科研之友
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摘要
Molecular biological methods such as PCR and LAMP, which are based on nucleic acid amplification, is becoming more and more popular because of their shorter reaction time and real-time detection ability. This study aimed to develop a micro-nanofluidic bio-reaction detecting system for isothermal DNA amplification and sensitive real-time detection of the amplification for sequence-special molecular identification. The identification system contains a plastic micro-nanofluidic chip and a real-time confocal detector. The DNA amplification method is based on loop-mediated isothermal amplification (LAMP). The primary chip was designed with the SolidWorks (TM) and fabricated from the optical disk production line with multiple parallel bioreactors in micro-nano liter in volume. Listeria moncytogenes, a bacterial species that causes diarrhea was chosen as the biological test object. The response time of the micro-nanofluidic system with a volume of 1.45 mu L was reduced to 45 minutes. The detection limit was as low as 1 DNA copy. The detector provided highly sensitive multi-target parallel detection in real time and low background noise. Chip's inert surface, UNG and dUTP in the reaction system were able to improve amplification stability in micro-nanoliter reaction assays. Our system can be developed to a novel high throughput lab-on-a-chip device and is promising to be used for single-molecule droplet amplification. It also has the potential for applications such as nanomedicine, clinical molecular diagnostics, and nanobiotechnology.
- 出版日期2015-5
- 单位南方医科大学; 清华大学; 中国人民解放军总医院
