Development of a novel point-of-care device detecting monkeypox virus using lyophilized microspheres and lateral flow strips

Real-time PCR has been used in clinical diagnostics as the gold standard for detecting monkeypox virus (MPXV), the cause of monkeypox disease (mpox). However, to better prevent the spread of mpox disease, especially in areas where PCR equipment is not available, fast and self-administered detection medical devices are urgently needed. Therefore, we attempted to detect mpox by loop-mediated isothermal amplification (LAMP) using a method involving lyophilized microspheres and reading results by color change and lateral flow immunoassay strips. We first tested the optimal temperature for our LAMP assay and found that 65°C presented optimal results. At this temperature, we then tested three different primer groups targeting different regions of the MPXV genes and found that the F3L-1 primer outperformed the others with a limit of detection (LoD) of 70 genome copies per reaction. Surprisingly, adding another primer group to the F3L-1 group only lowered the LoD to 60 genome copies per reaction. For interference assays, we selected substances and/or microorganisms that are commonly applied to or found on the skins of patients and showed that none of them interfered with the testing results at the indicated concentrations. Finally, we tested clinically collected human skin swabs containing MPXVs and demonstrated that our assay performed well with these samples at 1 ×, 2 ×, 3 ×, and 5 × LoD. Based on these findings, we propose a medical device that may be used as a point-of-care solution in areas where traditional PCR equipment is not available.