Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria

• Published in: Journal of clinical microbiology 2000-06, Vol.38 (6), p.2362-2365
• Main Authors: LYONS, S. R, GRIFFEN, A. L, LEYS, E. J
• Format: Article
• Language: English
• Subjects: Bacterial diseases; Bacteriology; Biological and medical sciences; Chronic Disease; Dental Plaque - microbiology; DNA Primers; DNA, Ribosomal - isolation & purification; Ent and stomatologic bacterial diseases; Human bacterial diseases; Humans; Infectious diseases; Medical sciences; Periodontitis - etiology; Periodontitis - microbiology; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Porphyromonas gingivalis; Porphyromonas gingivalis - cytology; Porphyromonas gingivalis - genetics; Porphyromonas gingivalis - isolation & purification; Species Specificity; Taq Polymerase
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/jcm.38.6.2362-2365.2000

Accurate quantitation of the number of cells of individual bacterial species in dental plaque samples is needed for understanding the bacterial etiology of periodontitis. Real-time PCR offers a sensitive, efficient, and reliable approach to quantitation. Using the TaqMan system we were able to determine both the amount of Porphyromonas gingivalis and the total number of bacterial cells present in plaque samples. Using species-specific primers and a fluorescent probe, detection of DNA from serial dilutions of P. gingivalis cells was linear over a large range of DNA concentrations (correlation coefficient = 0.96). No difference was observed between P. gingivalis DNA alone and the same DNA mixed with DNA isolated from dental plaque, indicating that P. gingivalis levels can be determined accurately from clinical samples. The total number of cells of all bacterial species was determined using universal primers and a fluorescent probe. Standard curves using four different bacterial species gave similar results (correlation coefficient = 0.86). Levels of both P. gingivalis and total bacteria were determined from a series of human plaque samples. High levels of P. gingivalis were observed in several of the samples from subjects with periodontitis and none of those from healthy subjects. Real-time quantitative PCR provided a sensitive and reliable method for quantitating P. gingivalis. In addition, it allowed the determination of the total number of bacterial cells present in a complex sample so that the percentage of P. gingivalis cells could be determined.