Viruses, Vol. 15, Pages 84: Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

More frequently recurring monkeypox outbreaks over the past years have made it necessary to develop a rapid, sensitive and specific diagnostic method for the surveillance of suspected cases. A main reason is because it is challenging to identify monkeypox only based on the clinical symptoms, especially for cases without any clinical presentations. In the absence of smallpox, the main clinical diagnostic obstacle is the discrimination of monkeypox from chickenpox [36]. Moreover, according to the WHO guidelines, any suspected monkeypox cases should be tested in properly equipped laboratories by relevant technical staff under safety regulations. At present, qPCR is the gold standard method for the confirmation of monkeypox virus infection. The challenges to develop robust mpox assays are to find specific, conserved targets due to other OPXV sharing substantial sequence homology to mpox (>90%), which limits the design of the molecular diagnostic assay. In a previous study, Li et al. developed a qPCR assay by using minor groove binding protein-based (MGB) probes to enhance assay sensitivity and specificity [22]. Moreover, both clades (West African and Congo Basin) of mpox have 99% sequence similarity [37], so it is challenging to develop a clade-specific qPCR detection approach due to the limited availability of unique sequences. In an effort to differentiate between isolates from two different clades, the terminal genomic sequences of mpox strains were analyzed [24]. Although sensitive and specific, the qPCR-based mpox detection approach is a time-consuming process and needs specialized instruments and trained staff, which limits their use in the field and clinic without sufficient laboratory support. These limitations need to be overcome. Currently, several isothermal methods have been developed for mpox molecular detection. 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]. However, RPA can be more expensive than LAMP, because it requires commercial multi-protein reaction mixes as well as chemically modified probes.Loop-mediated isothermal amplification (LAMP) is another promising isothermal molecular diagnostic technique for virus detection [40]. The LAMP technology relies on a set of six primers: two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). The reaction is carried out at an isothermal temperature between 60–65 °C. In our study, the target sequences were selected for LAMP assays detecting the terminal repetitions of the genome, which show greater variations among sequences of OPXV strains. After alignment, we selected the LAMP primer sets to identify the conserved region surrounding the N4R gene in the MPVX-FRA-2022-TLS67 reference strain. Based on this, we developed the fluorescent LAMP and the visual LAMP detection methods for mpox, which generates accurate results within 30 min. The results of both assays showed 100% consistency with real-time PCR results for mpox detection. The other LAMP assay was used to detect genome of Congo Basin (C-LAMP), West African (W-LAMP) and both Congo Basin and West African (COM-LAMP). The three different LAMP primer sets were designed according to the nucleotide sequences of the Congo Basin-specific D14L gene, the West African-specific partial ATI gene and the partial ATI gene that is shared by both groups. The sensitivity and specificity of COM-LAMP, C-LAMP, and W-LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively [41]. In addition, the recent LAMP assay for mpox detection used the A27L and F3L genes for designing of the two primer sets. However, the detection limits of these assays were both 20 copies/reaction mixture, which were 10-fold higher in terms of sensitivity, compared with our LAMP assay for mpox detection [42]. It is important to note that the 2022 mpox outbreak has been rapidly spreading in non-endemic countries, mainly in European countries and American countries, which have the resources to provide qPCR testing. However, to prevent future importation of cases, it is important to also enhance and simplify testing in endemic countries of Africa, which do not always have the required resources for laboratory diagnosis. There is an urgent need for modernization of the existing infrastructure and diagnostic facilities. LAMP provides a useful alternative for active surveillance of mpox, since it only requires a heat block and can be directly visualized with the naked eye. In addition, the recommended type of specimen for laboratory detecting monkeypox is skin material and/or swabs of surface lesions. Interestingly, mpox DNA can be found in the saliva of infected cases by using qPCR [43]. Previous reports showed that unpurified SARS-CoV-2 in saliva samples can be detected with no RNA extraction step via a RT-LAMP assay [44,45]. As the reaction temperature of our LAMP assay is 65 °C and mpox virus can be inactivated at 60 °C in under 15 min [46], this LAMP assay can theoretically be used to detect mpox from crude clinical saliva samples without prior DNA extraction. Another limitation in this research is that we only used clinical DNA samples belonging to mpox Clade I to test our LAMP assay, but not those from Clade IIa and Clade IIb. As such, our results could be supplemented with validation of these assays with samples from the 2022 mpox outbreak.

With high sensitivity and specificity, our LAMP assay is simple to use, low cost and time efficient. Moreover, the isothermal LAMP visual method does not require specialized instrument to observe the results. Since the 2022 mpox outbreak has now been classified as a PHEIC by the WHO, our results would extremely strengthen the public health interventions in providing much needed diagnostic services, especially in areas with limited laboratory capacities.

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