RPA coupled with cas12a as a simple, accurate and rapid method for diagnosing rickettsia in dogs

Abstract

Abstract Rickettsial pathogens including Ehrlichia canis and Anaplasma platys are bacteria that cause parasitic infections in dogs such as canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT), respectively affecting mortality and morbidity worldwide. An accurate, sensitive, and rapid method to diagnose these agents is essential for effective treatment. In this study, a recombinase polymerase amplification (RPA) coupled with CRISPR-Cas12a methods was established to detect E. canis and A. platys infection in dogs based on the 16S rRNA. The optimal condition for DNA amplification by RPA was 37°C for 20 min, followed by CRISPR-Cas12a digestion at 37°C for one hour. RPA coupled with cas12a detection showed no cross-reaction with other parasites, and offered high sensitivity, with a limit of detection at 102 copy numbers of both E. canis and A. platys 1,000 and 100 times higher than agarose gel electrophoresis detection, respectively. The RPA-assisted cas12a assay provides specific, sensitive, rapid, simple, and appropriate detection of rickettsia in canine blood at the point-of-care for diagnostics, disease prevention, and surveillance.