集成核酸提取的实时荧光PCR微全分析系统  

An Integrated Nucleic Acid Extraction Microchip for Real-time Polymerase Chain Reaction Micro Total Analysis System

作　　者：赵树弥 朱灵 朱灿灿 李阳 王华东 张龙 堵棣威 邓国庆[1,2] 王安 刘勇[1,2] 

机构地区：[1]中国科学院安徽光学精密机械研究所,安徽省生物医学光学仪器工程技术研究中心,合肥230031 [2]皖江新兴产业技术发展中心,铜陵244000

出　　处：《分析化学》2014年第10期1393-1399,共7页Chinese Journal of Analytical Chemistry

基　　金：中国科学院战略性先导科技专项基金(No.XDA08040109)资助项目

摘　　要：集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合，在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭，具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模，使用组合模具法和注塑法制作具有3D通道的PDMS基片，与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节（0～10 mL/min）的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品，采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行，以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗；以1 mL/min的流体驱动速度完成DNA洗脱，抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象，并以传统手工提取为对照，在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示，扩增曲线明显， Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致，均为89.9℃。结果表明，此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。A real-time polymerase chain reaction （ PCR ） micro total analysis system （μ-TAS ） integrated nucleic acid extraction, PCR amplification and real-time-fluorescent PCR detection on a same microfluidic chip was prepared for the fully automated and on-chip analysis of nucleic acid. The proposed method had the advantage such as low sample consumption, fast analysis and simple operation and so on. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A＆amp;nbsp;polydimethylsiloxane （PDMS） substrate with 3D channels was manufactured by a combination of molds and an injection molding method. The glass substrate and the chip were bonded together using a plasma treatment. The μ-TAS included a microfluidic control device whose micro fluidic velocity （ 0-10 mL/min ） could be adjusted, a TEC platform which the precision of temperature control was 0. 1℃ and a CCD detection module. The DNA of human blood was extracted by using a silica gel membrane method on the microfluidic chip. The processes of DNA extraction and detection were preset in the μ-TAS. Human blood lysate （ 20 μL ） were driven to the extraction chamber and was then washed. The fluidic drive speed was 2 mL/min. DNA and PCR reagents were mixed and then were driven into the PCR chamber. The fluidic drive speed was 1 mL/min. The GAPDH gene in extracted genome DNA was amplified by PCR and detected. The amplified product was verified by melting analysis. The results of nucleic acid extraction method on the chip were compared to those obtained using a standard manual centrifuge extraction method. The amplification curves were obvious. Ct values of the chip method were 25 . 3 and 26 . 9 . The denaturation temperature of all the melting was 89 . 9 ℃. The results validated that the chip-based method and device realized the extraction of nucleic acid, amplification and detection automatically.

关 键 词：核酸提取 微全分析系统 实时荧光PCR 微流控芯片 

分 类 号：O629.74[理学—有机化学] O657.3[理学—化学]