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 [21, 22]. 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 [23]. Although LAMP is a convenient, efficient, and inexpensive technique for detection [24, 25], 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 [26, 27]. 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 HAD 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 [23].
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. To verify specificity, we used MPX PV, CPX PV, HS PV, VZ PV and CMPV against amplified primers, and the reaction results indicated that the aforementioned three detection methods had excellent specificity. The sensitivity test results showed that the detection limits for HDA-LFT and RPA-LFT were ༜ 10 copies/µL and for qPCR ༜ 500 copies/µL.
Despite the fact that HDA-LFT and RPA-LFT techniques have a number of benefits, this research revealed their limitations: First, the dropwise addition of amplification products to the test strip can only provide a qualitative evaluation of the reaction and cannot detect its quantitative nature. Second, due to the phenomenon of non-specific amplification, which cannot be avoided during the reaction, a large number of primer designs and replicate experiments are required to avoid false positive results. Lastly, visual observation of whether the test strip is positive is subjective, and differences in vision and judgment between individuals may make it difficult to obtain accurate experimental results; consequently, this method may require the use of an LFT reading instrument. 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.