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In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, <jats:italic>S<\/jats:italic>. <jats:italic>aureus<\/jats:italic> (<jats:italic>spa<\/jats:italic>) and methicillin resistance (<jats:italic>mecA<\/jats:italic>). In addition, an internal control (lambda phage) was added to determine the assay\u2019s true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the <jats:italic>S<\/jats:italic>. <jats:italic>aureus<\/jats:italic> and coagulase-negative staphylococci (<jats:italic>CoNS<\/jats:italic>). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was \u223c 1 CFU\/mL from 2-5mL of whole blood and that of PCR was \u223c 1pg of NA. However, the combined protocol led to a lower detection limit of 100\u20131000 <jats:italic>MRSA<\/jats:italic> CFUs\/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study.<\/jats:p>","DOI":"10.1371\/journal.pone.0294782","type":"journal-article","created":{"date-parts":[[2023,11,27]],"date-time":"2023-11-27T19:12:25Z","timestamp":1701112345000},"page":"e0294782","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["A multiplex Taqman PCR assay for MRSA detection from whole blood"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1803-5389","authenticated-orcid":true,"given":"Suhanya","family":"Duraiswamy","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sushama","family":"Agarwalla","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khoi Sheng","family":"Lok","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yee Yung","family":"Tse","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1679-3087","authenticated-orcid":true,"given":"Ruige","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiping","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,11,27]]},"reference":[{"issue":"1","key":"pone.0294782.ref001","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1038\/nrmicro3161","article-title":"Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus","volume":"12","author":"TJ Foster","year":"2014","journal-title":"Nat Rev Microbiol"},{"issue":"9","key":"pone.0294782.ref002","doi-asserted-by":"crossref","first-page":"2464","DOI":"10.1172\/JCI38226","article-title":"Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era","volume":"119","author":"FR DeLeo","year":"2009","journal-title":"J Clin Investig"},{"issue":"5","key":"pone.0294782.ref003","doi-asserted-by":"crossref","first-page":"1640","DOI":"10.1128\/JCM.00143-07","article-title":"Reevaluation of Clinical and Laboratory Standards Institute disk diffusion breakpoints for tetracyclines for testing Enterobacteriaceae","volume":"45","author":"HSF Sader","year":"2007","journal-title":"J Clin Microbiol"},{"key":"pone.0294782.ref004","first-page":"384","article-title":"Manual of clinical microbiology","author":"TL Bannerman","year":"2003","journal-title":"Washington: American Society for Microbiology"},{"key":"pone.0294782.ref005","first-page":"384","author":"TL Bannerman","year":"2003","edition":"8"},{"issue":"4","key":"pone.0294782.ref006","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1007\/s11908-017-0576-7","article-title":"Challenges and strategies for prevention of multidrug-resistant organism transmission in nursing homes","volume":"19","author":"G Dumyati","year":"2017","journal-title":"Curr Infect Dis Rep"},{"issue":"11","key":"pone.0294782.ref007","article-title":"Infection control strategies for preventing the transmission of meticillin\u2010resistant Staphylococcus aureus (MRSA) in nursing homes for older people","author":"C Hughes","year":"2013","journal-title":"Cochrane Database Sys Rev"},{"issue":"4","key":"pone.0294782.ref008","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1128\/CMR.00109-13","article-title":"Coagulase-negative staphylococci","volume":"27","author":"K Becker","year":"2014","journal-title":"Clin Microbiol Rev"},{"issue":"1","key":"pone.0294782.ref009","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.diagmicrobio.2018.11.015","article-title":"Pathogen enrichment from human whole blood for the diagnosis of bloodstream infection: Prospects and limitations","volume":"94","author":"M Pilecky","year":"2019","journal-title":"Diagn Microbiol Infect"},{"key":"pone.0294782.ref010","first-page":"16","article-title":"Enrichment of bacterial DNA for the diagnosis of blood stream infections","author":"NT Trung","year":"2016","journal-title":"BMC Infect Dis"},{"key":"pone.0294782.ref011","first-page":"12","article-title":"A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections","author":"B Hu","year":"2021","journal-title":"Front Microbiol"},{"issue":"11","key":"pone.0294782.ref012","doi-asserted-by":"crossref","first-page":"4947","DOI":"10.1128\/JCM.42.11.4947-4955.2004","article-title":"New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci","volume":"42","author":"K Zhang","year":"2004","journal-title":"J. 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Oncol"},{"issue":"7","key":"pone.0294782.ref027","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets","volume":"33","author":"S Kumar","year":"2016","journal-title":"Mol Biol Evol"},{"issue":"2","key":"pone.0294782.ref028","doi-asserted-by":"crossref","first-page":"199","DOI":"10.11150\/kansenshogakuzasshi.84.199","article-title":"Evaluation of MRSA rapid detection by real-time PCR directly from positive blood cultures","volume":"84","author":"T Kimura","year":"2010","journal-title":"Kansenshogaku zasshi Jpn J Infect Dis"},{"issue":"1","key":"pone.0294782.ref029","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gks808","article-title":"Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies","volume":"41","author":"A Klindworth","year":"2013","journal-title":"Nucleic Acids Res"},{"issue":"1","key":"pone.0294782.ref030","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1099\/00221287-148-1-257","article-title":"Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set","volume":"148","author":"MA Nadkarni","year":"2002","journal-title":"Microbiol"},{"issue":"6","key":"pone.0294782.ref031","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1099\/00221287-146-6-1275","article-title":"Comparison of conserved structural and regulatory domains within divergent 16S rRNA\u201323S rRNA spacer sequences of cyanobacteria","volume":"146","author":"I Iteman","year":"2000","journal-title":"Microbiol"},{"issue":"2","key":"pone.0294782.ref032","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1128\/jb.173.2.697-703.1991","article-title":"16S ribosomal DNA amplification for phylogenetic study","volume":"173","author":"WG Weisburg","year":"1991","journal-title":"J Bacteriol"},{"issue":"2","key":"pone.0294782.ref033","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1111\/j.1574-6941.1998.tb00469.x","article-title":"Divergent members of the bacterial division Verrucomicrobiales in a temperate freshwater lake","volume":"25","author":"G Zwart","year":"1998","journal-title":"FEMS Microbiol. 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