RAA-CRISPR/Cas12a-mediated SERS magnetic biosensor combined with a portable Raman spectrometer for rapid and ultrasensitive detection of Lumpy skin disease virus.

Lumpy skin disease virus (LSDV) poses a severe threat to global cattle farming, causing significant economic losses. Current laboratory-based methods such as qPCR are accurate procedures; however, they are time-consuming and require sophisticated instrumentation, thus hindering onsite outbreak control. We report a novel magnetic biosensor integrating recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a, and surface-enhanced Raman scattering (SERS) for ultrasensitive and portable detection of LSDV. The biosensor operates through a synergistic cascade: (i) RAA isothermally amplifies the viral DNA target; (ii) the amplified product activates the trans-cleavage activity of CRISPR/Cas12a, thus generating numerous short ssDNA fragments; and (iii) these fragments cleave linker DNA, thereby releasing AuNS@DTNB SERS nanoprobes from the magnetic bead surface and enabling signal readout via a handheld Raman spectrometer. This triple-amplification strategy yielded an ultrahigh sensitivity at 1.2 copies/μL, with a total assay time of only 90 min. The platform exhibited exceptional specificity, discriminating LSDV from genetically homologous Capripoxviruses (up to 97% similarity). In blind tests on 30 beef samples spiked with LSDV nucleic acid, it achieved 100% concordance with qPCR results, thus demonstrating excellent robustness. By integrating a handheld Raman spectrometer and a portable thermostat, this platform delivers laboratory-grade performance in a field-deployable format. This biosensor represents a powerful new tool for onsite LSDV surveillance; given its modular design, it can be readily adapted for detecting other emerging nucleic acid targets.