Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance

Abstract

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping-or mutant-guided personalized medicine at emergency or source-limited regions.

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens

“…Recently, we have developed an ultra-fast one-pot method “FARPA” (FEN1-aided recombinase polymerase amplification) by combining RPA with invasive reactions for detecting respiratory pathogens, SNPs and ctDNA with one base mutation. 28,29 Although 25 min is enough for FARPA detection, more than 1 hour of turnover time is needed to report the results from samples due to the complicated DNA extraction. In addition, the low detection throughput of the previous FARPA method limits the clinical application for detecting real-world samples in which multiple pathogens, together with antibiotic-resistant genes should be screened at a time.…”