集成核酸提取的实时荧光PCR微全分析系统

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。

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

A real-time polymerase chain reaction (PCR) micro total analysis system (μ-TAS) was constructed by integrating nucleic acid extraction and PCR amplification with real-time fluorescent detection on a same microfluidic chip, allowing fully automated and on-chip analysis. This approach has the advantages such as low sample consumption, fast analysis and simple operation. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A 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 by which micro fluidic velocity (0–10 mL min^?1) could be adjusted, a TEC platform with a precision of temperature control of 0.1 °C, and a CCD detection module. The DNA of human blood was extracted using a silica gel membrane method on the microfluidic chip. The DNA extraction and detection were preset in the μ-TAS. Human blood lysate (20 μL) was loaded into the extraction chamber and then washed at a speed of 2 mL min^?1. DNA and PCR reagents were mixed and then driven into the PCR chamber at a speed of 1 mL min^?1. The reference gene GAPDH in extracted genome DNA was amplified by PCR and verified by melting curve analysis. The results of nucleic acid extraction method on the chip were compared with those obtained using a standard manual centrifuge extraction method. The on-chip PCR amplifications gave obvious amplification curves, with C_T values of 25.3 and 26.9 respectively. The melting temperature of all the amplification products was 89.9 °C. The results validated that the chip-based method and corresponding device could realize the extraction, amplification and detection of nucleic acid automatically.