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In dogs, it is considered the most clinically significant parasite and a known cause of acquired cardiomyopathy. Clinically, canine heartworm disease can manifest acutely as caval syndrome and chronically as heart failure, often resulting in death. Different types of diagnostic tests, such as antigen-detection and microfilariae-detection tests (MFDTs), including modified Knott\u2019s test and molecular assays, may be used to diagnose\n                      <jats:italic>D. immitis<\/jats:italic>\n                      infection. Although some molecular tools, such as real-time PCR (qPCR), exhibit high accuracy, cross-reactivity with other\n                      <jats:italic>Dirofilaria<\/jats:italic>\n                      species found in companion animals remains a concern. Following our laboratory\u2019s previously published protocol, cross-reactivity was found with\n                      <jats:italic>Dirofilaria striata<\/jats:italic>\n                      isolated from a subcutaneous nodule of a cat from Texas. The objectives of this study were to: (i) evaluate the performance of three newly designed probe-based qPCR assays for detecting\n                      <jats:italic>D. immitis<\/jats:italic>\n                      and (ii) assess overall detection with the newly designed protocols compared with the previously published protocol and other diagnostic tests.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We designed three new probes using accessioned\n                      <jats:italic>D. immitis<\/jats:italic>\n                      cytochrome\n                      <jats:italic>c<\/jats:italic>\n                      subunit 1 sequences from various countries worldwide to increase reliability. These new probes were then tested with genomic DNA extracted from\n                      <jats:italic>D. immitis<\/jats:italic>\n                      and\n                      <jats:italic>D. striata.<\/jats:italic>\n                      Following this, we further assessed the performance of these newly designed probes and previously developed probe using 136 archival shelter dog samples previously found to be positive for\n                      <jats:italic>D. immitis<\/jats:italic>\n                      by at least one diagnostic test.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Out of the three probes tested, two (probe 2 and probe 3) specifically detected\n                      <jats:italic>D. immitis<\/jats:italic>\n                      and showed no cross-reactivity with\n                      <jats:italic>D. striata.<\/jats:italic>\n                      Probe 3 showed the highest prevalence of\n                      <jats:italic>D. immitis<\/jats:italic>\n                      among all qPCR assays (68.4%;\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200993\/136 samples), followed by the original probe (66.9%;\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200991\/136), with probe 2 (59.6%;\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200981\/136) showing the lowest. Our analysis of agreement between probes using Cohen\u2019s kappa (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      ) statistic indicated almost perfect agreement between probe 3 and the previously published probe (\n                      <jats:italic>\u03ba<\/jats:italic>\n                      \u2009=\u20090.86). The shelter dog samples were also evaluated using other heartworm diagnostic methods, including the modified Knott\u2019s test, which was positive for 61.0% (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200983\/136) of the positive samples, and the previously published qPCR, which was positive for 66.9% (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200991\/136) of the samples. All samples were also tested in parallel with a commercially available antigen detection test (DiroCHEK\u00ae; Zoetis Inc., Parsippany, NJ, USA), yielding 80.1% positivity pre-heat treatment (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u2009109\/136) and 94.9% post-heat treatment (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u2009129\/136). Probe 3 paired with post-heat treatment achieved the highest positivity of the molecular diagnostic tests  (97.1%;\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u2009132\/136).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      This optimized probe-based qPCR assay (probe 3) provides an accurate and reliable method to detect\n                      <jats:italic>D. immitis<\/jats:italic>\n                      microfilariae in dogs and other carnivore hosts.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07323-2","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:18:08Z","timestamp":1787217488000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Improved specificity of a probe-based real-time PCR for the detection of Dirofilaria immitis in canine blood"],"prefix":"10.1186","volume":"19","author":[{"given":"Maureen A.","family":"Kelly","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiana L.","family":"Sanders","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexa","family":"Starnes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Orr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joe L.","family":"Luksovsky","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline","family":"Sobotyk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hassan","family":"Hakimi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pabasara","family":"Weerarathne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine M.","family":"Budke","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guilherme G.","family":"Verocai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"7323_CR1","doi-asserted-by":"publisher","DOI":"10.1186\/1756-3305-6-288","volume":"6","author":"F Dantas-Torres","year":"2013","unstructured":"Dantas-Torres F, Otranto D. 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