Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region

Abstract Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non- Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae .     

Diversity of Salmonella strains isolated from the aquatic environment as determined by serotyping and amplification of the ribosomal DNA spacer regions

Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186-194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.     

PCR amplification and characterization of the intergenic spacer region of the ribosomal DNA in Pyrenophora graminea

Successful amplification of the whole intergenic spacer region of the nuclear ribosomal repeat (IGS) in Pyrenophora graminea was obtained with a PCR-based assay. Single amplification products showed length differences. Depending on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates could be identified. The RFLP patterns of isolates obtained with the 6-base cutting enzymes ApaI, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups. Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length. In order to characterize the long and short IGS-PCR products the restriction map is shown. The long product shows an additional HindIII site and a BglII site that is lacking in the short product. However, the latter shows a SacI site that is not present in the long IGS-PCR product. Therefore, the described PCR-RFLP analysis of the IGS appears to be a useful tool to resolve genetic variation between P. graminea isolates.     

PCR amplification and polymorphism analysis of the intergenic spacer region of ribosomal DNA in Tuber borchii

PCR amplification of the complete intergenic spacer region (IGS) of the Tuber borchii nuclear ribosomal repeat was obtained using universal primers CNL 12 and NS1rev. In order to improve amplification yield a specific primer, T1, was selected from a partial sequence of the IGS product. IGS diversity was characterized both at the intraindividual and intraspecific level. The results obtained at the intraindividual level showed 10% varying repeats on ten screened colonies, while at the intraspecific level the IGS polymorphism was evident as difference in length amplification between mycelial strains and fruit bodies: 3.5 kb and 2 kb respectively.     

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.     

Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer

Abstract The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.     

DNA sequence analyses of the ribosomal spacer regions in theTriticeae

Two regions of the ribosomal DNA (rDNA) were sequenced from a range of species from the tribeTriticeae. One region, the central spacer, was found to be more divergent in sequence than the other, the 18 S-spacer junction. Both regions contained sequences 20–30 bp long which were more highly conserved than the remainder of the region and their possible significance in rDNA expression is discussed. Phenetic relationships based on the sequence data were generally consistent with the relationships based on other criteria. Species possessing the S, E, J₁J₂, D, and B genomes clustered together, with the H genome species being the most distinct of those examined. The R, P, and V genome species occupy an intermediate position in the overall pattern of relationships. Some relationships differed in detail from those established by other parameters, for example the position of the N genome species, and explanations for discrepancies of this type are discussed.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

MLST based serotype prediction for the accurate identification of non typhoidal Salmonella serovars

Molecular Biology Reports - Traditional serotyping based on the phenotypic variation of O- and H-antigen remains as the gold-standard for the identification and classification of Salmonella...