Benchtop to At-Home Test: Amplicon-Depleted Crispr-Regulated Loop Mediated Amplification at Skin-Temperature for Viral Load Monitoring

Abstract

CoRPLA (CRISPR-regulated One-pot Recombinase Polymerase Loop-mediated Amplification) is an amplicon-depleted skin-temperature operated iNAAT designed for at-home testing. It uses specially designed loop primers to enhance isothermal amplification, triggering Cas12 for in-situ amplicon depletion and signal amplification. This method addresses issues like amplicon-derived aerosol contamination and complex assay formats, enabling quantitative detection with sub-attomolar sensitivity (0.5 cps/μL). CoRPLA employs a DNA hydrogel wearable tape for real-time, colorimetric readout, allowing visual differentiation of pathogen loads. It was validated with clinical samples for SARS-CoV-2, RSV, influenza A, and HPV, successfully identifying multi-level viral loads of the positive cases with results consistent with qPCR. Offering high sensitivity while eliminating false positives from aerosol contamination, CoRPLA bridges the molecular assay from benchtop to home for daily viral infections monitoring.

Note:
Funding declaration: The work was financially supported by the Innovation and Technology Commission (MRP/077/20) and Research Grants Council (CRF 6107-20G, 16306519) of the Hong Kong SAR Government of China.

Conflict of Interests: The authors declare no competing interest. IMH and YMC filed a US provisional patent application on the CRISPR based amplicon-depleting strategy for home-based assay. The other authors declare no competing interests.

Ethical Approval: Clinical samples were provided by The Prince of Wales Hospital with approval from the Chinese University of Hong Kong Clinical Research Ethics Committee (CREC-2021-0188).

Keywords: Contamination-free, CRISPR-based reaction, Self-testing, Equipment-free, RPA, LAMP