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J Periodontal Res. 2003 Oct;38(5):518-24.

Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Author information

1
Laboratory of Oral Microbiology, Faculty of Odontology, University Complutense, Madrid, Spain.

Abstract

OBJECTIVE:

To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique.

MATERIAL AND METHODS:

Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species.

RESULTS:

A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number.

CONCLUSION:

A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.

PMID:
12941077
[Indexed for MEDLINE]

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