期刊
MOLECULAR AND CELLULAR PROBES
卷 23, 期 3-4, 页码 140-147出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.mcp.2009.02.001
关键词
PCR; Pseudomonas detection; rpoD; Species identification
类别
资金
- CICYT (Spain) [CGL 00838/BOS, CGL 2008-03242/BOS]
- FEDER
- Pla Balear de Recerca i Desenvolupament Tecnologic de les Illes Balears (PRIB)
A polymerase chain reaction-based approach was developed for species identification of Pseudomonas strains and for the direct detection of Pseudomonas populations in their natural environment. A highly selective set of primers (PsEG30F and PsEG790R), giving an amplicon of 760 nucleotides in length, was designed based on the internal conserved sequences of 33 selected rpoD gene sequences (the sigma 70 factor subunit of the DNA polymerase) of Pseudomonas type strains, representing the entire intrageneric phylogenetic clusters described in the genus. The utility of the primer set was verified on 96 Pseudomonas type strains and on another 112 recognised Pseudomonas strains. The specificity of the primer set was also tested against strains from species not belonging to the genus Pseudomonas. These primers were also shown to be useful for the direct detection of Pseudomonas species in environmental DNA after a cloning procedure. These results were compared in parallel with other cloning procedures described previously, based on the analysis of other genes (16S rDNA and ITS1) and also by using primers designed for rpoD on sequences from gamma-proteobacteria. All of the cultured Pseudomonas strains tested could be amplified with these novel primers, indicating that this method is also a useful tool for the specific analysis of Pseudomonas populations from environmental samples without the need for cultivation. (C) 2009 Elsevier Ltd. All rights reserved.
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