高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (3): 545-550.doi: 10.7503/cjcu20120902

• 分析化学 • 上一篇    下一篇

一种可绝对定量核酸的数字PCR微流控芯片

朱强远, 杨文秀, 高一博, 于丙文, 邱琳, 周超, 金伟, 金钦汉, 牟颖   

  1. 浙江大学工业控制技术国家重点实验室, 智能系统与控制研究所, 分析仪器研究中心, 杭州 310058
  • 收稿日期:2012-09-29 出版日期:2013-03-10 发布日期:2013-02-18
  • 通讯作者: 牟颖,女,博士,教授,博士生导师,主要从事生命科学新技术、新仪器研究.E-mail:muying@zju.edu.cn E-mail:muying@zju.edu.cn
  • 基金资助:

    国家自然科学基金(批准号:31070772,31270907)和教育部博士学科点基金(批准号:20090101110136)资助.

Microfluidic Digital Chip for Absolute Quantification of Nucleic Acid Amplification

ZHU Qiang-Yuan, YANG Wen-Xiu, GAO Yi-Bo, YU Bing-Wen, QIU Lin, ZHOU Chao, JIN Wei, JIN Qin-Han, MU Ying   

  1. Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310058, China
  • Received:2012-09-29 Online:2013-03-10 Published:2013-02-18

摘要:

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.

关键词: 数字聚合酶链式反应, 微流控芯片, 单分子扩增, 核酸定量, 绝对定量

Abstract:

A novel microfluidic digital polymerase chain reaction(PCR) chip for single molecule amplification and absolute quantification of nucleic acid was fabricated by multilayer soft lithography technique and composed of three layers with valves controlling liquid, the material of silicone elastomer polydimethylsiloxane(PDMS) and glass coverslip. The microfluidic chip is equal to a piece of glass coverslip in size, which contains 4 separate panels, and each panel contains 640 independent 6 nL-chambers; the chip is capable of detecting 4 samples simultaneously. Digital PCR on the microfluidic chip was tested quantitatively using 18sRNA cDNA from A549. The sample was serially diluted and target DNA molecules were randomly distributed in chip, which can be described by Poisson distribution. If a panel contains template DNA much less than on average 0.5 template molecules per chamber, then there would be 0 or 1 copy in a chamber; the chambers containing template DNA are amplified by PCR and analyzed to be positive, while the chambers without template molecule are analyzed to be negative, the copy number of target DNA molecules of the sample can be read out accurately just by counting positive reactions. The result has proved the feasibility and flexibility of the microfluidic chip that single molecule amplification and absolute quantification of nucleic acid amplification can be succeeded. The design of the chip has the potential to meet the requirements for the general labs: inexpensive, sensitive, economizing labor time and reagent, and simple operation. It is possible to make the digital PCR technology into ordinary laboratory, and make it become one of the common tools in biology research, especially in the developing world. This technique is useful for molecular genetic analysis in cancer and infectious diseases, single cell analysis, bacterial determination, non-invasive prenatal diagnosis in which many biologists are interested.

Key words: Digital polymerase chain reaction(PCR), Microfluidic chip, Single molecular amplification, Nucleic acid quantification, Absolute quantification

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