An Integrated, Real-Time Convective PCR System for Isolation, Amplification, and Detection of Nucleic Acids
Guijun Miao,
Meng Guo,
Ke Li,
Xiangzhong Ye,
Michael G. Mauk,
Shengxiang Ge,
Ningshao Xia,
Duli Yu,
Xianbo Qiu
Affiliations
Guijun Miao
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Meng Guo
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Ke Li
Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing 102206, China
Xiangzhong Ye
Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing 102206, China
Michael G. Mauk
Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104, USA
Shengxiang Ge
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen 361005, China
Ningshao Xia
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen 361005, China
Duli Yu
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Xianbo Qiu
Institute of Microfluidic Chip Development in Biomedical Engineering, College of Information Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China
Convective PCR (CPCR) can perform rapid nucleic acid amplification by inducing thermal convection to continuously, cyclically driving reagent between different zones of the reactor for spatially separate melting, annealing, and extending in a capillary tube with constant heating temperatures at different locations. CPCR is promoted by incorporating an FTA membrane filter into the capillary tube, which constructs a single convective PCR reactor for both sample preparation and amplification. To simplify fluid control in sample preparation, lysed sample or wash buffer is driven through the membrane filter through centrifugation. A movable resistance heater is used to heat the capillary tube for amplification, and meanwhile, a smartphone camera is adopted to monitor in situ fluorescence signal from the reaction. Different from other existing CPCR systems with the described simple, easy-to-use, integrated, real-time microfluidic CPCR system, rapid nucleic acid analysis can be performed from sample to answer. A couple of critical issues, including wash scheme and reaction temperature, are analyzed for optimized system performance. It is demonstrated that influenza A virus with the reasonable concentration down to 1.0 TCID50/mL can be successfully detected by the integrated microfluidic system within 45 min.
