A Novel Oligonucleotide Pair for Genotyping Members of the Pseudomonas Genus by Single-Round PCR Amplification of the gyrB Gene

AGARAS, BETINA; VALVERDE, CLAUDIO

Methods and Protocols

MDPI

Lugar: Basilea; Año: 2018 vol. 1

Pseudomonas is a phylogenetically diversebacterial genus which is broadly distributed in different ecological niches,and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genesroutinely used for multilocus sequence analysis (MLSA). As we noticed thatthere was not a single primer pair available in the literature suitable fordirect sequencing of this gene, we decided to design a unique oligonucleotidepair and to set up a polymerase chain reaction (PCR) protocol to obtain asingle amplicon for the entire Pseudomonasgenus. Based on the available gyrBsequence from 148 Pseudomonasspecies, we identified highly conserved regions to design oligonucleotideswithout fully degenerate positions. We then set up cycling conditions forachieving high specificity and yield of the PCR protocol. Then, we showed thatthe amplicons produced with this procedure were appropriate for directsequencing with both primers, obtaining more than 95% of amplicons coverage.Finally, we demonstrated that a PCR-RFLP (Restriction Fragment Length Polymorphism)approach served to differentiate among Pseudomonasspecies, and even between members of the same species.