Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics
Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics
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DOI：
10.1039/b812515e
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发表时间：
2008-01-01
期刊：
影响因子：
6.1
通讯作者：
Lee, Luke P.
中科院分区：
文献类型：
--
作者：
Dimov, Ivan K.;Garcia-Cordero, Jose L.;Lee, Luke P.
我们展示了第一个集成微流控 tmRNA 纯化和基于核酸序列的扩增 (NASBA) 设备，并结合了实时检测。实时扩增和检测步骤可在 3 分钟内从 100 个裂解细菌的芯片纯化 RNA 中产生病原体特异性反应。芯片上 RNA 纯化采用新的硅珠固定方法。片上扩增采用定制设计的高选择性引物，实时检测采用分子信标荧光探针技术；两者都与 NASBA 集成在片上。 tmRNA (10Sa RNA) 存在于所有细菌中，包含生物体特异性识别序列，相对于 mRNA 表现出异常高的稳定性，并且每个生物体具有高拷贝数；后两个因素提高了检测限，加快了阳性反应的时间，并且非常适合这种方法来检测少量细菌。通过在整个过程 30 分钟内对 100 μL 粗裂解液中的 100 个大肠杆菌进行集成芯片纯化、扩增和实时检测，证明了设备的功效。
We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 mu L of crude lysate in under 30 min for the entire process.