Self-amplifying CRISPR-based one-pot ultrasensitive testing for rapid SARS-CoV-2 and its variant detection

Abstract

Rapid, accessible molecular tests that can resolve viral variants remain a critical unmet need. We report a onetube clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) assay that couples target recognition to a T7-promoter-driven self-amplifying loop, thereby achieving exponential fluorescence amplification at a single temperature (37 degrees C) within 40 min. Without separate preamplification, the assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open-reading frame 1a (ORF1a), nucleocapsid (N), spike (S), and envelope (E) RNAs with limits of detection (LoDs) of 0.32-0.96 copies mu L- 1, corresponding to attomolar level sensitivity. A compact 16-well reader and a smartphone application enable real-time quantification and field-deployable operation. The system discriminates S mutations (D614G, H69-70del, D80A, L452R, P26S, A67V, and A27S) and maintains specificity in mixed-variant samples. In a clinical study (n = 105; 75 positives and 30 negatives), assay calls are concordant with routine reverse transcription quantitative polymerase chain reaction (RT-qPCR). These results establish a minimal-handling, extraction-free workflow that quantitatively detects SARS-CoV-2 and resolves key mutations, suggesting a generalizable architecture for point-of-care (POC) nucleic-acid testing.