In recent years, MPX has evolved into a global epidemic and a “public health emergency of international concern” [14]. Early in its discovery, MPX was frequently misdiagnosed as smallpox due to the similarity of its symptoms to those of smallpox. Since smallpox has been eradicated, the routine diagnosis of MPX primarily involves distinguishing it from varicella caused by varicella-zoster virus (VZV) [15]. Currently, MPXV is spreading rapidly in non-endemic countries and regions, and confirmed cases of monkeypox have been found successively in Europe and the North. As a crucial step in the process of epidemic prevention and control, the diagnosis and detection of monkeypox require the support of numerous types of diagnosis and detection technologies, rapidity of detection, on-site detection in special locations, clinical diagnosis, laboratory identification, and other work. In addition to developing a gold standard qPCR for the detection of monkeypox virus, we have developed two novel detection methods with high sensitivity and specificity in this study: HDA-LFT and RPA-LFT. The two aforementioned methods are simple to operate; moreover, the instrument is simple to use, test results can be determined by observing the number of lines displayed on the LFT, and the entire testing procedure can be performed without the need for specialized training. Therefore, these two types of methods are anticipated to become ideal diagnostic tools for detecting MPXV in settings with inadequate medical care and limited resources.

Although the definition of a suspected case of MPX and the manner in which MPXV is diagnosed varies slightly across the globe, it can be categorized as follows: (1), Electron microscopy. Th primary advantage of this method is that the results can be observed directly without the use of specific biological reagents; however, the cycle is longer, sample preparation and operation are more complicated, and a laboratory employing an electron microscope must be equipped with qualified technical personnel. (2), molecular diagnosis [16]. These include qPCR and next-generation sequencing (NGS). qPCR is the method of choice for the routine diagnosis of pathogenic microorganisms and the gold standard for the positive diagnosis of MPXV. It has high sensitivity and specificity for detecting MPXV, MPXV branches can be effectively identified, and other OPXV can be distinguished, but the amplification process requires three steps: Denaturation, annealing, and extension, this process requires temperature control equipment, a considerable amount of time, and skilled labor [17]. By detecting the DNA sequence of MPXV, NGS can help us better understand the epidemiology, source of infection, and mode of transmission of the virus. However, NGS is not suitable for large-scale testing because it is expensive and requires the capability to process sequencing data downstream [18]. (3), the detection of serum antibodies, ELISA is the preferred method for serum antibody detection, utilizing antigen-antibody specific binding reaction for immune reaction qualitative and quantitative analysis; thus, the MPXV specific antibody can be detected in either animal or human serum. However, this method is incapable of achieving early diagnosis and can only be used for late auxiliary diagnosis with poor compliance [19]. (4), MPXV’s isolation and culture. Although isolation and culture of MPXV is the gold standard for diagnosing viral disease, the isolation and culture of MPXV is limited and requires a high level of laboratory biosafety; isolation and culture operations must be performed in a Level III or higher laboratory by experienced personnel [20]. Therefore, it is essential to develop a rapid detection method with high sensitivity for MPXV detection in environments with limited resources.

While using the HDA method to detect MPXV for the first time, we also combined it with RPA technology and LFT to determine the sensitivity and specificity of the three detection methods in conjunction with our own qPCR. Other INAAT have been tested for the monkeypox virus before [7, 21]. Loop-mediated isothermal amplification (LAMP) is a novel technique for the amplification of nucleic acids that does not involve thermal cycling. Using LAMP technology, Iizuka et al., established a real-time quantitative amplification system for the monkeypox virus genome. Primers were designed to target the type A inclusion body (AT1), the D14L gene specific to Congo Basin monkeypox virus, and a portion of the AT1 gene from West African MPXV. LAMP was used to distinguish the strain from the Congo Basin from the strain from West Africa [22]. Although LAMP is a convenient, efficient, and inexpensive technique for detection [23, 24], the technique itself requires a significant amount of optimization and validation to identify specific primers and enzymes, as well as the design of many pairs of primers and enzymes. In addition, DNA polymerases are more sensitive to changes in temperature, prone to false positive results, and prone to the formation of difficult to eliminate aerosols [25, 26]. Compared to the LAMP assay, our HDA-LFT and RPA-LFT assays offer significant advantages. Simple primer composition, which eliminates the need for multiple sets of primers, effectively reduces the formation of dimers. LAMP technology’s high temperature amplification increases the likelihood of cross-contamination, whereas the reaction temperature of RPA is closer to that of the human body, can effectively avoid aerosol pollution, and can be used outside of the PCR laboratory. Combining HDA technology and RPA technology with LFT not only has high sensitivity and specificity but also simplifies the operation steps, does not require complex equipment or professional training, and allows for more direct observation of reaction results; therefore, this study has the potential to become an ideal tool for POCT [22].

