Development of a CRISPR/Cas12a‐recombinase polymerase amplification assay for visual and highly specific identification of the Congo Basin and West African strains of mpox virus

Abstract

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a nuclease (CRISPR/Cas12a)-mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR-RPA).Specific RPA primers targeting D14L and ATI were designed. CRISPR-RPA assay was performed using various target templates. In the designed CRISPR-RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans-cleavage of a single-stranded DNA probe. The limit of detection for the CRISPR-RPA assay was 10 copies per reaction for D14L-and ATI-plasmids. No cross-reactivity was observed with non-mpox strains, confirming the high specificity of the CRISPR-RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR-RPA assay can be completed within 45 min using real-time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource-limited laboratories.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing

“…When analyzing with a pseudotyped virus, the LoD level reached down to 12.5 copies per microliter. Although the sensitivity of multiplex ET-PCR assay was marginally lower than that of previous isothermal amplification-based studies, such as MCDA, 26 LAMP, 24 and RPA 36 techniques, as well as the established real-time PCR methods, 19,20 the newly developed method was able to identify generic, clade I and clade II MPXV strains in a single reaction rather than one or two of them. In addition, the combination of generic and clade-specific testing improves detection accuracy and aids in new clade identification.…”

“…9 Thus, NAATs are preferred laboratory tests to identify and characterize MPXV with rapidness, high sensitivity and specificity. 16 Recently, many NAATs have been developed to confirm MPXV infection, including conventional PCR, 17,18 real-time PCR, 19–21 recombinase polymerase amplification (RPA), 22,23 loop-mediated isothermal amplification (LAMP), 24,25 multiple cross displacement amplification (MCDA), 26 and restriction length fragment polymorphism (RFLP), 10,27 all of which commonly target one specific region of the monkeypox virus genome and possess varying sensitivities and limits of detection (LoD) for MPXV diagnosis. 28 Although the whole genome sequencing technique has also been reported to be deployed for MPXV strain identification, it mainly occurred in centralized and specialized laboratories, 29,30 which were usually not available for large-scale and timely testing.…”

A modified multiple cross displacement amplification linked with a gold nanoparticle biosensor for the detection of Epstein-Barr virus in clinical applications

“…Recently, isothermal amplification techniques such as multiple cross displacement amplification (MCDA; Wang et al, 2015 ), loop-mediated isothermal amplification (LAMP; Notomi et al, 2000 ; Yang et al, 2022 ), recombinase polymerase amplification (RPA; Yang et al, 2023c ), and cross-priming amplification (CPA; Wang et al, 2021 ) were devised and applied successfully in many pathogens detection. These isothermal techniques overcome the disadvantages of PCR-based assays that require a thermal cycler because they only require a single temperature ( Yang et al, 2023a ).…”