Development of a single-tube RPA/CRISPR-cas12a detection platform for monkeypox virus

Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks primarily occurring in West and Central Africa. The recent global MPXV outbreak underscores the urgent need for effective detection methods. Currently, qPCR is considered the gold standard for MPXV detection; however, it requires specialized personnel and costly equipment. This study introduces a CRISPR-Cas12a-based detection system targeting the MPXV A27L gene, achieving a detection limit as low as 10 aM. This system exhibits high specificity, with no cross-reactivity with other orthopoxviruses, and delivers results in under 40 min. To support point-of-care testing (POCT), we developed a lateral flow assay (LFA) strip for easy result visualization. The detection system was validated using six different clinical sample types, revealing that herpes fluid and saliva are the most suitable sources. The findings of this study align with qPCR results. Additionally, we lyophilized the RPA and CRISPR reagents to improve transport, storage, and field deployment. In conclusion, this study presents a reliable molecular diagnostic approach for early MPXV detection and point-of-care testing, contributing to epidemic prevention and control.