CRISPR/Cas12a assisted specific detection of monkeypox virus

Abstract

AbstractObjectiveTo identify monkeypox-specific sequence and distinguish it from related orthopoxviruses followed by development of a CRISPR-Cas12a-based, specific and sensitive detection of monkeypox virus.MethodsA common detection mixture (CDM) was constituted comprising of CRISPR RNAs (crRNAs) for general orthopoxviruses and monkeypox virus-specific targets along with LbCas12a and fluorescent reporter. Recombinase polymerase amplification (RPA) of target loci was carried out followed by detection using the CRISPR-Cas12 CDM complex. Fluorescence-based read out can be monitored by a fluorescent reader and alternatively can also be visualized under a blue light illuminator by naked eye.ResultsMonkeypox-specific conserved sequences were identified inpolAgene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in genus Orthopoxvirus. Our Cas12a-based assay was capable of specifically distinguishing monkeypox virus from other related orthopoxviruses with an LOD of 60 copies in 1 hour after the initiation of the reaction.ConclusionOur assay exhibits sensitive and specific detection of monkeypox virus which can prove to be of practical value for surveillance in areas infected with multiple orthopoxviruses, especially in hotspots of monkeypox virus infections.