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We developed and validated a SYBR Green-based real-time PCR assay targeting the <jats:italic>msp1<\/jats:italic> gene to detect and differentiate <jats:italic>P. knowlesi, P. cynomolgi<\/jats:italic>, and <jats:italic>P. inui<\/jats:italic>. Species-specific amplification was confirmed through distinct melting temperature (Tm) profiles. The assay demonstrated high analytical sensitivity, with a limit of detection of 10 copies\/\u00b5L, excellent specificity with no cross-reactivity, and strong reproducibility, with low coefficients of variation for both cycle threshold (Ct) and Tm values. Amplification efficiency was within acceptable ranges, with R<jats:sup>2<\/jats:sup> &gt; 0.90 across standard curves. The assay was evaluated using 191 archived blood samples from wild <jats:italic>M. fascicularis<\/jats:italic> collected across three provinces in Thailand, with <jats:italic>P. knowlesi<\/jats:italic> detected in two samples. Both positive detections were confirmed by nested PCR and sequencing. This assay offers a rapid, cost-effective, and reliable tool for detecting simian malaria parasites in laboratory analyses and has potential for further application in field surveillance.<\/jats:p>","DOI":"10.1038\/s41598-025-07337-3","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T01:47:13Z","timestamp":1751420833000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and validation of a multiplex PCR assay with melt curve analysis for detecting simian Plasmodium in wild Macaca fascicularis"],"prefix":"10.1038","volume":"15","author":[{"given":"Thitiluck","family":"Swangsri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rucksak","family":"Rucksaken","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanat","family":"Sricharern","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wang","family":"Nguitragool","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naowarat","family":"Saralamba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,2]]},"reference":[{"key":"7337_CR1","doi-asserted-by":"publisher","DOI":"10.30875\/9789287076717","volume-title":"World Malaria Report 2024","author":"World Health Organization","year":"2024","unstructured":"World Health Organization. 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