The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria.

Abstract

Variableregions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms. In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms. This study presents a general method to obtain species-specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes. The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes. Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacterla.Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest. In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail. A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus. Theapplication of DNA probe methodology to the identification and detection of microorganisms is becoming well established in the field of diagnostic bacteriology. Fundamental to such a technology is the ability to define suitable nucleic acid sequences that identify a particular microorganism or group of related microorganisms. DNA probes have been used successfully in the identification and detection of microorganisms./1,21 In general, nucleic acid sequences that are used as DNA probe targets for microorganisms fall into five main categories: (1) DNA sequences that code for antigens, (2) DNA sequences that code for toxins, (3) DNA sequences identified by differential hybridization using total DNA probes from related species against a DNA bank made from the microorganism of interest, (4) unique plasmid-borne DNA sequences, and (5) ribosomal RNA (rRNA) sequences.In the case of the first four categories, the means by which the DNA targets are selected involve laborious cloning methodologies and frequently a signficant amount of biochemical or immunological data. Subsequently, cross-reactivity, nonspecificity, and the possibility of the loss of the target sequences due to recombination and deletion events, especially with relation to plasmid-borne sequences, results, in some cases, in a limited usefulness of these probes, rRNAs in general have been the main targets for the generation of DNA markers for microorganisms, and we have used this region as the target for DNA probes for a number of microorganisms. (2) The major disadvantage these sequences have as candidates for DNA markers is that the "variable" regions can almost be identical when closely related microorganisms are examined, resulting in the need for finely defined stringency conditions when using such probes to detect a microorganism of...

Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera

“…The length variability of the ITS region has been used for identification of prokaryotic species including cyanobacteria (Barry et al 1991;Jensen et al 1993;Lu et al 1997). In this study we amplified the tRNA ile , the ITS region and the 5′ end of the 23S rRNA (Wilmotte et al 1993).…”

Sequence Diversity in the 16S–23S Intergenic Spacer Region (ISR) of the rRNA Operons in Representatives of the Escherichia coli ECOR Collection

“…Although recently the stress of phylogeny studies has been centered on 16S rRNA, this molecular chronometer may not be appropriate for close relationships in some taxa. The gene coding for 23S rRNA and also the 16S-23S spacer are now considered as candidates for studying variation within and between closely related species (Barry et al 1991;Dolzani et al 1994;Emond et al 1993;Kostman et al 1992;Leys et al 1994;Matar et al 1993;McLaughlin et al 1993;Whiley et al 1995).…”

Oxacillin Resistance and Antimicrobial Susceptibility Profile of Staphylococcussaprophyticus and Other Staphylococci Isolated from Patients with Urinary Tract Infection

“…isolates were genotyped using conserved primer sequences adjacent to the 16S and 23S genes. This method described by Barry et al [39] and Couto et al [40] is known as internal transcribed spacer-PCR. In the present study, genotyping was performed as described by Couto et al [40] using the G1 (5′ GAA GTC GTA ACA AGG 3′) and L1 (5′ CAA GGC ATC CAC CGT 3′) primers.…”