Background-Free Rolling Circle Amplification for SERS Bioassay Using a Chimeric Hairpin-Integrated CRISPR/Cas12a System

Sep 9, 2025Analytical chemistry

Background-Free DNA Amplification for Sensitive SERS Detection Using a Combined Hairpin and CRISPR/Cas12a System

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Abstract

The SCOPE-RCA system can detect African swine fever virus (ASFV) at levels as low as 59 copies/mL.

A strategy involving CRISPR/Cas12a cleaves designed DNA/RNA chimeric hairpin preprimers to initiate RCA.
This system achieves 99% cleavage of single-stranded DNA (ssDNA) within 20 minutes, reducing nonspecific amplification.
The conversion of double-stranded DNA (dsDNA) to single-stranded RNA (ssRNA) is facilitated by the chimeric hairpin primer.
The SCOPE-RCA system integrates catalytic hairpin assembly and surface-enhanced Raman scattering for signal amplification.

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Rolling circle amplification (RCA) has revolutionized nucleic acid detection owing to its isothermal simplicity. However, over two decades of clinical application have been hampered by off-target amplification and incompatibility with double-stranded DNA (dsDNA). Herein, a strategy, specifically cleavage of rationally designed DNA/RNA chimeric hairpin preprimer by dsDNA-targeted CRISPR/Cas12a to rlease ssRNA for initiating RCA (SCOPE-RCA), is proposed for nucleic acid identification of African swine fever virus (ASFV). Leveraging the transcleavage capability of CRISPR/Cas12a, the SCOPE-RCA system achieves 99% single-stranded DNA (ssDNA) cleavage within 20 min, thus largely suppressing nonspecific amplification. Simultaneously, dsDNA-to-ssRNA conversion is realized using a DNA/RNA chimeric hairpin primer, where RNA functions as an RCA activator, enabling dsDNA-compatible RCA. Eventually, cascaded with catalytic hairpin assembly (CHA) coupled with surface-enhanced Raman scattering (SERS) signal amplification, the SCOPE-RCA system can detect ASFV in mock-infected samples at levels as low as 59 copies/mL. With its ease of use and ultrasensitive detection capability, the SCOPE-RCA system demonstrates significant potential for nucleic acid research and clinical applications. e

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Background-Free Rolling Circle Amplification for SERS Bioassay Using a Chimeric Hairpin-Integrated CRISPR/Cas12a System

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