Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria

McLaughlin, Gerald L.; Howe, Daniel K.; Biggs, David R.; Smith, Amanda R.; Ludwinski, Penny H.; Fox, Barry C.; Tripathy, Deoki N.; Frasch, Carl E.; Wenger, Jay D.; Carey, Roberta B.; Hassan-King, Musa; Vodkin, Michael H.

doi:10.1006/mcpr.1993.1002

Cited by 37 publications

(35 citation statements)

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

1998

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The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

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Section: Discussionmentioning

confidence: 99%

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

1998

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“…Multicopy genes are still the optimal choice providing the potential for highly sensitive assays. PCR assays based upon multicopy ribosomal genes such as the 16S rRNA have been described but still rely on ethidium bromide stained gel analysis [16,17] or Southern hybridisation [18].…”

Section: Discussionmentioning

confidence: 99%

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

Borrow¹

1998

FEMS Microbiology Letters

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At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reactionenzyme-linked immunosorbent assay (PCR ELISA)) has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

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“…Sequence analysis of bacterial ribosomal (r) DNA loci was used to design primer pairs which specifically detected N meningitidis [56]. An amplicon of 600 bp obtained from the 16s-23s internal transcribed spacer region of N meningitidis has been digested with AZuI to generate restriction fragments which permit molecular typing [57].…”

Section: Pcr-based Methodsmentioning

confidence: 99%

Molecular typing methods for Neisseria meningitidis

Yakubu¹,

Abadi²,

Pennington³

1999

Journal of Medical Microbiology

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Neisseria rneningifidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningifidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods -PFGE, MLEE and MLST -is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.

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Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria

Cited by 37 publications

References 0 publications

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

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

False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA

Molecular typing methods for Neisseria meningitidis

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