A rapid isothermal RPA–CRISPR/Cas12a assay for detection of Rickettsia rickettsii

Introduction Rocky Mountain spotted fever (RMSF) resulting from the tick-borne Rickettsia rickettsii infections is a potentially fatal tick-borne disease affecting humans and dogs in the Americas. It is difficult to confirm the diagnosis early in the laboratory on account of low-level and inconsistent rickettsemia in whole blood. Methods Here we established a fast, fully isothermal RPA–CRISPR/Cas12a assay that targeted the vitamin uptake transporter ( vut ) gene of R. rickettsii . Two independent crRNA primer sets (104 bp and 92 bp) were developed to independently amplify the gene target to enhance the reliability and specificity of the assay. Results Using quantified synthetic DNA (gBlock) standards derived from the R. rickettsii Sheila Smith vut target region, the assay was able to detect target DNA in about 40 min at 37 °C with an observed analytical detection limit of 60–70 copies per reaction. Specificity testing on four R. rickettsii strains and a panel of non-target spotted fever group rickettsiae and tick-borne bacteria of clinical importance demonstrated no cross-reactivity of the assay with these pathogen nucleic acids. Applied to archived longitudinal canine whole-blood DNA extracts from experimentally infected dogs ( n = 6), the performance was intermittent for known limitations of whole blood testing in RMSF and limited to specific post-infection days. Discussion The findings of this study support the feasibility of RPA–Cas12a as a rapid molecular workflow for R. rickettsii detection, while indicating that broader validation in additional clinically relevant and removed tick sample types is still needed.