Quantification of Periodontopathic Bacteria in Saliva Using the Invader Assay

Abstract

SUMMARY: When quantifying periodontopathic bacteria, it is important to use a convenient method that does not produce false negative results. The Invader assay is a convenient method because it does not involve gene amplification. The purpose of this study was to evaluate the validity of the Invader assay to quantify periodontopathic bacteria. The Invader technology was applied in quantifying five periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola). The Invader assay produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of 10 3.7 copies/tube and intra-day and inter-day variance of 0.1z to 4.7z and 0.1z to 3.4z, respectively, in quantifying five periodontopathic bacteria. We compared the results of the Invader assay with those of real-time polymerase chain reaction (PCR) performed for quantifying five periodontopathic bacteria in 22 patients with periodontitis. Among the Invader-detectable bacterial strains of each species, significant correlations were observed in the counts of concerned bacterial species between these two methods, with correlation coefficients ranging from 0.757 to 0.996. This study validated repeatability and reproducibility of the Invader assay in quantifying periodontopathic bacteria and demonstrated consistent agreement between the Invader assay and real-time PCR in quantifying periodontopathic bacteria.

Characterization of specimens obtained by different sampling methods for evaluation of periodontal bacteria

“…Each sample thus collected was centrifuged at 3,000 rpm for 5 min at 4ºC. All the samples were collected in the order described above and then immediately sent to a medical examination laboratory (BML Corporation, Tokyo, Japan) for bacterial analysis (7)(8)(9)12).…”

“…Briefly, bacterial DNA was extracted using a commercial kit (MagNA Pure LC Total Nucleic Acid Isolation Kit, Roche, Basel, Switzerland). Primary probes and Invader oligos were used as described previously (7,12). Each tube contained 15 μL of reaction mixture containing 50 μM dNTP, 700 nM primary probe, 70 nM Invader oligo, 2.5 U polymerase chain reaction (PCR) enzyme (EagleTaq DNA polymerase, Roche), and the Invader core reagent kit (Cleavase XI Invader core reagent kit, HOLOGIC, Marlborough, MA, USA) containing a fluorescence resonance energy transfer (FRET) mix, an enzyme/MgCl 2 solution and template DNA.…”

Evaluation of a novel immunochromatographic device for rapid and accurate clinical detection of Porphyromonas gingivalis in subgingival plaque

“…The remaining sample solution in the tube was stored at −80°C and then sent to a microbiological testing laboratory (BML, Tokyo, Japan) for quantitative analysis of periodontal pathogens including P. gingivalis, using PCR-Invader method (Mein et al, 2000;Tada et al, 2012).…”

Changes in antimicrobial susceptibility profile and prevalence of quinolone low-sensitive strains in subgingival plaque from acute periodontal lesions after systemic administration of sitafloxacin

“…For the detection of periodontal pathogens (Aggregatibacter actinomycetemcomitans, P. gingivalis, T. forsythia and T. denticola) by PCR-Invader method [14,15], the subgingival plaque samples were sent to a commercial laboratory (BML, Tokyo) [12].…”