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In this study, a real-time SYBR Green PCR assay was developed to detect <jats:italic>Babesia<\/jats:italic> and <jats:italic>Theileria<\/jats:italic> species in hard ticks (<jats:italic>N<\/jats:italic>\u00a0=\u00a065) and cattle blood samples (<jats:italic>N<\/jats:italic>\u00a0=\u00a0143) from Thailand. Using primers targeting the mitochondrial cytochrome b gene for <jats:italic>Babesia<\/jats:italic> and the nuclear 18S rRNA gene for <jats:italic>Theileria<\/jats:italic>, the assay measured specific melting temperatures (Tm) for each species. The results showed distinct Tm values for <jats:italic>Babesia bigemina<\/jats:italic> (74.38\u00a0\u00b1\u00a00.04\u00a0\u00b0C), <jats:italic>Babesia bovis<\/jats:italic> (75.7\u00a0\u00b1\u00a00.06\u00a0\u00b0C), <jats:italic>Theileria orientalis<\/jats:italic> (74.61\u00a0\u00b1\u00a00.03\u00a0\u00b0C), <jats:italic>Theileria sinensis<\/jats:italic> (75.84\u00a0\u00b1\u00a00.03\u00a0\u00b0C), and <jats:italic>Theileria annulata<\/jats:italic> (74.06\u00a0\u00b1\u00a00.03\u00a0\u00b0C). The assay demonstrated high specificity, with a cutoff cycle threshold of\u00a0&lt;\u00a035 cycles and a minimum detectable concentration of 10 copies\/\u03bcL. Significant species differences in melting curves were confirmed using Tukey\u2019s HSD test (<jats:italic>p<\/jats:italic>\u00a0&lt;\u00a00.05). <jats:italic>Theileria orientalis<\/jats:italic> was detected in 8.4% of cattle blood samples, while <jats:italic>T. sinensis<\/jats:italic> was found in 25.9%, and <jats:italic>B. bigemina<\/jats:italic> in 0.7%. <jats:italic>Theileria orientalis<\/jats:italic> was also detected in 7.7% of tick samples, <jats:italic>T. sinensis<\/jats:italic> in 16.9%, and <jats:italic>B. bigemina<\/jats:italic> in 6.1%. The assay returned negative results for all non-target blood and tissue pathogens tested for specificity. This robust, high-throughput assay is highly effective for monitoring <jats:italic>Babesia<\/jats:italic> and <jats:italic>Theileria<\/jats:italic> infections, facilitating close surveillance and intervention efforts against tick-borne diseases in cattle.<\/jats:p>","DOI":"10.1051\/parasite\/2025040","type":"journal-article","created":{"date-parts":[[2025,8,26]],"date-time":"2025-08-26T07:53:01Z","timestamp":1756194781000},"page":"54","source":"Crossref","is-referenced-by-count":4,"title":["Development and validation of a real-time SYBR green PCR method for the detection and differentiation of <i>Babesia<\/i> and <i>Theileria<\/i> species (Apicomplexa: Piroplasmida) in hard ticks and cattle blood from Thailand"],"prefix":"10.1051","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-3745-5972","authenticated-orcid":false,"given":"May 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