A high resolution DNA melting curve analysis for the rapid and efficient molecular diagnostics of extended spectrum β-lactamase determinants from foodborne escherichia coli

dc.contributor.authorNjage, Patrick Murigu Kamau
dc.contributor.authorBuys, E.M. (Elna Maria)
dc.contributor.emailelna.buys@up.ac.zaen_ZA
dc.date.accessioned2020-06-02T08:30:30Z
dc.date.available2020-06-02T08:30:30Z
dc.date.issued2020-01-09
dc.description.abstractThe accurate identification of Extended-Spectrum β-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present β-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.en_ZA
dc.description.departmentConsumer Scienceen_ZA
dc.description.departmentFood Scienceen_ZA
dc.description.librarianam2020en_ZA
dc.description.sponsorshipThe National Research Foundation Free-standing Postdoctoral Fellowshipen_ZA
dc.description.urihttp://www.mdpi.com/journal/microorganismsen_ZA
dc.identifier.citationNjage, P.M.K. & Buys, E. 2020, 'A high resolution DNA melting curve analysis for the rapid and efficient molecular diagnostics of extended spectrum β-lactamase determinants from foodborne escherichia coli', Microorganisms, vol. 8, art. 90, pp. 1-11.en_ZA
dc.identifier.issn2076-2607 (online)
dc.identifier.other10.3390/microorganisms8010090
dc.identifier.urihttp://hdl.handle.net/2263/74819
dc.language.isoenen_ZA
dc.publisherMDPI Publishingen_ZA
dc.rights© 2020 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.en_ZA
dc.subjectEscherichia colien_ZA
dc.subjectAntibiotic resistanceen_ZA
dc.subjectGenotypingen_ZA
dc.subjectHigh-resolution DNA melting curveen_ZA
dc.subjectReal-time PCRen_ZA
dc.subjectExtended spectrum β-lactamase (ESBL)en_ZA
dc.subjectPolymerase chain reaction (PCR)en_ZA
dc.subjectDeoxyribonucleic acid (DNA)en_ZA
dc.titleA high resolution DNA melting curve analysis for the rapid and efficient molecular diagnostics of extended spectrum β-lactamase determinants from foodborne escherichia colien_ZA
dc.typeArticleen_ZA

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