{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T17:09:00Z","timestamp":1792084140312,"version":"4.4.0"},"reference-count":11,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,2,14]],"date-time":"2023-02-14T00:00:00Z","timestamp":1676332800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004595","name":"Universiti Sains Malaysia Research University Grant (RUI)","doi-asserted-by":"publisher","award":["1001.PPSP.8012259"],"award-info":[{"award-number":["1001.PPSP.8012259"]}],"id":[{"id":"10.13039\/501100004595","id-type":"DOI","asserted-by":"publisher"}]},{"name":"USM Fellowship","award":["1001.PPSP.8012259"],"award-info":[{"award-number":["1001.PPSP.8012259"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background: Enterococci are Gram-positive cocci found in the guts of humans and animals. The goal of this research is to develop a multiplex PCR assay that can detect the Enterococcus genus, four VRE genes, and three LZRE genes simultaneously. Methods: Primers used in this study were specifically designed for the detection of 16S rRNA of Enterococcus genus, vanA\u2014vanB\u2014vanC\u2014vanD for vancomycin, cfr methyltransferase, and optrA, and poxtA, as well as an adenosine triphosphate-binding cassette (ABC) transporter for linezolid. A Vibrio cholerae\u00a0ctxA (internal amplification control) was included. Optimization of primer concentrations and PCR components was also done. This was followed by evaluating the sensitivity and specificity of the optimized multiplex PCR. Results: Final Primer concentrations were optimized as follows: 16S rRNA is 1.0 pmol\/\u03bcL, vanA is 1.0 pmol\/\u03bcL, optrA is 1.0 pmol\/\u03bcL, cfr is 1.0 pmol\/\u03bcL, poxtA is 0.1 pmol\/\u03bcL, vanB is 0.08 pmol\/\u03bcL, ctxA is 0.07 pmol\/\u03bcL, vanC is 0.8 pmol\/\u03bcL, and vanD is 0.1 pmol\/\u03bcL. Further, the optimized concentrations for MgCl2, dNTPs and Taq DNA polymerase were 2.5 mM, 0.16 mM, and 0.75 units respectively, and an annealing temperature of 64.5 \u00b0C. Conclusions: The developed multiplex PCR is sensitive and species-specific. The development of a multiplex PCR assay that will take into account all known VRE genes and linezolid mutation is highly recommended.<\/jats:p>","DOI":"10.3390\/diagnostics13040722","type":"journal-article","created":{"date-parts":[[2023,2,15]],"date-time":"2023-02-15T03:37:24Z","timestamp":1676432244000},"page":"722","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["A Nanoplex PCR Assay for the Simultaneous Detection of Vancomycin- and Linezolid-Resistant Genes in Enterococcus"],"prefix":"10.3390","volume":"13","author":[{"given":"Yusuf","family":"Wada","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"},{"name":"Department of Zoology, Faculty of Life Sciences, Ahmadu Bello University, Zaria 810211, Nigeria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azian","family":"Harun","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"},{"name":"Hospital Universiti Sains Malaysia, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0034-7624","authenticated-orcid":false,"given":"Chan Yean","family":"Yean","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6612-8360","authenticated-orcid":false,"given":"Abdul Rahman","family":"Zaidah","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"},{"name":"Hospital Universiti Sains Malaysia, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.17582\/journal.aavs\/2019\/7.11.1021.1034","article-title":"Vancomycin-Resistant Enterococcus: Issues in Human Health, Animal Health, Resistant Mechanisms and the Malaysian Paradox","volume":"7","author":"Wada","year":"2019","journal-title":"Adv. Anim. Vet. Sci."},{"key":"ref_2","unstructured":"(2022, December 19). Antimicrobial Resistance. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/antimicrobial-resistance."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e893","DOI":"10.1016\/S2542-5196(21)00280-1","article-title":"Global Antibiotic Consumption and Usage in Humans, 2000\u20132018: A Spatial Modelling Study","volume":"5","author":"Browne","year":"2021","journal-title":"Lancet Planet. Health"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/S0140-6736(21)02724-0","article-title":"Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis","volume":"399","author":"Murray","year":"2022","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Yean, C.Y., Yin, L.S., Lalitha, P., and Ravichandran, M. (2007). A Nanoplex PCR Assay for the Rapid Detection of Vancomycin and Bifunctional Aminoglycoside Resistance Genes in Enterococcus Species. BMC Microbiol., 7.","DOI":"10.1186\/1471-2180-7-112"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.mimet.2019.03.025","article-title":"Development of a Multiplex-PCR to Simultaneously Detect Acquired Linezolid Resistance Genes Cfr, OptrA and PoxtA in Enterococci of Clinical Origin","volume":"160","author":"Bender","year":"2019","journal-title":"J. Microbiol. Methods"},{"key":"ref_7","unstructured":"Zangenberg, G., Saiki, R.K., and Reynolds, R. (1999). PCR Applications, Academic Press."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e26","DOI":"10.4142\/jvs.2019.20.e26","article-title":"Multiplex PCR Analysis of Virulence Genes and Their Influence on Antibiotic Resistance in Enterococcus Spp. Isolated from Broiler Chicken","volume":"20","author":"Song","year":"2019","journal-title":"J. Vet. Sci."},{"key":"ref_9","first-page":"361","article-title":"Malaysian Journal of Microbiology Linezolid-resistant Enterococcus casseliflavus and Enterococcus gallinarum isolated from poultry farms in Kelantan, Malaysia","volume":"17","author":"Chan","year":"2021","journal-title":"Malays. J. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"307","DOI":"10.3389\/fpubh.2017.00307","article-title":"Sensitivity, Specificity, and Predictive Values: Foundations, Pliabilities, and Pitfalls in Research and Practice","volume":"5","author":"Trevethan","year":"2017","journal-title":"Front. Public Health"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Nik Zuraina, N.M.N., Goni, M.D., Amalina, K.N., Hasan, H., Mohamad, S., and Suraiya, S. (2021). Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens. Diagnostics, 11.","DOI":"10.3390\/diagnostics11050753"}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/13\/4\/722\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:35:17Z","timestamp":1760121317000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/13\/4\/722"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,14]]},"references-count":11,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["diagnostics13040722"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics13040722","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,2,14]]}}}