A portable all-in-one microfluidic platform integrated with CRISPR-based extraction-free assay for rapid and on-site detection of monkeypox and lumpy skin disease

Rapid and accurate identification of infectious disease pathogens plays a key role in emergency preparedness and respond to a wide range of biosecurity threats. However, current strategies require dedicated laboratories, expensive instruments and skilled operators, which are not suitable for point-of-care testing. Here, we developed an integrated CRISPR-based microfluidic platform that consisted of 3D printed bioreactors and a homemade miniaturized heater for rapid and sensitive nucleic acids detection. The all-in-one, containment-free bioreactors combined recombinase polymerase amplification, CRISPR and lateral flow dipsticks (RPA-CRISPR-LFD), allowing user-friendly operation and eliminating the need for complex laboratory equipment. The RPA-CRISPR-LFD assay was able to efficiently detect plasmids of monkeypox virus (MPXV) and lumpy skin disease virus (LSDV) down to 1 copy $\mu L^{-1}$. The platform can identify 5 extraction-free samples in parallel within 30 min, significantly reducing time consumption. In addition, we validated its clinical utility and versatility by testing 67 simulated blood or swab samples of MPXV and LSDV, yielding results consistent with real-time PCR. Thus, the platform enables rapid and convenient detection of diverse pathogens, and with the future integration of lyophilized reagents, it offers a promising tool for point-of-care testing of major biosecurity threats, especially in source-limited areas.