Molecular Detection of ESBL Factors

In order to detect extra-chromosomally encoded ESBL factors, plasmid DNA was isolated by commercially available kit (Thermo-Scientific Gene Jet plasmid Miniprep Kit). ESBL factors including bla_-TEM, bla_-SHV and genes bla_-OXA, bla_-PSE were PCR amplified as described elsewhere [18 (link)]. Amplified products were then purified (Gel Band Purification Kit, Amersham, USA) and sequencing was done by automated DNA sequencer (ABI 3130, Perkin-Elmer Applied Biosystems, Foster City, California). Sequences were reported to the Gene Bank database (accession number; KX171170–171195). PCR amplifications and sequencing of bla_-CTX-M allele was carried out, bla_-CTX-M types were determined by comparing DNA sequences available in the database [19 ]. A total of 18 different virulence factors (VF) corresponding to the main classes of extra-intestinal virulence associated genes (VAGs) including adhesins, toxins, siderophores, capsular proteins and uropathogenic-specific protein (usp) were scrutinized in all 155 isolates. VF genes were amplified by previously reported sets of primers and amplification conditions [20 (link)].

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Ali I., Rafaque Z., Ahmed I., Tariq F., Graham S.E., Salzman E., Foxman B, & Dasti J.I. (2019). Phylogeny, sequence-typing and virulence profile of uropathogenic Escherichia coli (UPEC) strains from Pakistan. BMC Infectious Diseases, 19, 620.

Publication 2019

Gene Jet plasmid Miniprep Kit Gel Band Purification Kit ABI 3130

Adhesins Allele Capsular Dna sequences Genes Genes classes Intestinal Plasmid Primers Protein Siderophores Toxins Virulence Virulence factors

Corresponding Organization : Quaid-i-Azam University

Other organizations : University of Haripur, University of Michigan–Ann Arbor

Top 5 similar protocols

Variable analysis

independent variables

Isolation of plasmid DNA using commercially available kit (Thermo-Scientific Gene Jet plasmid Miniprep Kit)
PCR amplification of ESBL factor genes including bla-TEM, bla-SHV, bla-OXA, and bla-PSE
Amplification and sequencing of bla-CTX-M allele

dependent variables

Presence/absence of ESBL factor genes
Specific bla-CTX-M allele types

control variables

Amplification and sequencing conditions for ESBL factor genes and bla-CTX-M allele

positive controls

No positive controls are explicitly mentioned.

negative controls

No negative controls are explicitly mentioned.

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