Molecular Characterization of Carbapenemase-Producing Isolates

To perform the molecular characterization of the isolates recovered, total DNA extraction was carried out using Wizard® Genomic DNA Purification Kit according to manufacturer instructions (Promega, Madison, WI). PCR reactions using GoTaq® enzyme (Promega, Madison, WI) were carried out for the most common carbapenemase genes (bla_IMP, bla_VIM, bla_KPC and bla_NDM), as well as, the presence of mobile elements related with them. Plasmid extractions were carried using two different techniques, the QIAprep Spin Miniprep Kit following manufacturer instructions (Qiagen Germantown, MD, USA) and a manual plasmid extraction design for megaplasmids(23 (link), 24 (link)).

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Hoard A., Montaña S., Moriano A., Fernandez J.S., Traglia G.M., Quiroga C., Franchi A., Cohen E., Corigliano C., Almuzara M, & Ramirez M.S. (2020). Genomic analysis of two NDM-1 Providencia stuartii strains recovered from a single patient. Current microbiology, 77(12), 4029-4036.

Publication 2020

Wizard® Genomic DNA Purification Kit GoTaq® enzyme QIAprep Spin Miniprep Kit

Carbapenemase Enzyme Genes Genomic Plasmid Promega

Corresponding Organization :

Other organizations : California State University, Fullerton, Hospital de Clínicas "José de San Martín", University of Buenos Aires

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Variable analysis

independent variables

DNA extraction using Wizard® Genomic DNA Purification Kit
PCR reactions using GoTaq® enzyme
Plasmid extractions using QIAprep Spin Miniprep Kit and manual plasmid extraction for megaplasmids

dependent variables

Molecular characterization of the isolates recovered
Presence of carbapenemase genes (blaIMP, blaVIM, blaKPC, and blaNDM)
Presence of mobile elements related with carbapenemase genes

control variables

Manufacturer instructions for Wizard® Genomic DNA Purification Kit
Manufacturer instructions for QIAprep Spin Miniprep Kit
Manual plasmid extraction design for megaplasmids as described in references 23 and 24

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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