Dong Xiaobin, Tang Zhiqian, Jiang Xiaodan, Fu Qiang, Xu Donglin, Zhang Lulu, Qiu Xianbo
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Talanta. 2023 Oct 16;268(Pt 1):125319. doi: 10.1016/j.talanta.2023.125319.
A real-time centrifugal microfluidic chip with a companion analyzer was developed for highly sensitive, multiplexed nucleic acid detection based on RPA (recombinase polymerase amplification) isothermal amplification. In order to improve the detection sensitivity, two different optimization strategies were systematically studied. Witnessing the high viscosity of RPA reagent, one way was to improve the amplification efficiency by intentionally introducing active mixing based on centrifugal actuation. While the other way was to improve the detection sensitivity by utilizing two-stage amplification. The templates were pre-amplified in the first-stage amplification chamber before they were aliquoted and distributed into a couple of second-stage ones for multiplexed detection. Different mixing methods relative to different actuation time were studied and compared. Similarly, different two-stage amplification modes relative to different time protocols were compared as well. Totally four different amplification modes including with or without mixing, and with or without two-stage amplification, were systematically analyzed and compared. It was found that, the detection sensitivity could be significantly improved by the two-stage amplification with active mixing. Furthermore, as a proof of concept, the performance of the developed microfluidic chip was demonstrated by successfully detecting different genes of African swine fever virus (ASFV) in parallel with high sensitivity.
开发了一种带有配套分析仪的实时离心微流控芯片，用于基于重组酶聚合酶扩增（RPA）等温扩增的高灵敏度、多重核酸检测。为了提高检测灵敏度，系统研究了两种不同的优化策略。鉴于RPA试剂的高粘度，一种方法是基于离心驱动有意引入主动混合来提高扩增效率。而另一种方法是通过利用两级扩增来提高检测灵敏度。模板在第一阶段扩增室中进行预扩增，然后再分装到几个第二阶段扩增室中进行多重检测。研究并比较了相对于不同驱动时间的不同混合方法。同样，也比较了相对于不同时间方案的不同两级扩增模式。系统地分析和比较了总共四种不同的扩增模式，包括有或没有混合以及有或没有两级扩增。结果发现，主动混合的两级扩增可以显著提高检测灵敏度。此外，作为概念验证，通过成功并行高灵敏度检测非洲猪瘟病毒（ASFV）的不同基因，证明了所开发微流控芯片的性能。
