Rapid detection of mpox virus using recombinase aided amplification assay

Abstract

A recent, unprecedented outbreak of human mpox virus infection has led to cases in non-African nations, and the number of confirmed or suspected cases outside of Africa has exceeded 1,000 within 5 weeks. Mpox may pose a double threat to public health in the context of the ongoing COVID-19 pandemic. It is difficult to distinguish mpox virus infection from other diseases in the early stages, and patients are contagious from the onset of nonspecific symptoms; therefore, it is crucial to develop rapid and specific diagnostic methods. The diagnosis of mpox relies on real-time polymerase chain reaction, a time-consuming method that requires a highly sophisticated thermal cycler, which makes it unsuitable for widespread use in underdeveloped areas, where the outbreak is still severe. In this study, we developed a recombinase-aided amplification (RAA) assay that can detect mpox virus within 5–10 minutes. The conserved regions of the A27L gene and F3L gene were selected as targets, as they amplify well from different mpox virus clades with no cross-reaction from other pathogens. The sensitivity of this RAA assay is 10 copies/reaction for the A27L gene and 102 copies/reaction for the F3L gene. When applied to simulated clinical samples, both targets showed 100% specificity, and the detection limits were consistent with the sensitivity results. Moreover, through clinical blinded sample detection, RAA exhibits the same detection power as RT-PCR. In summary, the RAA mpox assay described here exhibits rapid detection, high sensitivity and specificity, and low operational difficulty, making it suitable for mpox virus detection in less developed countries and regions.

Evaluation of analytical performance of the STANDARD TM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus

“…A different approach was used by Cui et al, who developed a Recombinase-aided amplification (RAA)-based isothermal assay, which would benefit from a significant reduction of analysis time (10 min), costs (5$/sample), and laboratory requirements (i.e. : expertise, cold-chain, RT–PCR instruments); however, a minor analytical sensitivity, compared to RT–PCR, could impair the potential of this technology, especially in a low prevalence context [ 25 ]. The epidemiological scenario extremely changed starting in late 2022, with a highly significant drop in new cases [ 26 ]; nevertheless, the virus continues its circulation under the radar, requiring a new approach: smaller numbers and the need to perform tests in limited expertise settings represent two important issues for surveillance.…”

Development of a real-time recombinase-aided amplification assay for rapid and sensitive detection of Edwardsiella piscicida

“…Compared with the general molecular detection methods, RAA possesses several advantages, such as low-cost, simplicity of operation (as it does not necessitate complex equipment or highly skilled personnel, can be conducted using portable device), low and constant reaction temperature (39°C), and rapid reaction speed (within 20 min) ( Xie et al., 2012 ). As a result, RAA has been widely used in identifying viruses, bacteria, and parasites ( Cui et al., 2022 ; Fu et al., 2022 ; Lin et al., 2022 ; Cui H et al., 2023 ; Cui X et al., 2023 ). Consequently, it represents a rapid and user-friendly method suitable for clinical use, particularly in primary laboratories.…”

“…Previous studies on viral and bacterial pathogens have indicated the detection limit of the RAA assay varied from 10 to 142 copies per reaction when tested against standard plasmids ( Zhang et al., 2017 ; Cui et al., 2022 ; Fu et al., 2022 ; Lin et al., 2022 ; Cui H et al., 2023 ; Cui X et al., 2023 ). In this study, a novel and simple method for E. piscicida detection was proposed based on RAA technique.…”

Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris

“…18 Recombinase-aided amplification (RAA) is a isothermal amplification technology that can complete rapid DNA or RNA amplification at a constant temperature in a short time with accurate results. 19 Here, we developed a RAA assay to detect C. auris, then confirmed its sensitivity, specificity, and application for clinical sample detection, and compared it with real-time PCR to enable timely diagnosis and treatment of C. auris infections and improve patient outcomes. Figure 1C shows the detection mechanism for the RAA assay.…”