FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Ma, Yi; Wu, Haiping; Chen, Shan; Xie, Chao; Hu, Jingjing; Qi, Xinxin; Ma, Xin; Chu, Yanan; Shan, Jingwen; Lü, Yan; Cui, Lunbiao; Zou, Bingjie; Zhou, Guohua

doi:10.1016/j.bios.2023.115456

Cited by 7 publications

(4 citation statements)

References 43 publications

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“…Previously, we found that the concentrations of FEN1 and probe DP affected the specificity and sensitivity of FARPA, 29 therefore, it is necessary to optimize the reaction conditions. FARPA with a series of concentrations of FEN1 and DP was performed individually to detect targets with different amounts.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Huang,

Yue,

Wei

et al. 2024

Analyst

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Huang,

Yue,

Wei

et al. 2024

Analyst

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“…Using this RPA primer pair, 300 U/μL FEN1 for conventional FARPA 37 was employed to detect the L858R mutant in the artificial samples with low mutation abundances (0, 0.1, and 1%). Unfortunately, the sample with the 1% mutant did not give a high signal (Figure S3A).…”

Section: Logic Of Farpa For Single-base Mutation Detectionmentioning

confidence: 99%

“…We have proved that invasive reactions could be used to detect single nucleotide polymorphisms (SNPs) when coupling RPA to amplify the targets; however, it is not a one-pot format, and open-tube operation is necessary after RPA due to the incompatibility between the two reaction systems. Recently, we have developed a novel method called FEN1-aided RPA (FARPA) to solve this issue, and the two reaction systems could be compatible in a homogeneous way. Although FARPA has been demonstrated to be very effective in pathogen detection as a POCT tool, it is unclear whether FARPA could be well used for liquid biopsy.…”

Section: Introductionmentioning

confidence: 99%

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

Ma,

Chu,

et al. 2023

Anal. Chem.

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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.

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A point-of-care single nucleotide variation assay based on strand-displacement-triggered recombinase polymerase amplification

Zhang,

Xu,

Zhang

et al. 2024

Sensors and Actuators B: Chemical

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FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Cited by 7 publications

References 43 publications

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

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

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

A point-of-care single nucleotide variation assay based on strand-displacement-triggered recombinase polymerase amplification

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