Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system

ABSTRACT The monkeypox virus, which was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO), continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner and isolate patients to prevent outbreaks so that the vulnerable population is protected and the emergence of dangerous monkeypox variants is restrained. In this study, a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox based on a sucrose-aided multiphase aqueous system has been established with an ultra-high sensitivity of a single copy, which is 10 times higher than the existing RPA-CRISPR one-pot method. The entire reaction was completed within 60 min at 37°C. The detection method exhibited good specificity, effectively distinguishing monkeypox from other orthopoxviruses. The detection results could be observed by the naked eye under ultraviolet or blue light, making it highly suitable for home or limited healthcare settings. The assay has solved the incompatibility between the Cas12a cleavage reaction and the RPA reaction and shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox and sets a technical example of using a multiphase aqueous system to establish one-pot RPA-CRISPR detection. IMPORTANCE The monkeypox virus was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) and continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner to prevent outbreaks. This study establishes a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox virus with an ultra-high sensitivity. The assay shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox, which is useful for the prevention of an epidemic.