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As populations of\n                      <jats:italic>Ixodes ricinus<\/jats:italic>\n                      , the principal vector, expand geographically, including into urban environments, there is a growing need for rapid, sensitive, and field-deployable surveillance tools. In this study, we aim to developed a Recombinase Polymerase Amplification Lateral Flow (RPA-LF) assay for the detection of\n                      <jats:italic>B. divergens<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      RPA assays targeting\n                      <jats:italic>B. divergens hsp70<\/jats:italic>\n                      ,\n                      <jats:italic>cox1<\/jats:italic>\n                      , and\n                      <jats:italic>18S rRNA<\/jats:italic>\n                      genes were designed and evaluated for sensitivity and specificity using serial dilutions and control samples, including other apicomplexan parasites and tick samples spiked with\n                      <jats:italic>B. divergens<\/jats:italic>\n                      DNA. To assess field performance, 279\n                      <jats:italic>I. ricinus<\/jats:italic>\n                      ticks were collected from urban parks in Greater London and screened by qPCR and a subset also tested using the new RPA-LF assays.\n                      <jats:italic>Anaplasma phagocytophilum<\/jats:italic>\n                      was screened by qPCR but not detected.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The\n                      <jats:italic>cox1<\/jats:italic>\n                      and\n                      <jats:italic>18S rRNA<\/jats:italic>\n                      RPA-LF assays demonstrated high specificity, with detection limits of at least 10\n                      <jats:sup>\u22125<\/jats:sup>\n                      \u00a0ng\/\u03bcl and 10\n                      <jats:sup>\u22123<\/jats:sup>\n                      \u00a0ng\/\u03bcl, respectively, under isothermal conditions (39\u00a0\u00b0C for 30\u00a0min). No cross-reactivity was observed with non-target DNA. Field validation using pooled samples revealed a single sample positive for\n                      <jats:italic>Babesia divergens<\/jats:italic>\n                      by qPCR, indicating an estimated nymphal infection prevalence of 0.4\u20131.2%, as previously observed in UK tick studies. The\n                      <jats:italic>18S rRNA<\/jats:italic>\n                      RPA-LF assay successfully detected this pool, whereas the\n                      <jats:italic>cox1<\/jats:italic>\n                      RPA-LF did not. Sanger sequencing confirmed the presence of\n                      <jats:italic>B. divergens<\/jats:italic>\n                      \/\n                      <jats:italic>B. capreoli<\/jats:italic>\n                      -like DNA, although species-level discrimination was limited due to high sequence similarity.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      We report the first successful development of an RPA-LF assay for the detection in ticks of\n                      <jats:italic>B. divergens<\/jats:italic>\n                      or the closely related species\n                      <jats:italic>B. capreoli<\/jats:italic>\n                      . The assay demonstrates high sensitivity and specificity under isothermal conditions and provides a portable and reliable tool for field-based molecular surveillance of emerging tick-borne pathogens. Field validation revealed\n                      <jats:italic>B. divergens<\/jats:italic>\n                      \/\n                      <jats:italic>B. capreoli<\/jats:italic>\n                      in questing ticks from urban green spaces in London, underscoring the importance of this tool for early detection and ongoing surveillance of tick-borne parasites in expanding urban ecosystems.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07549-0","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T10:03:00Z","timestamp":1785146580000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A recombinase polymerase amplification lateral flow assay (RPA-LF) for detection of Babesia divergens-like parasites"],"prefix":"10.1186","volume":"19","author":[{"given":"Ernest Chin Jia","family":"Bin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keir","family":"Hughes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheryl","family":"Whitehorn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mia","family":"White","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kayleigh","family":"Hansford","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jolyon","family":"Medlock","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen","family":"Knuepfer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taane G.","family":"Clark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mojca","family":"Kristan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susana","family":"Campino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"key":"7549_CR1","doi-asserted-by":"publisher","first-page":"140","DOI":"10.1093\/biohorizons\/hzr016","volume":"4","author":"BPJ Greenfield","year":"2011","unstructured":"Greenfield BPJ. 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