Detection of monkeypox virus using helicase dependent amplification and recombinase polymerase amplification combined with lateral flow test.

The monkeypox virus (MPXV) is a zoonotic DNA virus that belongs to the poxvirus family. Conventional laboratory methods for detecting MPXV are complex and expensive, making them unsuitable for detecting the virus in regions with limited resources. In this study, we using the Helicase dependent amplification (HDA) method and the Recombinase polymerase amplification (RPA) technique in combination with the lateral flow test (LFT), together with a self-designed qPCR technique for the detection of the MPXV specific conserved fragment F3L, to compare the sensitivity and specificity of the three assays. By analyzing the sensitivity detection results using Probit, it can be seen that the limit of detection (LOD) of the HDA-LFT detection target is 9.86 copies/µL (95% confidence interval, CI 7.52 copies/µL lower bound), the RPA-LFT detection target is 6.97 copies/µL (95% CI 3.90 copies/µL lower bound), and the qPCR detection target is 479.24 copies/mL (95% CI 273.81 copies/mL lower bound). The specificity test results showed that the specificity of the three methods mentioned above was higher than 90% in detecting pseudoviruses of the same genus of MPXV. The simple, highly sensitive, and specific MPXV assay developed in this study is anticipated to provide a solid foundation for future applications in the early screening, diagnosis, and evaluation of the efficacy of MPXV. This is the first time the HDA-LFT assay has been utilized to detect MPXV infection.