CRISPR/Cas12a assisted specific detection of monkeypox virus

Abstract

ObjectiveTo 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.

Clinical and laboratory diagnosis of monkeypox (mpox): Current status and future directions

“…In response to the current outbreak, CRISPR-based systems have been developed for MPXV diagnostics. 122 , 123 In one of the first detection methods, Sui and co-workers developed a CRISPR system that was able to detect the MPXV DNA by using fluorescence readout. 122 In this study, the authors found that the FAM fluorescent signal was detectable in 2 min and a strong signal was achieved within 10 min, indicating that the system has potential to apply in the field.…”

“… 122 In another similar report, Singh and co-workers designed a CRISPR-Cas12a-based system to detect MPXV, achieving a high sensitivity and specificity to detect synthetic DNA. 123 Despite these promising findings, CRISPR/Cas-based diagnostic methods are not currently used in reference laboratories and need further implementation.…”

Electrochemical Immunoassay Platform for Human Monkeypox Virus Detection

“…Until now, there are some monkeypox diagnostic methods that have been used for diagnosing the infections. Some of these techniques are polymerase chain reaction, , virus culture-based diagnosis, protein Cas-based diagnosis, , loop-mediated isothermal amplification, , enzyme-linked immunosorbent assay, , and recombinase polymerase amplification assay. ,, Considering the point-of-care (POC) nature of rapid tests, antibody-based immunoassays seem to be stronger diagnostic tool candidates for MPXV detection. However, due to the antigen context of Orthopoxvirus genomes, most human samples become cross-reactive, and this situation makes it impossible to differentiate different kinds of Orthopoxvirus viruses. − …”