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Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of <jats:italic>B. canis<\/jats:italic> and <jats:italic>A. phagocytophilum<\/jats:italic> in canine clinical samples.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Sequence-based polymorphism analysis of genes encoding <jats:italic>B. canis<\/jats:italic>-specific merozoite surface protein Bc28.1 (<jats:italic>Bc28.1<\/jats:italic>) and <jats:italic>A. phagocytophilum<\/jats:italic> malate dehydrogenase (<jats:italic>mdh<\/jats:italic>) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The presence of three <jats:italic>B. canis Bc28.1<\/jats:italic> gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of <jats:italic>A. phagocytophilum mdh<\/jats:italic> genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for <jats:italic>B. canis<\/jats:italic> and <jats:italic>A. phagocytophilum<\/jats:italic>. The analytical sensitivity of this assay was ten gene copies\/ reaction for both pathogens.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>A novel duplex RT-PCR molecular method was developed for the detection of <jats:italic>B. canis<\/jats:italic> and <jats:italic>A. phagocytophilum<\/jats:italic> in canine clinical samples. Sequence variability of <jats:italic>Bc28.1<\/jats:italic> and <jats:italic>mdh<\/jats:italic> genes indicated the genetic variability of <jats:italic>B. canis<\/jats:italic> and <jats:italic>A. phagocytophilum<\/jats:italic> isolates occurring in Latvian domestic dogs.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical Abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-021-04756-9","type":"journal-article","created":{"date-parts":[[2021,5,20]],"date-time":"2021-05-20T13:03:07Z","timestamp":1621515787000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis"],"prefix":"10.1186","volume":"14","author":[{"given":"Agnija","family":"Kivrane","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agne","family":"Namina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maija","family":"Seleznova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarmite","family":"Akopjana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina","family":"Capligina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renate","family":"Ranka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,5,20]]},"reference":[{"key":"4756_CR1","doi-asserted-by":"publisher","first-page":"336","DOI":"10.1186\/s13071-016-1596-0","volume":"9","author":"L Solano-Gallego","year":"2016","unstructured":"Solano-Gallego L, Sainz A, Roura X, Estrada-Pena A, Miro G. 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