{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T16:00:53Z","timestamp":1792166453975,"version":"4.4.0"},"reference-count":42,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,5,27]],"date-time":"2026-05-27T00:00:00Z","timestamp":1779840000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key R&amp;D Program of China","doi-asserted-by":"publisher","award":["2024YFD1800200"],"award-info":[{"award-number":["2024YFD1800200"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Three pathogenic species of the genus Yersinia, including Plague-associated Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, are commonly associated with human infection. Current qPCR detection methods are mainly limited to the identification of one or two Yersinia species in a single reaction tube, while multiplex assays for multiple genera have been more commonly reported. Therefore, the present study aimed to establish a multiplex TaqMan qPCR assay for the simultaneous detection of these three pathogenic Yersinia species. Primer and probe sets were designed based on the inv gene for Y. pseudotuberculosis, the caf1 gene for Y. pestis, and the foxA gene for Y. enterocolitica. Under the optimized reaction conditions, the standard curve slopes for the caf1, inv, and foxA genes were \u22123.046, \u22122.968, and \u22122.948, respectively. The correlation coefficients (R2) ranged from 0.993 to 0.996, while the amplification efficiencies ranged from 109% to 115%. The limits of detection (LOD) were determined to be 5 \u00d7 102 copies\/\u03bcL for inv (FAM), 1 \u00d7 101 copies\/\u03bcL for caf1 (ROX), and 1 \u00d7 101 copies\/\u03bcL for foxA (CY5). The sensitivity of the multiplex qPCR assay was 10- to 100-fold higher than that of conventional PCR, depending on the target. Specificity experiments demonstrated that no cross-reactivity was observed with non-target bacteria, including Francisella tularensis, Brucella spp., Vibrio cholerae, Salmonella Typhi, and Shigella spp. The intra-assay coefficients of variation (CVs) ranged from 0.13% to 0.79%, whereas the inter-assay CVs ranged from 0.62% to 2.61%. Among 173 spleen samples collected from wild rodents, no positive signal for Y. pestis or Y. pseudotuberculosis was detected. In contrast, Y. enterocolitica was detected in three samples (1.73%, 3\/173). In conclusion, the multiplex qPCR assay developed in this study provides a sensitive and specific tool for the simultaneous detection of three pathogenic Yersinia species and has the potential to improve detection efficiency in clinical and epidemiological investigations.<\/jats:p>","DOI":"10.3390\/vetsci13060520","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T08:00:36Z","timestamp":1779955236000},"page":"520","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development of a Multiplex TaqMan Real-Time PCR Assay for the Simultaneous Detection and Differentiation of Three Pathogenic Yersinia Species"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-9994-3060","authenticated-orcid":false,"given":"Xue","family":"Yang","sequence":"first","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Lei","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongjun","family":"Luo","sequence":"additional","affiliation":[{"name":"Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, Urumqi 830002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiali","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiuyu","family":"Fan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofang","family":"Yan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haohong","family":"Zheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ciren","family":"Zhuoma","sequence":"additional","affiliation":[{"name":"Shuanghu County Agricultural and Animal Husbandry Science and Technology Service Station, Nagqu 853300, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunhan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7495-9332","authenticated-orcid":false,"given":"Haifeng","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziyao","family":"Zhou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhijun","family":"Zhong","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jialiang","family":"Xin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9850-528X","authenticated-orcid":false,"given":"Zhengli","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9898-5754","authenticated-orcid":false,"given":"Guangneng","family":"Peng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1128\/CMR.10.1.35","article-title":"Yersinia 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