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

Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 31 January 2025 V1 Latest version Share on Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts Authors : Shradha Gemini , Gururaj Kumaresan 0000-0002-1461-1692 [email protected] , and S.V Singh Authors Info & Affiliations https://doi.org/10.22541/au.173833013.32359097/v1 220 views 102 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Brucellosis is a zoonotic disease that is caused by the species of bacteria, Brucella affecting goats and other livestock, that leads to economic deprivation and the likelihood of human transmission. This study sought to develop a highly sensitive and specific TaqMan® real-time PCR assay integrating hydrolysis probes for multiplex assay to differentiate active Brucella infection from passive shedding in goats targeting specific genes associated with Brucella melitensis . Using discontinuous 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. The sensitivity of the multiplex assay for determining the Limit of Detection was evaluated by log 10 serial dilutions of Brucella nucleic acid (DNA, showing the presence of bacteria) and (RNA showing the presence of live bacteria), for each gene and optimized by probe, primer, and template titrations. LOD calculated for DNA was 8.53×10 10 (dil.1) to 8.53×10 7 (dil.3) for omp25- HEX, = 1.32×10 11 (dil.1) to 1.32×10 8 (dil.3) for omp31- FAM, 1.08×10 11 (dil.1) to 1.08×10 7 (dil.4) for IS711- Texas Red. The lowest detectable limits were 8.53×10 7 , 1.32×10 8 , and 1.32×10 8 copies, respectively. For RNA (cDNA) LOD calculated was 1.58×10 10 (dil.1) to 1.58×10 7 (dil.3) for omp25- HEX, 2.45×10 10 (dil.1) to 1.8×10 6 (dil.4) for omp31- HEX, 2.0×10 10 (dil.1) to 2.0×10 6 (dil.4) for IS711- Texas Red. The lowest detectable limits were 1.58×10 7 , 1.8×10 6 and 2.0×10 6 copies, respectively.LOD calculated from the cloned plasmid (target genes were cloned into pGEM-T Easy vector) was 2.6×10 4 , 1.8×10 5 , 1.6×10 4 in simplex qPCR assay and 2.6×10 4 , 1.8×10 5 , 1.6×10 5 in multiplex QPCR assay for omp31, omp21, IS711 genes respectively, making it easier to distinguish between animals that are actively shedding and those that are passively releasing non-viable Brucella . The specificity of the assay was confirmed through validation by comparing the nucleic acids from Brucella melitensis culture with Escherichia coli (Gram-negative) and Staphylococcus aureus, (Gram-positive) with the latter generating no specific response. This assay has the potential to supplement epidemiological research to ascertain the true prevalence of Brucella infection in goats. It also shows promise as a replacement for culture-based tests. Supplementary Material File (research artical-shradha(ted).docx) Download 3.36 MB Information & Authors Information Version history V1 Version 1 31 January 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords brucella melitensis limit of detection multiplex taqman outer membrane proteins real-time pcr sensitivity and specificity testing Authors Affiliations Shradha Gemini ICAR - Central Institute for Research on Goats View all articles by this author Gururaj Kumaresan 0000-0002-1461-1692 [email protected] ICAR - Central Institute for Research on Goats View all articles by this author S.V Singh GLA University View all articles by this author Metrics & Citations Metrics Article Usage 220 views 102 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shradha Gemini, Gururaj Kumaresan, S.V Singh. Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts. Authorea . 31 January 2025. DOI: https://doi.org/10.22541/au.173833013.32359097/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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