Exploiting the orthogonal CRISPR-Cas12a/Cas13a trans-cleavage for dual-gene virus detection using a handheld device

Although CRISPR-Cas12a and CRISPR-Cas13a systems work individually effective on gene detection, their multiplex detection capability is limited due to the lack of specific probe cleavage mechanism. Herein we present a high-efficient dual-gene diagnostic technique based on the orthogonal DNA/RNA collateral cleavage mechanism of Cas12a/Cas13a system. In this design, dual-gene amplified products from the multiplex recombinase polymerase amplification (RPA) were simultaneously detected by Cas12a and Cas13a assay in a single tube. The resulting orthogonal DNA/RNA collateral cleavage can specifically illuminate two spectral differentiated DNA and RNA probes, respectively. By integrating with the smartphone-based fluorescence readout, a portable detection platform is achieved. As a proof-of-concept, reliable dual-gene detection of SARS-CoV-2 and African Swine fever virus (ASFV) were demonstrated, exhibiting 100% sensitivity and specificity for clinical samples analysis (32 swab specimens for SARS-CoV-2 and 35 ASFV suspected swine blood samples). This developed portable dual-gene detection platform can provide accurate point-of-care screening of infectious diseases in resources-limited settings.