{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T05:00:41Z","timestamp":1790226041348,"version":"4.1.0"},"reference-count":38,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T00:00:00Z","timestamp":1776297600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      Rocky Mountain spotted fever (RMSF) resulting from the tick-borne\n                      <jats:italic>Rickettsia rickettsii<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Here we established a fast, fully isothermal RPA\u2013CRISPR\/Cas12a assay that targeted the vitamin uptake transporter (\n                      <jats:italic>vut<\/jats:italic>\n                      ) gene of\n                      <jats:italic>R. rickettsii<\/jats:italic>\n                      . 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Using quantified synthetic DNA (gBlock) standards derived from the\n                      <jats:italic>R. rickettsii<\/jats:italic>\n                      Sheila Smith\n                      <jats:italic>vut<\/jats:italic>\n                      target region, the assay was able to detect target DNA in about 40\u202fmin at 37 \u00b0C with an observed analytical detection limit of 60\u201370 copies per reaction. Specificity testing on four\n                      <jats:italic>R. rickettsii<\/jats:italic>\n                      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\u2002canine whole-blood DNA extracts from experimentally infected dogs (\n                      <jats:italic>n<\/jats:italic>\n                      \u202f=\u202f6), the performance was intermittent for known limitations of whole blood testing in RMSF and limited to specific post-infection days.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>\n                      The findings of this study support the feasibility of RPA\u2013Cas12a as a rapid molecular workflow for\n                      <jats:italic>R. rickettsii<\/jats:italic>\n                      detection, while indicating that broader validation in additional clinically relevant and removed tick sample types is still needed.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1823193","type":"journal-article","created":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T05:25:19Z","timestamp":1776317119000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["A rapid isothermal RPA\u2013CRISPR\/Cas12a assay for detection of Rickettsia rickettsii"],"prefix":"10.3389","volume":"17","author":[{"given":"Sezayi","family":"Ozubek","sequence":"first","affiliation":[{"name":"Department of Pathobiology and Integrative Biomedical Sciences, College of Veterinary Medicine, Bond Life Sciences Center, University of Missouri","place":["Columbia, MO, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huitao","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pathobiology and Integrative Biomedical Sciences, College of Veterinary Medicine, Bond Life Sciences Center, University of Missouri","place":["Columbia, MO, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roman R.","family":"Ganta","sequence":"additional","affiliation":[{"name":"Department of Pathobiology and Integrative Biomedical Sciences, College of Veterinary Medicine, Bond Life Sciences Center, University of Missouri","place":["Columbia, MO, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,4,16]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"e00628","DOI":"10.1128\/IAI.00628-18","article-title":"Rickettsia rickettsii whole-cell antigens offer protection against Rocky Mountain spotted fever in the canine host","volume":"87","author":"Alhassan","year":"2019","journal-title":"Infect. 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