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Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system.
- Source :
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Sensors & Actuators B: Chemical . May2023, Vol. 383, pN.PAG-N.PAG. 1p. - Publication Year :
- 2023
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Abstract
- Circulating tumor DNA (ctDNA) is a promising biomarker for real-time, minimally invasive diagnostics and monitoring in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. However, the current methods for ctDNA detection are cumbersome and expensive. While advanced CRISPR/Cas-based assays offer advantages of simplicity, low cost and high sensitivity, their application for ctDNA detection is restricted by the requirement of a protospacer adjacent motif (PAM) near the mutation site and off-target cleavages (i.e., false-positive results) due to the extreme similarities between the mutant and wild-type sequences, especially single nucleotide variants. Herein, we propose a novel strategy comprising recombinase polymerase amplification (RPA) and CRISPR/Cas12a to detect ctDNA with high universality and accuracy. The use of artificially inserted PAMs by modified RPA primers or suboptimal PAMs unlocks the PAM restriction; introducing single- or double-base mismatches in CRISPR RNA effectively reduces the off-target effects and improves the specificity to single-base resolution. Under optimized conditions, this method detected ctDNA mutations with a limit of detection at 100 aM and identified mutations down to 0.02% variant allele frequency in 50 min, requiring only isothermal control. We successfully applied this method to multiple clinical samples of NSCLC and the results were validated using real-time polymerase chain reaction analysis. In summary, we established a rapid, sensitive, universal and highly accurate method for ctDNA detection that has great potential application in the early diagnosis, therapy guidance and prognosis prediction of NSCLC. • The universal CRISPR/Cas12a detection method uses an inserted or suboptimal PAM. • Highly accurate ctDNA mutation detection is ensured using base mismatches in crRNA. • The strategy showed a detection limit of 100 aM with high selectivity (0.02% VAF). • The strategy was used to successfully detect ctDNA in clinical samples. • The accuracy of the proposed strategy was comparable to that of the commercial kit. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 09254005
- Volume :
- 383
- Database :
- Academic Search Index
- Journal :
- Sensors & Actuators B: Chemical
- Publication Type :
- Academic Journal
- Accession number :
- 162388105
- Full Text :
- https://doi.org/10.1016/j.snb.2023.133493