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Quantitative real‐time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis
Author(s) -
Morillo Juan M,
Lau Laura,
Sanz Mariano,
Herrera David,
Silva Augusto
Publication year - 2003
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1034/j.1600-0765.2003.00684.x
Subject(s) - porphyromonas gingivalis , amplicon , actinobacillus , polymerase chain reaction , biology , real time polymerase chain reaction , microbiology and biotechnology , repeatability , sybr green i , periodontitis , gene , chemistry , bacteria , genetics , chromatography , dentistry , medicine
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 destilled 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 repeatibility of cycle threshold ( C T ) 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 repeatibility will allow its application to analysis of subgingival plaque samples.