An Integrated, Real-Time Convective PCR System for Isolation, Amplification, and Detection of Nucleic Acids

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.

Automated summary

Convective PCR (CPCR) utilizes thermal convection to continuously drive reagent in a capillary tube, enabling spatially separate melting, annealing, and extending. By incorporating an FTA membrane filter, CPCR constructs a single reactor for both sample preparation and amplification. The system performance is optimized by analyzing critical issues such as wash scheme and reaction temperature.