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.
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.
Subject(s)
COVID-19 , Monkeypox , Humans , Sensitivity and Specificity , Monkeypox virus , Recombinases , PandemicsABSTRACT
Monkeypox virus (MPXV) is a human pathogenic virus that belongs to the genus Orthopoxvirus. In 2022, MPXV caused an unprecedented number of infections in many countries. As it is difficult to distinguish MPXV from other pathogens by its symptoms in the early stage of infection, a rapid and reliable assay for MPXV detection is needed. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of MPXV and evaluated its application in simulated clinical samples. The A27L-1 and F3L-1 primer sets were identified as the optimal primers, and 63°C was the most appropriate reaction temperature for sequence amplification. The detection limits of the LAMP assay using primer sets A27L-1 and F3L-1 were both 20 copies/reaction mixture, which were >100-fold higher in terms of sensitivity, compared with conventional PCR. The LAMP assay findings were negative for all 21 non-MPXV pathogens, confirming the high specificity of our assay. All three types of simulated clinical samples were clearly identified by our LAMP assay, and the detection limits were consistent with the sensitivity results, indicating efficient clinical sample identification. Our rapid and reliable MPXV LAMP assay could be useful for MPXV detection and on-site diagnosis, especially in primary hospitals and rural areas. IMPORTANCE MPXV outbreaks rapidly grew in the first half of 2022, and this virus has been recognized as an increasing public health threat, particularly in the context of the COVID-19 pandemic. Thus, developing reliable and fast detection methods for MPXV is necessary.