Rapid and visual RPA-CRISPR/Cas12a detection for Staphylococcus pseudintermediusand methicillin-resistant S. pseudintermediusin clinical samples of dogs and cats

Staphylococcus pseudintermediuscan cause severe infections of the skin, ear and other tissues in dogs and cats. Methicillin-resistant S. pseudintermedius(MRSP) has recently become more prevalent, posing a severe threat to companion animals and public health. Therefore, rapid and accurate diagnosis of S. pseudintermediusand MRSP infections in dogs and cats is essential for timely controlling infections. The development of CRISPR/Cas technology offers an innovative solution for rapid diagnosis. Here, we established an assay combining recombinant polymerase amplification (RPA) and CRISPR/Cas12a. By separately detecting the spsJgene, the specific gene of S. pseudintermedius, and the mecAgene, the methicillin resistance gene, this method allows for the direct detection of methicillin-susceptible S. pseudintermedius(MSSP) and MRSP in clinical samples at 37 °C for a total of 40 min, The results can be directly visualized by the naked eye under blue light. The limits of detection of the RPA-CRISPR/Cas12a assay were 103copies per reaction for the spsJgene and 104copies per reaction for the mecAgene. The RPA-CRISPR/Cas12a detection successfully detected and differentiated clinical isolates of MSSP and MRSP without cross-reactivity with other tested bacteria species. The evaluation of the detection performance of RPA-CRISPR/Cas12a with 47 clinical samples (without culture) from dogs and cats showed that the results of detection were 100% consistent with those of clinical culture and colony sequencing, which was more sensitive than PCR. RPA-CRISPR/Cas12a assay can quickly and sensitively detect S. pseudintermediusand MRSP in clinical samples without expensive instruments, making it suitable for small veterinary clinics.