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Protocol publication

[…] To determine which bla KPC variants were circulating in patients from hospitals in São Luis, Maranhão, the 882-bp amplified PCR products were purified using a commercial Wizard® SV Gel and PCR Clean-Up System Kit (both from Promega Corporation, Madison, USA), according to the manufacturer’s instructions. PCR purified products were sequenced by Myleus Biotechnology (Belo Horizonte, Minas Gerais, Brazil) using an ABI 3730XL DNA analyzer (Applied Biosystems, Carlsbad, CA, USA). Amplicons were bidirectionally sequenced at least three times; thus, each PCR product was sequenced six times.The quality of the sequence electropherograms obtained during the sequencing process was analyzed with ChromasPro (http://www.technelysium.com.au/chromas.html) software. At least two sequences of each type of KPC were chosen from the database for alignments using MEGA 4.0 []. Similarities between the nucleotide sequences obtained were verified using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi).To identify the bla KPC gene variants, all sequences were translated into amino acids using the software ExPASy [translate tool (http://web.expasy.org/translate/) in the six reading frames]. The correct translation was chosen based on the available data in GenBank. Deduced amino acid sequences were compared with KPC protein sequences from GenBank using BLASTX. Similarity values for amino acid sequences ranged from 99 to 100%, indicating a highly conserved region. […]

Pipeline specifications

Software tools ChromasPro, MEGA, BLASTN, Expasy translate tool, BLASTX
Databases ExPASy
Organisms Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Homo sapiens
Diseases Infection, Sprains and Strains, Wiskott-Aldrich Syndrome, Precursor Cell Lymphoblastic Leukemia-Lymphoma
Chemicals Amikacin, Gentamicins