Computational protocol: Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh

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Protocol publication

[…] Chromosomal DNA from representative strains was prepared and purified by procedures described previously. Sequencing was performed to identify specific TEM and OXA type ESBL genes. Sequencing of gyrA and parC genes were performed in the qnr positive ESBL isolates according the procedure described elsewhere –. After PCR, the amplicons were purified with the GFX PCR DNA and gel band purification kit (Amersham Pharmacia, USA), and sequenced using the dideoxy-nucleotide chain termination method with an ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit (Perkin-Elmer Applied Biosystems, Foster City, California) on an automated sequencer (ABI PRISM 310). The chromatogram sequencing files were inspected using Chromas 2.23 (Technelysium, Queensland, Australia), and contigs were prepared using SeqMan II (DNASTAR, Madison, WI). Nucleotide and protein sequence similarity searches were performed using the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD) BLAST (Basic Local Alignment Search Tool) server on GenBank database, release 138.0 . Multiple sequence alignments were developed using CLUSTALX 1.81 . Sequences were manually edited in the GeneDoc version 2.6.002 alignment editor. Sequences of OXA and TEM genes reported in this paper were submitted to the Genbank using the National Center for Biotechnology Information (NCBI, Bethesda, MD) Sequin, version 7.77 under the accession number EU752482-86 and EU752487-91, respectively. […]

Pipeline specifications

Software tools Chromas, BLASTN, Clustal W
Application Sanger sequencing
Organisms Escherichia coli, Homo sapiens
Diseases Infection, Zellweger Syndrome
Chemicals Aztreonam, Cefotaxime, Ceftazidime, Ceftriaxone, Ciprofloxacin, Nalidixic Acid