A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus Pseudomonas (Sensu Stricto) in Environmental Samples

Abstract

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection ofPseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection ofPseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool forPseudomonas population structure analyses and taxonomic confirmations.

PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods

“…PCR products from soils, using the Ps-for/Ps-rev, were not obtained when the annealing temperature was 68³C but were present at 65³C. This observation is an agreement with Widmer et al [39].…”

“…In this study, and others [39], ampli¢cation using Ps-for/ Ps-rev with soil-extracted DNA as a template could only be achieved when the annealing temperature was lowered to 65³C with resultant lowering of ampli¢cation stringency. Widmer et al [39] showed that 92% of Ps-for/Psrev clones ampli¢ed directly from soil were Pseudomonas in origin, with 2.7% represented by a clone closely related to a Nitrosospira sp. (GenBank accession number AJ012107) which displays three mismatches with Ps-for and two mismatches with Ps-rev.…”

“…Ps-for/Ps-rev PCR was conducted according to Widmer et al [39]. Ps-for/Ps-rev products were separated from primers on QIAquick columns according to the manufacturer (Qiagen, Crawley, UK).…”

Influence of Biological Treatment and Ultraviolet Disinfection System on Pseudomonas spp. Diversity in Wastewater as Assessed by Denaturing Gradient Gel Electrophoresis

“…(Sampling: EB, raw input; DTI, distribution tank input; D1, disc tank 1; Bio1, biofilm of disc 1; Bio2, biofilm of disc 2; D2, disc tank 2 and SD, output secondary clarifier). and PsR, reported by Widmer et al [21]. The PCR mixture consists of MgCl 2 (2.5 mM), dNTPs (200 mmol), 2.5 U Taq DNA polymerase in 1Â Taq buffer and 20 pmol of each primer.…”

The impact of grassland management regime on the community structure of selected bacterial groups in soils

“…For the ammonia oxidisers, the ¢rst round of PCR was carried out using the CTO primers [15] with a cycle of 95 ‡C for 5 min followed by 35 cycles of 95 ‡C for 1 min, 57 ‡C for 1 min, 72 ‡C for 2 min, and a ¢nal extension of 72 ‡C for 10 min. Pseudomonad (sensu stricto) 16S rDNA was ampli¢ed using the primers Ps-for and Ps-rev [16], and the cycle, 95 ‡C for 5 min followed by 35 cycles of 95 ‡C for 1 min, 62 ‡C for 1 min, 72 ‡C for 2 min., and a ¢nal extension time of 10 min at 72 ‡C was employed. The products from the ¢rst round of PCR were then run on a 1% low melting point agarose gel (Roche Diagnostics), stained in ethidium bromide and visualised under ultraviolet light.…”