Subgingival plaque and saliva samples were collected from 81 male and female subjects, aged from 7 to 81, recruited from the Department of Dental Disease Prevention (University of Cagliari), who had given informed consent to take part in the microbiological analysis. The patient's conditions were: non-diseased (n = 26.32%), with gingivitis (n = 31, 38.3%), with chronic periodontitis (n = 17.21%) and with aggressive periodontitis (n = 7, 8.7%) 35. Each patient's health status and background were recorded: age, sex, probing pocket depth, clinical attachment level, bleeding on probing (measured in six sites on all teeth present in the mouth). None of the patients who took part in the experiments was under antibiotic therapy during the previous 6 months. At first, 400 μl of saliva was collected from each patient and placed into a sterile tube, afterwards the subgingival plaque samples were obtained (one site per patient) from the deepest periodontal sites in all subjects. The sample area was isolated using sterile cotton rolls and air-dried to avoid saliva contamination; supragingival plaque was removed by using a sterile curette. A sterile paper point ISO 45 (Roeko Dental, Langenau, Germany) was inserted into the pocket and held in place for 30 seconds 36. The paper point was then removed and placed into a vial containing 800 μl of sterile saline solution, NaCl 0.9%, with 15 glass beads (about 100 mg Bio-Spec Products, Bartesville, USA). After vigorous vortexing, 200 μl of the suspension was immediately used for culture analysis, while the remaining suspension (600 ul) was stored at -20°C and 400 ul of this were used for DNA extraction.

Genomic DNA from positive controls and clinical samples was obtained by the CTAB modified method. 400 μl of sample were added to 70 μl of 10% sodium dodecyl sulphate (SDS) and 5 μl of proteinase K at 10 mg/ml concentration (SIGMA – Aldrich, ST. Louis, Missouri, USA); after vigorous vortexing, this mixture was incubated for 10 minutes at 65°C. Next, 100 μl of NaCl [5 M] and 100 μl of CTAB/NaCl (0.274 M CTAB, Hexadecyl trimetylammonium bromide and 0.877 M NaCl, Sigma-Aldrich) were added to the tube, which was vortexed briefly and incubated at 65°C for 10 minutes. 750 μl of SEVAG (Chloroform: Isoamyl Alcohol 24:1, Sigma-Aldrich) were added and the mixture was vortexed for 10 sec. After centrifuging for 5 min (at 12000 rpm) 0.6 volumes of isopropanol (Sigma-Aldrich) were added to the supernatant. After 30 min at -20°C and after being centrifuged for 30 min at 12.000 rpm, the pellet was dried at room temperature for 20 min and suspended in 20 μl of molecular biology grade distilled water (Gibco, Invitrogen Paisley, Scotland, UK). 2 μl of this were used as DNA suspension for conventional PCR and real time PCR reaction.

Primers for real time PCR were designed using the promoter region on the Leukotoxin operon sequences extracted from the NCBI database GenBank with S68133 and M27399 accession numbers. Figure 1 shows primer sequence and position and the real time PCR mechanism with the two different (652/JP2) Aa genotypes. Possible oligonucleotide dimer formation, self-complementarity and the annealing temperatures of the real time PCR were calculated using the Oligo program vers. 4 (MedProbe, Oslo, Norway). The real time PCR primers (OG155 and OG156) amplified a region of 696 bp in the 652 genotype and 195 bp in the JP2 genotype (Figure 1). The theoretic melting temperatures of the different PCR amplicons (Tms) were calculated using module 1 of the DNA hybridization prediction algorithm program "HYTHER" 3750 with the following sets of parameters: (i) monovalent cation concentration at 0.05 mol/L, (ii) Mg²⁺ at 0.004 mol/L, (iii) a concentration of PCR products (Top/Bottom strands) at 10^-7 mol/L and (iv) hybridisation temperature at 37°C. The same procedures have been used to design all the oligonucleotides used in traditional PCR (Table 1).

By using two primers (OG155 and OG156) flanking an ltx promoter region, we obtained two different length PCR products: 696 bp for 652 and 195 bp for the JP2 genotype. These amplicons showed two different T_ms melting peaks after the PCR real time reaction with SYBR green I dye. To evaluate product specificity, ltx PCR amplicons were also sequenced by a conventional automated sequencer as described in literature by Ianelli et al.,1998, 38. The results were edited and analyzed with nucleotide-nucleotide BLAST (blastn) 51 and compared with sequences deposited in the DNA data bank.

Real time PCR standard curves were performed on different DNA extracts, obtained by different Aa genotype suspensions with a concentration range of 10⁷-10¹ cells/ml. The number of bacteria was calculated by the interpolation of the clinical sample threshold cycle 39 with a standard curve obtained for each genotype (Figure 4). Bacterial concentration in specimens was expressed in: i) Aa cells/ml paper point suspension for subgingival plaque and ii) Aa cells/ml saliva in saliva samples. We used the following equation to calculate Aa concentration for the paper point specimens.

a) [cells/ml paper point suspension].= ([Aa Rt cells]*2,5.

[Aa Rt cells]= Bacterial cells, calculated by PCR real time standard curve interpolation.

