(上海);上海海洋大学食品学院食品热加工工程技术研究中心;
【基金】：国家自然科学基金(21405167,81472751,61271162) 上海晨光计划(15CG54) 国家科技重大专项(2017ZX10302201-005-006)
【分类号】：R155.3
，
本文编号：2224498
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