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Infected animals may remain asymptomatic for years while intermittently shedding MAP into their environment through feces, contributing to ongoing transmission. To develop a robust multiplex qPCR assay, we reviewed all TaqMan qPCR studies published since 1990 and selected 18 primer-probe combinations targeting the MAP-specific F\n                      <jats:italic toggle=\"yes\">57<\/jats:italic>\n                      gene and the repetitive sequence elements ISMAP\n                      <jats:italic toggle=\"yes\">02<\/jats:italic>\n                      and IS\n                      <jats:italic toggle=\"yes\">900<\/jats:italic>\n                      . In samples with moderate to high MAP levels, all combinations performed well, with only minor differences in analytical performance. However, in low-abundance samples, several TaqMan designs yielded unreliable results, indicating limited specificity in complex matrices. Among the evaluated assays, the IS\n                      <jats:italic toggle=\"yes\">900<\/jats:italic>\n                      -Herthnek design demonstrated significantly higher diagnostic sensitivity, detecting MAP in 81% of low-abundance samples, compared to 0% and 3% for the IS\n                      <jats:italic toggle=\"yes\">900<\/jats:italic>\n                      -Kim and IS\n                      <jats:italic toggle=\"yes\">900<\/jats:italic>\n                      -Slana assays, respectively. For ISMAP\n                      <jats:italic toggle=\"yes\">02<\/jats:italic>\n                      , only the ISMAP\n                      <jats:italic toggle=\"yes\">02<\/jats:italic>\n                      -Sevilla assay produced reliable results. For F\n                      <jats:italic toggle=\"yes\">57<\/jats:italic>\n                      , Herthnek provided the most consistent and accurate quantification. Similar trends were observed in environmental sample testing. Based on these findings, we recommend a multiplex qPCR assay incorporating the IS\n                      <jats:italic toggle=\"yes\">900<\/jats:italic>\n                      -Herthnek, ISMAP\n                      <jats:italic toggle=\"yes\">02<\/jats:italic>\n                      -Sevilla, and F\n                      <jats:italic toggle=\"yes\">57<\/jats:italic>\n                      -Herthnek TaqMan designs for the detection of MAP in fecal and environmental samples. This combination offers high analytical sensitivity and specificity, making it a valuable and accurate tool for the diagnosis of paratuberculosis and for environmental surveillance on dairy farms to identify herds potentially harboring MAP-infected animals.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Mycobacterium avium<\/jats:italic>\n                        subsp.\n                        <jats:italic toggle=\"yes\">paratuberculosis<\/jats:italic>\n                        (MAP) is the etiological agent of Johne\u2019s disease (JD) in ruminant livestock industries and has been associated with Crohn\u2019s disease in humans. Emerging scientific evidence also links MAP to other human conditions, including inflammatory bowel disease, autoimmune disorders, colorectal cancer, and Alzheimer\u2019s disease. This potential public health threat has intensified interest in developing more sensitive diagnostic tools and effective control strategies to eradicate MAP from dairy herds. Infected ruminants typically remain in the subclinical stage of the disease for 2\u20135 years, during which they shed MAP in their feces and contaminate the environment. Diagnosis during this stage is particularly challenging, as the pathogen evades the host\u2019s immune response, rendering serological tests insufficiently sensitive. In contrast, fecal PCR offers greater sensitivity than serum ELISA and traditional culture methods. Multiplex quantitative PCR is especially promising due to its high specificity and sensitivity for detecting MAP-infected animals and identifying herds with active shedders. Herd-level environmental screening, followed by individual animal testing, represents a robust national biosecurity strategy. This approach is a critical step toward reducing MAP transmission and improving herd health within the dairy industry.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00872-25","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T13:00:52Z","timestamp":1767013252000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Systematic evaluation and optimization of TaqMan qPCR assays targeting F\n                    <i>57<\/i>\n                    , ISMAP\n                    <i>02<\/i>\n                    , and IS\n                    <i>900<\/i>\n                    for multiplex detection of\n                    <i>Mycobacterium avium<\/i>\n                    subsp.\n                    <i>paratuberculosis<\/i>"],"prefix":"10.1128","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3281-209X","authenticated-orcid":true,"given":"Nathalie","family":"Bissonnette","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00zywfh80","id-type":"ROR","asserted-by":"publisher"}],"name":"Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre","place":["Sherbrooke, Canada"]},{"id":[{"id":"https:\/\/ror.org\/0161xgx34","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universit\u00e9 de Montr\u00e9al","place":["Saint-Hyacinthe, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2299-3835","authenticated-orcid":true,"given":"S\u00e9verine","family":"Ollier","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00zywfh80","id-type":"ROR","asserted-by":"publisher"}],"name":"Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre","place":["Sherbrooke, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1897-0221","authenticated-orcid":true,"given":"Jean-Philippe","family":"Brousseau","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00zywfh80","id-type":"ROR","asserted-by":"publisher"}],"name":"Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre","place":["Sherbrooke, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4084-5673","authenticated-orcid":true,"given":"Alexander Stephan","family":"Byrne","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04haebc03","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Memorial University of Newfoundland","place":["St. John's, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Savard","sequence":"additional","affiliation":[{"name":"Biovet","place":["Saint-Hyacinthe, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2436-682X","authenticated-orcid":true,"given":"Kapil","family":"Tahlan","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04haebc03","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, Memorial University of Newfoundland","place":["St. John's, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.1007\/s10620-024-08508-4"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms8122054"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.18103\/mra.v12i7.5541"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.1017\/S095026881500076X"},{"key":"e_1_3_5_6_2","doi-asserted-by":"publisher","DOI":"10.2903\/j.efsa.2017.4960"},{"key":"e_1_3_5_7_2","doi-asserted-by":"publisher","DOI":"10.1111\/zph.12221"},{"key":"e_1_3_5_8_2","doi-asserted-by":"publisher","DOI":"10.1079\/9781789243413.0266"},{"key":"e_1_3_5_9_2","first-page":"519","volume-title":"86th Annual Meeting of the United States Animal Health Association","author":"Merkal RS","year":"1982","unstructured":"Merkal RS, Lyle P, Whipple DL. 1982. 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