Dataset Information

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

ABSTRACT: By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.

SUBMITTER: Leys EJ

PROVIDER: S-EPMC263671 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

Leys E J EJ Griffen A L AL Strong S J SJ Fuerst P A PA

Journal of clinical microbiology 19940501 5

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosoma ...[more]

PMID: 8051258

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Project description:Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.

Dataset Information

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

Publications

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

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