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Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect <jats:italic>T. gondii<\/jats:italic> and <jats:italic>N. caninum<\/jats:italic> while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. <jats:italic>T. gondii<\/jats:italic> and <jats:italic>N. caninum<\/jats:italic>. The <jats:italic>T. gondii<\/jats:italic> assay is based on a 529-bp repetitive (REP) element and the <jats:italic>N. caninum<\/jats:italic> assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either <jats:italic>T. gondii<\/jats:italic> or <jats:italic>N. caninum<\/jats:italic>. The <jats:italic>T. gondii<\/jats:italic> and <jats:italic>N. caninum<\/jats:italic> multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.<\/jats:p>","DOI":"10.1007\/s00436-023-07796-5","type":"journal-article","created":{"date-parts":[[2023,2,19]],"date-time":"2023-02-19T07:13:41Z","timestamp":1676790821000},"page":"1043-1047","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum"],"prefix":"10.1007","volume":"122","author":[{"given":"Marcus","family":"Truong","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan","family":"\u0160lapeta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,2,18]]},"reference":[{"key":"7796_CR1","doi-asserted-by":"publisher","first-page":"2177","DOI":"10.1111\/jvim.16565","volume":"36","author":"F Allan","year":"2022","unstructured":"Allan F, Blake D, Miller Z, Church D (2022) Biliary protozoa in a dog with acute cholangiohepatitis fed a raw food diet. 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