Rapid Detection and Quantification of Five Periodontopathic Bacteria by Real‐Time PCR

Abstract

Abstract:The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LighrCycler" system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from IlY to more than lOS cells (r=O.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.Key words: Periodontopathic bacteria, Periodontitis, Quantitative PCR Periodontal disease, a polymicrobial mixed infection, is one of the major oral diseases. It is caused by several microbial species, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii. Proper diagnosis of the disease depends on early detection of the above bacteria. Currently, the l6S rRNA-based polymerase chain reaction (PCR) method seems to be the most sensitive and rapid method for determining the prevalence of such microorganisms (l, 6, 10). Accurate quantification of periodontal pathogens in subgingival plaque is needed for understanding the etiologic role of these bacteria. The conventional PCR (endpoint PCR) method detects the plateau phase of the reaction, however, is difficult to quantify. Recently, a real-time PCR using the Lightf'ycler" system (Roche Molecular Biochemicals, Mannheim, Germany) that allows monitoring of the exponential phase has been used to detect Borrelia burgdorferi in tissue samples (5). This method allows rapid detection and quantification of the above bacteria in clinical samples. In addition, a real-time PCR using the TaqMan system (PElABI) has been used to quantitate B. forsythus (7) givalis (3) in subgingival plaque. This system, however, requires more time for analysis than the Lightf'ycler" system.In this study, we compared three methods, the conventional PCR method, the real-time PCR method using the Lightf'ycler" system, and the culture method, for the detection and quantification of periodontopathic bacteria including A. actinomycetemcomitans, B. forsythus, P. gingivalis, T. denticola, and T. socranskii in saliva and subgingival plaque. Materials and MethodsSample collection and nucleic acid extraction. Saliva samples were collected in sterile plastic tubes from five patients [three subjects with rapidly progressive periodontitis (RP), mean age ± standard deviation (SD) of 32 ± 8.7 years, and two subjects with adult periodontitis (AP), 60±5.7 years]. The saliva samples were washed four times with sterile distilled water. After the final wash, bacterial cell p...

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

“…This finding indicated that the use of saliva as well as subgingival plaque in the analysis of the oral bacterial community is effective. In addition, periodontopathic bacteria such as Actinobacillus actinomycetemcomitans, Tannerella forsythensis (formerly Bacteroides forsythus) (Sakamoto et al, 2002), Porphyromonas gingivalis, Treponema denticola and Treponema socranskii were more frequently detected in saliva than in subgingival plaque samples by the real-time PCR (Sakamoto et al, 2001). These results confirmed those of a previous study (Umeda et al, 1998).…”

Use of quantitative PCR to evaluate methods of bacteria sampling in periodontal patients

“…The sequences are shown in Table 1. The number of total bacteria was determined using the SYBR GREEN I format, and the primers were designed according to a previously reported method (17). In the PCR reaction to determine the number of total bacteria, 5 µl DNA was reacted in a 20-µl reaction solution containing SYBR Premix Ex Taq (Takara Bio Inc., Kyoto, Japan) and 0.5 µM primers.…”

Changes in salivary periodontal pathogens after orthodontic treatment: An in vivo prospective study

“…In particular, real-time PCR is simple, rapid, and useful for detecting uncultured or extremely anaerobic microorganisms. 6 Many studies have reported quantitative changes in bacterial levels related to orthodontic treatment and periodontal pathogens related to oral hygiene during orthodontic treatment. 7,8 However, few studies have investigated quantitative changes in periodontal pathogens after orthodontic treatment, specifically in saliva.…”