An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples

An rpoD gene sequence based evaluation of cultured Pseudomonas diversity on different growth media

“…Ultimately, we were able to base our analyses on a larger set of 129 strains of Pseudomonas species, while due to the limited amount of sequences available at that time, Yamamoto & Harayama (1998) had to restrict their analyses to 20 sequences. Mulet et al (2009) developed primers that allow specific targeting of the rpoD gene in a wide range of Pseudomonas species. Their primers were designed based on rpoD sequences of 35 species representing the different intrageneric phylogenetic Pseudomonas clusters.…”

“…DNA was extracted according to Pitcher et al (1989). rpoD gene amplification PCR was performed in triplicate on each DNA extract according to Mulet et al (2009). PCR amplicons were purified with the Nucleofast 1 96 PCR system (Millipore).…”

Pseudomonas helleri sp. nov. and Pseudomonas weihenstephanensis sp. nov., isolated from raw cow's milk

“…Seven isolates (WS4917 T , WS4995, WS4999, WS5001, WS5002, WS4993 T and WS4994) from seven different samples were assigned to the genus Pseudomonas but could not be identified to the species level. The phylogeny of the isolates was determined by 16S rRNA gene sequence and partial rpoD nucleotide sequence analysis, since the 16S rRNA gene cannot be used alone to discriminate related species of the genus Pseudomonas (Mulet et al, 2009(Mulet et al, , 2010. DNA extraction for PCR and PCR conditions have been described elsewhere .…”

“…As a modification, the annealing temperature for partial rpoD nucleotide sequences was increased to 51 8C to avoid unspecific primer binding. Primers used for PCR were 16S_27f (59-AGAGTTTGATCCTGGCTCA-39) and 16S_1492r (59-CGGCTACCTTGTTACGAC-39), leading to almost-complete 16S rRNA gene sequences, and PsEG30F (59-ATYGAAATCGCCAARCG-39) and PsEG790R (59-CGG-TTGATKTCCTTGA-39), resulting in partial rpoD gene sequences of ,750 bp (Mulet et al, 2009). Nucleotide sequencing was performed at LGC Genomics GmbH (Berlin, Germany) using primer PsEG30F for the partial rpoD gene and primers 16S_926r (59-CCGTCAATTCCTTT-GAGTTT-39) and 16S_519F (59-CAGCAGCCGCGGTA-ATAC-39) for the 16S rRNA gene.…”

The Study and Identification of Bacterial Spoilage Species Isolated from Catfish during Refrigerated Storage

“…These results compared to other samples with just one match at 100% accuracy raised concern. Results obtained by Mulet et al [11] showed the 16S rDNA gene sequence is a good tool for phylogenetic studies, however, in many cases it does not discriminate sufficiently to permit resolution of Pseudomonas spp. intrageneric relationships because of its slow rate of evolution.…”