Fig. 6

Workflow of two-step one-tube dRPA-Cas12a/Cas13a assay. (A) The nucleic acid extracts are prepared from swab samples collected from feline patients with URTD. Reverse transcription is implemented for RNA templates preparation in an independent tube. After that, the target templates are transferred to another tube for immediate duplex RPA reaction (the first step). Then, the prepared dRPA products are added into the premixed hybrid Cas12-crRNA and Cas13a-crRNA complex to trigger the one-tube reaction system (the second step). Before that, the target FCV-ORF1 cDNAs are synchronously transcribed to RNAs for subsequent crRNA recognition using T7 RNA polymerase. In the one-tube hybrid reaction system, Cas12a/crRNA/target DNA oligonucleotides triplets or Cas13a/crRNA/target RNA oligonucleotides triplets are assembled, respectively. Then, hybrid LbaCas12 and (or) LwaCas13a trans-cleavage activities are triggered immediately once specific crRNA-guided recognition of target FHV-1 and (or) FCV templates in the reaction system. Subsequently, the ssDNA and ssRNA reporters are cleaved to generate visible signals. (B) Four types of visual readouts based on the dRPA-Cas12a/Cas13a assay are displayed by multi-channel fluorescence detectors or lateral flow dipsticks