In this study, the specific sequence F3L of MPXV was detected using HDA-LFT, RPA-LFT, and qPCR techniques. The reaction temperature, reaction time, primer concentration, and dNTPs concentration were optimized for HDA technology. As can be seen from the optimisation results, the concentration of dNTPs has an impact on the outcome of the reaction and when it is high, it inhibits the reaction and leads to the production of primer dimers. Additionally, we have optimized the reaction time, temperature, concentration of primers, MgOAc, and Nfo in the RPA technique. The Nfo enzyme, the probe shear enzyme of the RPA technique, has a significant impact on the efficacy of the reaction, with higher Nfo concentrations resulting in false positive results. Moreover, in designing conventional qPCR reactions, we optimized annealing temperature, primer concentration, and probe concentration. Due to our inability to obtain clinical samples related to MPX for our research, we simulated the method of adding recombinant plasmids to normal blood samples to detect real clinical samples to address this issue. This is due to the fact that the virus can be detected in the blood at an early stage of the infection process. This stage is called the prodromal stage and can occur before visible skin lesions. The results of the sensitivity test showed that the LOD of the HDA-LFT detection target is 9.86 copies/µL (95% CI 7.52 copies/µL lower bound), the RPA-LFT detection target is 6.97 copies/µL (95% CI 3.897 copies/µL lower bound), and the qPCR detection target is 479.24 copies/mL (95% CI 273.81 copies/mL lower bound). We used MPX PV, CPX PV, HS PV, VZ PV and CMPV as targets to verify the specificity of the above three methods. The reaction results showed that the specificity of the above three methods in detecting Monkeypox virus homologues was more than 90%.

Although this study demonstrated many advantages of the HDA-LFT and RPA-LFT techniques in the detection of MPXV, we acknowledge that there are some limitations: firstly, the results of the LFT are usually judged by visual observation of the appearance of bands, which not only results in the assay providing only a qualitative assessment of the response, but also makes it difficult to obtain accurate experimental results due to inter-individual variability in judgement. Secondly, although HDA/RPA-LFT is simpler than the traditional PCR method, and does not need a complex temperature change process, it can be detected in a water bath or at room temperature, but it can only be used after the sample is pretreated with nucleic acid extraction reagents and special Laboratory equipment, which may weaken the applicability in this field. In future research, we will conduct more in-depth research on how to use HDA/RPA-LFT technology to directly detect MPXV clinical samples, hoping to provide effective reference value for promoting the application of MPXV detection technology in POCT in the future. Finally, the potential contamination is a drawback of the above detection methods, which may be caused by opening the reaction tube when analyzing the product through LFT. Therefore, it is crucial to take sealing measures during the analysis process to prevent potential pollution. Besides, a point of concern is that the sensitivity LOD of the qPCR technique in our experiments as a control group showed some differences compared with the sensitivity of the recently reported qPCR assays on MPXV [27], which may be related to the quality of the reagents, the accuracy of the instruments and the technical proficiency of the operators as well as the design of the primers. We will continue to explore and optimise these differential factors in depth in subsequent experiments. In the future biosecurity prevention and control process, it will be necessary to further establish a monkeypox virus detection platform, develop efficient and accurate detection products, continually improve detection efficiency and accuracy, and establish strategic technical reserves to prevent safety accidents. It can prevent the occurrence of major public health incidents.