One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection.

Abstract

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.