A temporally controlled isothermal amplification-CRISPR/Cas12a platform for the rapid detection of monkeypox virus

Given the continuous threat posed by emerging and re-emerging infectious diseases worldwide, a rapid, sensitive, and practical molecular detection technology is urgently required for timely point-of-care (POC) diagnosis. Although the quantitative real-time PCR (qPCR) based technologies exhibit high sensitivity and specificity compared with traditional pathogen detection methods, they are not applicable for POC detection scenarios.

Based on the photocontrolled principle, this study established a universal photoactivation strategy for LbCas12a by modifying four sites in the repeat region of LbCas12a crRNA with the photocleavable protecting group 6-nitropiperonyloxymethyl (NPOM). This strategy was integrated with multienzyme isothermal rapid amplification (MIRA) to develop a photocontrolled one-pot rapid detection method for monkeypox virus (MPXV), termed the temporally controlled MIRA-CRISPR/Cas12a (TC-MIRA-CRISPR/Cas12a) assay.

The TC-MIRA-CRISPR/Cas12a assay exhibited a 100-fold improvement in detection sensitivity over the conventional one-step assay, achieving a limit of detection (LOD) of 3.9 copies per reaction, which was comparable to stepwise detection assay. The entire assay can be completed within 40 min, faster than the widely used qPCR. In clinical sample detection, this method showed good consistency with qPCR, with a Kappa coefficient of 0.980 (P < 0.001), a sensitivity of 98.4%, and a specificity of 100%.

This method effectively avoids amplicon contamination while maintaining high sensitivity, and exhibits excellent universality and expandability. It enables detection of other pathogens simply by modifying the spacer sequence of crRNA, providing a novel technical approach and research perspective for POC detection of MPXV and other pathogens.