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Published on: October 18, 2022
Background-Free Rolling Circle Amplification for SERS Bioassay Using a Chimeric Hairpin-Integrated CRISPR/Cas12a
Xian-Ming Guo1, Shi-Ying Fu1, Yu-Qing Wang1
1State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, College of Energy, Discipline of Intelligent Instrument and Equipment, State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory, Xiamen University, Xiamen 361005, China.
A new method, SCOPE-RCA, uses CRISPR/Cas12a to enable isothermal rolling circle amplification (RCA) for detecting African swine fever virus (ASFV) DNA. This approach overcomes limitations of traditional RCA, improving nucleic acid detection sensitivity.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Rolling circle amplification (RCA) is a powerful isothermal nucleic acid detection method.
- Clinical applications of RCA are limited by off-target amplification and inability to detect double-stranded DNA (dsDNA).
- African swine fever virus (ASFV) poses a significant threat to swine populations globally.
Purpose of the Study:
- To develop a novel nucleic acid detection strategy for ASFV.
- To overcome the limitations of traditional RCA, including off-target amplification and dsDNA incompatibility.
- To enable ultrasensitive and specific detection of ASFV DNA.
Main Methods:
- CRISPR/Cas12a-mediated cleavage of a DNA/RNA chimeric hairpin preprimer to release ssRNA.
- Initiation of RCA using the released ssRNA for dsDNA-compatible amplification.
- Cascading with catalytic hairpin assembly (CHA) and surface-enhanced Raman scattering (SERS) for signal amplification.
- Detection of ASFV in mock-infected samples.
Main Results:
- The SCOPE-RCA system demonstrated efficient ssDNA cleavage (99% within 20 min) using CRISPR/Cas12a, suppressing nonspecific amplification.
- dsDNA-to-ssRNA conversion was achieved using a chimeric hairpin primer, enabling dsDNA-compatible RCA.
- The system achieved ultrasensitive detection of ASFV with a limit as low as 59 copies/mL.
- High specificity and efficiency were observed in mock-infected samples.
Conclusions:
- SCOPE-RCA effectively addresses limitations of traditional RCA, enabling dsDNA detection.
- The developed method offers a highly sensitive and specific approach for ASFV nucleic acid identification.
- SCOPE-RCA shows significant potential for nucleic acid research and clinical diagnostics.
