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

• Published in: Angewandte Chemie International Edition Vol. 59; no. 27; pp. 10981 - 10988
• Main Authors: Ouyang, Wei, Han, Jongyoon
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 26.06.2020
• Edition: International ed. in English
• Subjects: Acid resistance; Acids; Amplification; Deoxyribonucleic acid; disease diagnosis; DNA; DNA, Bacterial - isolation & purification; Electrokinetics; Humans; Limit of Detection; Microfluidics; Mycobacterium tuberculosis - genetics; Nucleic acids; Nucleic Acids - analysis; Nucleic Acids - isolation & purification; Oligonucleotide Array Sequence Analysis - methods; Optimization; Polymerase chain reaction; Polymerase Chain Reaction - methods; Purification; Tuberculosis; Workflow; electrokinetics; disease diagnosis; nucleic acids; microfluidics; polymerase chain reaction
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201915788

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. Concentrate (and reconcentrate): Two‐stage selective electrokinetic concentration enables one‐step purification of nucleic acids and microfluidic PCR resistant to non‐specific amplification, thereby significantly shortening the development cycle and simplifying the workflow of nucleic acid amplification tests for point‐of‐care disease diagnosis.