WITHDRAWN: Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts

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This study developed a multiplex qPCR assay targeting <italic>Brucella</italic> genes <italic>omp25</italic>, <italic>omp31</italic>, and <italic>IS711</italic> to differentiate active from passive brucellosis in goats, demonstrating high sensitivity and specificity.

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This withdrawn Research Square preprint (v2) concerns the development of an mRNA-based hydrolysis probe TaqMan real-time multiplex qPCR assay intended to distinguish active versus passive brucellosis in caprine (goat) hosts. However, the full text was withdrawn because it was submitted in error, and the authors state that the work should not be cited as a reference, leaving no substantive methods, data, or results available in the provided material. The only explicit caveats presented are the withdrawal status and the associated instruction not to cite. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

Abstract Background An anthropozoonotic disease, brucellosis is caused by the species of bacteria, Brucella melitensis, affecting small ruminants and other livestock, leading to fiscal losses, and the likelihood of human transmission. Methods This study sought to develop a highly sensitive and specific TaqMan® real-time PCR assay integrating hydrolysis probes for a multiplex assay to differentiate active Brucella infection from passive shedding in goats, targeting specific genes associated with Brucella melitensis . Using discontiguous conserved sequences, primers were designed for B. melitensis genes i.e., omp25, omp31, and IS711 with their compatible fluorescent dyes (Hex, FAM, Texas Red), and quencher molecules (BHQ1, BHQ1, BHQ2), respectively enabling multi-target detection within the same assay. Results The sensitivity of the multiplex assay was evaluated using serial log10 dilutions of Brucella nucleic acid (DNA, showing the presence of bacteria) and (RNA showing the presence of live bacteria), with LOD expressed as absolute copy numbers per reaction. For DNA templates, omp25 was detectable from 8.53×10¹⁰ to 8.53×10⁷ copies, omp31 from 1.32×10¹¹ to 1.32×10⁸, and IS711 from 1.08×10¹¹ to 1.08×10⁷. For RNA (cDNA): omp25 was detected from 1.58×10¹⁰ to 1.58×10⁷ copies, omp31 from 2.45×10¹⁰ to 1.8×10⁶, and IS711 from 2.0×10¹⁰ to 2.0×10⁶. Plasmid-based LODs were 2.6×10⁴, 1.8×10⁵, and 1.6×10⁴ (simplex), and 2.6×10⁴, 1.8×10⁵, and 1.6×10⁵ copies (multiplex). Logistic regression analysis confirmed that omp25 (HEX) had the highest sensitivity with a 95% LOD of approximately 1.95×10⁻⁸ copies. No amplification was observed with Escherichia coli (Gram-negative) or Staphylococcus aureus (Gram-positive), confirming assay specificity. Conclusion However, other abortion-causing microorganisms, such as Listeria monocytogenes , Campylobacter fetus , and Chlamydia abortus , were not tested in this study. Future research should include these pathogens to further validate the specificity of the assay.
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WITHDRAWN: Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article WITHDRAWN: Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts Shradha Gemini, K. Gururaj, S. V Singh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6115135/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Editorial Note The full text of this preprint has been withdrawn, as it was submitted in error. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Editorial notes are used to provide important context regarding the topic of a preprint or to alert readers to potential issues concerning that preprint or a downstream publication associated with it. For more information on editorial notes, see our Editorial Policies . Abstract The full text of this preprint has been withdrawn, as it was submitted in error. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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