Specific PCR amplification for the genus Pseudomonas targeting the 3' half of 16S rDNA and the whole 16S-23S rDNA spacer.
A PCR protocol was developed for the selective amplification of a segment of the ribosomal RNA operon in Pseudomonas strains. Two specific conserved sequences suitable for PCR priming were identified in the middle of the 16S rDNA and at the very beginning of the 23S rDNA respectively. As a result, amplified region includes the 3' half of the 16S rDNA with the whole 16S-23S rRNA Internal Transcripted Spacer (ITS1) sequence. The specificity of the primer set was checked on sequence databases and validated on collection strains and on one hundred soil bacterial isolates. Our results showed that both collection, soil-inhabiting Pseudomonas and some Pseudomonas-related Azotobacter DNAs could be amplified. This specific PCR for the detection of Pseudomonas strains was in good agreement with colony hybridisation using a Pseudomonas-specific probe. The targeted segment is relevant for a characterisation at the species (16S rDNA) as well as at the infraspecific (ITS1) levels. This PCR-based approach offers promising potential for the characterisation of environmental Pseudomonas populations.
D024509:3' Flanking Region; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D020563:Gammaproteobacteria / Q000145:classification / Q000235:genetics; D005798:Genes, Bacterial; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000032:analysis*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012338:RNA, Ribosomal, 23S / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013045:Species Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S