Rapid detection of monkeypox virus by multiple cross displacement amplification combined with nanoparticle‐based biosensor platform

The current outbreak of monkeypox virus (MPXV) has become a public health emergency of international concern that highlights the need for rapid, sensitive MPXV diagnostic assays. Here, we combined isothermal multiple cross displacement amplification (MCDA) with nanoparticle‐based lateral flow biosensor (LFB) to devise a diagnostic test for the diagnosis of MPXV infection (called MPXV‐MCDA‐LFB) and differentiation of West and Central African MPXV isolates. The MPXV‐MCDA‐LFB protocol conducts isothermal MCDA reaction for DNA templates followed by LFB detection of preamplification target sequences. Two MCDA primer sets were designed targeting the D41L and ATI genes of Central and West African MPXV isolates, respectively, and the optimal condition of two MCDA reactions was 64°C for 30 min. The two MCDA reactions were decoded by LFB, which was devised for detecting three targets, including two amplicons yielded from two MCDA reactions and a chromatography control. Thus, the MPXV‐MCDA‐LFB assay could be completed within 50 min including rapid template preparation (15 min), MCDA reaction (30 min) and reporting of result (