{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T03:29:26Z","timestamp":1789615766421,"version":"build-2803163510"},"reference-count":37,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2000,7,1]],"date-time":"2000-07-01T00:00:00Z","timestamp":962409600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2000,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title><jats:p>A multiplex amplification and detection platform for the diagnosis of<jats:italic>Mycobacterium bovis<\/jats:italic>and<jats:italic>Brucella abortus<\/jats:italic>infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of<jats:italic>B. abortus<\/jats:italic>and a repeat-sequence region in the<jats:italic>hsp65<\/jats:italic>gene of<jats:italic>M. bovis<\/jats:italic>, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents\/ml of milk for<jats:italic>B. abortus<\/jats:italic>and as low as 4 CFU equivalents per ml of milk for<jats:italic>M. bovis<\/jats:italic>. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with<jats:italic>B. abortus<\/jats:italic>. The assay sensitivity, based on culture status as a \u201cgold standard,\u201d was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of<jats:italic>B. abortus<\/jats:italic>(samples from India) and<jats:italic>M. bovis<\/jats:italic>(samples from Mexico) was identified by BPDA-PCR. In samples from India,<jats:italic>B. abortus<\/jats:italic>shedding was identified in 86% of milk ring test-positive animals (<jats:italic>n<\/jats:italic>= 15) and 80% of milk ring test-negative cows (<jats:italic>n<\/jats:italic>= 5). In samples from Mexico,<jats:italic>M. bovis<\/jats:italic>was identified by PCR in 32.6% of pools (<jats:italic>n<\/jats:italic>= 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (<jats:italic>n<\/jats:italic>= 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (<jats:italic>n<\/jats:italic>= 70) had a modest prevalence of<jats:italic>M. bovis<\/jats:italic>shedding in nasal swabs (2.9%) and milk (1.4%) and of<jats:italic>B. abortus<\/jats:italic>in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.<\/jats:p>","DOI":"10.1128\/jcm.38.7.2602-2610.2000","type":"journal-article","created":{"date-parts":[[2019,12,31]],"date-time":"2019-12-31T17:57:00Z","timestamp":1577815020000},"page":"2602-2610","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":42,"title":["A Multiplex Approach to Molecular Detection of<i>Brucella abortus<\/i>and\/or<i>Mycobacterium bovis<\/i>Infection in Cattle"],"prefix":"10.1128","volume":"38","author":[{"given":"Srinand","family":"Sreevatsan","sequence":"first","affiliation":[{"name":"<!--label omitted: 1-->ClinCyte, LLC, San Diego, California 921211;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jack B.","family":"Bookout","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->ClinCyte, LLC, San Diego, California 921211;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fidel","family":"Ringpis","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->ClinCyte, LLC, San Diego, California 921211;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veera S.","family":"Perumaalla","sequence":"additional","affiliation":[{"name":"<!--label omitted: 2-->Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&amp;M University, College Station, Texas 77843-44672;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas A.","family":"Ficht","sequence":"additional","affiliation":[{"name":"<!--label omitted: 2-->Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&amp;M University, College Station, Texas 77843-44672;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"L. Garry","family":"Adams","sequence":"additional","affiliation":[{"name":"<!--label omitted: 2-->Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&amp;M University, College Station, Texas 77843-44672;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sue D.","family":"Hagius","sequence":"additional","affiliation":[{"name":"<!--label omitted: 3-->Department of Veterinary Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 708033;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip H.","family":"Elzer","sequence":"additional","affiliation":[{"name":"<!--label omitted: 3-->Department of Veterinary Science, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 708033;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Betsy J.","family":"Bricker","sequence":"additional","affiliation":[{"name":"<!--label omitted: 4-->Division of Zoonotic Disease Research, National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 500104;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Girish K.","family":"Kumar","sequence":"additional","affiliation":[{"name":"<!--label omitted: 5-->Department of Genetics and Plant Breeding, University of Agricultural Sciences, Bangalore, India 5600655; and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Rajasekhar","sequence":"additional","affiliation":[{"name":"<!--label omitted: 6-->Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore, India 5600246"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Srikrishna","family":"Isloor","sequence":"additional","affiliation":[{"name":"<!--label omitted: 6-->Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore, India 5600246"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raj R.","family":"Barathur","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->ClinCyte, LLC, San Diego, California 921211;"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","unstructured":"Acha P. 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