Development and Characterization of Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of Monkeypox Virus

Abstract

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), in which outbreaks mainly occurred in West and Central Africa, with only sporadic spillovers to countries outside Africa due to international travel or close contact with wildlife. During May 2022, multiple countries in Europe, North and South America, Australia, Asia, and Africa reported near-simultaneous outbreaks of MPXV, the first time that patient clusters were reported over such a large geographical area. Cases have no known epidemiological links to MPXV-endemic countries in West or Central Africa. Real-time PCR is currently the gold standard for MPXV diagnostics, but it requires trained laboratory personnel and specialized equipment, and results can only be obtained after several hours. A rapid and simple-to-operate point-of-care diagnostic test for MPXV is crucial for limiting its spread and controlling outbreaks. Here, three recombinase-based isothermal amplification assays (RPA/RAA) for the rapid detection of MPXV isolates were developed. These three assays target the MPXV G2R gene, and the limit of detection for these systems is approximately 100 copies of DNA per reaction. The assays were found to be specific and non-cross reactive against other pox viruses, such as vaccinia virus, and the results can be visualized within 20–30 min. The assays were validated with DNA extracted from 19 clinical samples from suspected or confirmed MPXV patients from Central Africa, and found to be consistent with findings from traditional qPCR. These results provide a solid platform for the early diagnosis of potential MPXV cases, and will help with the control and prevention of current and future outbreaks.

Mpox diagnostics: Review of current and emerging technologies

“…In addition, these assays can be readily applied using existing qPCR platforms for high‐throughput testing, without going through extensive assay validation to meet regulatory requirements. Clade‐specific assays in the format of multiplexed PCR and LAMP had also been described 31,32 . These assays designed their primers and probes to detect MPXV genomic regions (F3L and G2R) with a few nucleotide differences between Clades I, IIa, and IIb.…”

CRISPR/Cas12a assisted specific detection of monkeypox virus

“…This is well suited for detection in remote areas as expensive instrumentation such as RT-PCR machine is not required. Further, the assay is also compatible with the CRISPR-CUBE [11], a highly portable low-cost battery-operated incubator-cum-detector, in which could be an advantage over previous CRISPR-based monkeypox detection reports [19], [31]. Use of CRISPR-CUBE eliminates the recurring cost of lateral flow strips.…”

Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

“…Davi et al established a mpox-recombinase polymerase amplification (RPA) assay targeting the G2R gene, which produced diagnostic results within 10 min and the results are comparable with qPCR results [ 38 ]. Mao et al developed RPA combined with CRISPR-Cas12a (RPA-Cas12a), real-time RPA, and recombinase-aided amplification (RAA) combined with lateral flow strips (RAA-LFS) against mpox [ 39 ]. These assays work by detecting the tumor necrosis factor (TNF) binding protein gene, G2L gene and G2R gene of the mpox genome, respectively [ 38 , 39 ].…”

“…Mao et al developed RPA combined with CRISPR-Cas12a (RPA-Cas12a), real-time RPA, and recombinase-aided amplification (RAA) combined with lateral flow strips (RAA-LFS) against mpox [ 39 ]. These assays work by detecting the tumor necrosis factor (TNF) binding protein gene, G2L gene and G2R gene of the mpox genome, respectively [ 38 , 39 ]. However, RPA can be more expensive than LAMP, because it requires commercial multi-protein reaction mixes as well as chemically modified probes.…”