Molecular Characterization of MRSA Isolates

Positive S. aureus isolate genomic DNA was isolated using Wizard Genomic DNA preparation kit (Promega, Madison, WI). Polymerase chain reaction (PCR) was used to amplify the presence of methicillin resistance gene (mecA) and PVL genes (lukS, lukF) [24 (link), 25 (link)]. Furthermore, Staphylococcus protein A (spa; FOR 5’-GAACAA-CGTAACGGCTTCATCC-3′ and REV 5’-CAGCAGTAGTGCCGTTTGCCT) was used for molecular typing [26 (link)–28 (link)]. Ridom StaphType software was used to assign spa types (v2.2.1; Ridom GmbH, Wurzburg, Germany). The Based upon Repeat Pattern (BURP) algorithm was used to group spa types based on their genetic proximity [29 (link)], as well as Bionumerics software (version 7.6.2). Only spa typing was conducted, since previous studies have found high congruence and discriminatory power compared to MLST sequence data [28 (link), 30 (link), 31 ]. A positive (USA300) and negative control were used for all biochemical and molecular assays.

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Dalman M., Bhatta S., Nagajothi N., Thapaliya D., Olson H., Naimi H.M, & Smith T.C. (2019). Characterizing the molecular epidemiology of Staphylococcus aureus across and within fitness facility types. BMC Infectious Diseases, 19, 69.

Publication 2019

Wizard Genomic DNA preparation kit StaphType

Assays Genes Genomic Meca Methicillin resistance Polymerase chain reaction Promega Proximity Staphylococcus protein a

Corresponding Organization : Kent State University

Other organizations : Kabul University

Top 5 similar protocols

Variable analysis

independent variables

Genomic DNA isolation method (Wizard Genomic DNA preparation kit)
PCR amplification of methicillin resistance gene (mecA) and PVL genes (lukS, lukF)
Molecular typing method (spa typing using Ridom StaphType software)

dependent variables

Presence/absence of methicillin resistance gene (mecA)
Presence/absence of PVL genes (lukS, lukF)
Spa types and their grouping using BURP algorithm

control variables

Positive control (USA300)
Negative control

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