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Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other <jats:italic>Babesia<\/jats:italic> species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both <jats:italic>T. equi<\/jats:italic> and <jats:italic>B. caballi<\/jats:italic>. In this method, primers and probes targeting the <jats:italic>18S rRNA<\/jats:italic> gene of both <jats:italic>T. equi<\/jats:italic> and <jats:italic>B. caballi<\/jats:italic>, the <jats:italic>ema-1<\/jats:italic> gene of <jats:italic>T. equi<\/jats:italic> and the <jats:italic>bc48<\/jats:italic> gene of <jats:italic>B. caballi<\/jats:italic> were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R<jats:sup>2<\/jats:sup> value was 0.983 for the concentration range 0.2 ng (4.1 \u00d7 10<jats:sup>7<\/jats:sup> DNA copies) to 2.0 fg (4.1 \u00d7 10<jats:sup>1<\/jats:sup> DNA copies). For the pUC57-<jats:italic>ema<\/jats:italic> gene DNA, the R<jats:sup>2<\/jats:sup> value was 0.993 for the concentration range 0.2 ng (5.26 \u00d7 10<jats:sup>7<\/jats:sup> DNA copies) to 2.0 fg (5.26 \u00d7 10<jats:sup>2<\/jats:sup> DNA copies). For the pUC57-Bc18S gene DNA the R<jats:sup>2<\/jats:sup> value was 0.976 for the concentration range 2.0 ng (4.21 \u00d7 10<jats:sup>8<\/jats:sup> DNA copies) to 2.0 fg (4.21 \u00d7 10<jats:sup>2<\/jats:sup> DNA copies). For the pUC57-Te18S gene DNA, the R<jats:sup>2<\/jats:sup> value was 0.952 (Fig.\u00a0S3b) for the concentration range 2.0 ng (4.16 \u00d7 10<jats:sup>8<\/jats:sup> DNA copies) to 2.0 fg (4.16 \u00d7 10<jats:sup>2<\/jats:sup> DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the <jats:italic>bc48<\/jats:italic> gene of <jats:italic>B. caballi<\/jats:italic> and the <jats:italic>18S rRNA<\/jats:italic> gene of <jats:italic>T. equi<\/jats:italic> is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing <jats:italic>T. equi<\/jats:italic> and <jats:italic>B. caballi<\/jats:italic>, showing great potential for on-site EP screening of horses.<\/jats:p>","DOI":"10.1038\/s41598-020-60997-1","type":"journal-article","created":{"date-parts":[[2020,3,5]],"date-time":"2020-03-05T11:06:26Z","timestamp":1583406386000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis"],"prefix":"10.1038","volume":"10","author":[{"given":"Rong","family":"Lei","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyi","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Di","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yize","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qijun","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,3,5]]},"reference":[{"key":"60997_CR1","doi-asserted-by":"publisher","first-page":"1334","DOI":"10.1111\/jvim.12168","volume":"27","author":"LN Wise","year":"2013","unstructured":"Wise, L. 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