Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis

Abstract

Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.