For a sensitivity study, molecular methods were compared with traditional culture analysis for all the clinical samples: 200 μl of saliva and paper point suspensions were diluted 10 and 100 fold in Schaedler Broth (Microbiol, Uta, Cagliari, Italy). 100 μl of each dilution were plated in Columbia agar blood (Microbiol, Uta, Cagliari, Italy) and incubated in a 15% CO₂ atmosphere at 37°C in a jar using the Genbox system (Biomérieux Marcy l'Etoile, France). After 6 days of incubation the typical Aa colonies were identified by a biochemical test using API 20A (Biomérieux Marcy l'Etoile, France) according to the manufacturer's instructions.

For the 81 analyzed samples we used traditional PCR able to detect: (i) A. actinomycetemcomitans by different DNA target (16S rRNA) and (ii) P. gingivalis, P. intermedia, T. forsythensis, T. denticola. For all bacteria, the reaction was performed in 25 μl reaction volumes using MegaMix 2MM-5 (Microzone Limited, West Sussex, UK) according to the manufacturer's instructions. The primer and target sequences used are shown in Table 1. The mixture contained 7 pmol of each primer, 3.8 mM MgCl₂ and 2 μl of DNA suspension. The thermocycler profile was as follows: an initial denaturation at 95° C for 5 min; 35 cycles consisting of 50°C for 1 min, 68° C for 3 min and 40 sec and 95° C for 1 min. PCR products were analysed by electrophoresis on a 1.2 % agarose gel containing ethidium bromide (0.5 mg/ml). With reconstruction experiments this method showed a detection limit range of 10-50 cells/PCR.

A reconstruction experiment confirmed the PCR real time selectivity in the DNA extracts of three different suspensions: (i) Aa 652, (ii) Aa JP2 and (iii) a mixture of the two genotypes. Figures 2, 3 shows melting curves and the 1% agarose gel electrophoresis results of PCR real time products. In our study, with reconstruction and with clinical samples, we demonstrated melting temperatures of 76 ± 2 °C in the JP2 positive samples, and 82 ± 2 °C in the 652 positive samples; a double melting peak was obtained with both genotype mixtures tested (Figure 3). The 6 °C difference in T_ms allows an easier differentiation of two genotype profiles; no cross-reaction in T_ms peaks was detected with strains 652 and JP2, indicating the good selectivity of the method. Only one size band for each genotype was verified and confirmed in agarose gel electrophoresis, respectively 696 bp for 652 and 192 bp for the JP2 strain (Figure 2); this suggests a total absence of non-specific products. Real-time PCR, as evaluated here, has been shown to have a detection limit of 40 Aa 652 or JP2 cells/PCR (100 cells/ml). The wide linear range (10⁷-10²cells obtained for two genotypes) as illustrated in Figure .4, indicates the efficiency of this real time PCR. Standard curves showed a similar slope and a good correlation regression coefficient R2 of 0.98–0.97 40, (Figure 4). In these experiments negative control contained DNA saliva extracts recovered from a non-diseased patient with PCR/negative culture for Aa, the total DNA amount of these samples was about 50 μg/μl. C lanes/curves do not contain aspecific products from previous interaction with human or non-specific bacterial genomic DNA as shown in Figures 2, 3. The DNA sequencing results of the PCR amplicons were a perfect match, with Blast program analysis 51, for the 652 genotype (Gen Bank accession n. S68133) or the JP2 genotype (Gen Bank accession n. X16829).

We analyzed plaque and saliva samples collected from 81 patients following the protocols just described. In comparison with the cultural procedure, real time resulted as being a little more sensitive (1 saliva and 2 paper point samples were positive only with molecular methods), while real time and conventional PCR were in accordance. All negative samples with the real time PCR procedure also resulted negative with cultural and conventional PCR methods too. Only one subject in the non-diseased group (26 in total) resulted 652 Aa genotype-positive for the periodontal pocket specimen, with 10³ cells/ml bacteria in the paper point suspension and negative in the saliva sample. Table 2 shows that 9 out of 55 diseased patients (16.3%) were infected with Aa and the disease severity was associated with a high bacteria title and/or leukotoxic genotype presence. In accordance with scientific data, we only identified the leukotoxic clone (JP2) in patients with severe forms of periodontitis 41. However, whenever a similar degree of clinical infection was present, and only the leukotoxic 652 genotype was identified, high bacteria titles were present (about ≥ 10⁴ cells/ml in the paper point suspension). Two patients, with aggressive periodontitis, showed bacteria in saliva samples too; one was found positive for the 652 strain and the other for JP2, the same genotypes were detected respectively in periodontal pockets.

Five periodontopathic bacteria: A. actinomycemcomitans P. gingivalis, P. intermedia, T. forsytensis and T. denticola were detected by a conventional PCR method from the subgingival plaque of all 81 patients. No periodontopathogenic bacterial DNA was observed in 39 of the samples (51%). The percentage of PCR positive samples was: Aa 12.2%, Pg 14.8%, Pi 22%, Tf (the most representative bacteria in these samples) 40.8% and Td 8.7 %. Six specimens resulting positive for Aa, (C2, C5, C6, C7, C75, C80) were associated with at least another periodontal bacteria. The principal pathogen was T. forsythensis present in 50% of the Aa positive samples. Sample C80, isolated in a non-diseased patient (2 mm subgingival pocket), contained all 4 tested pathogens (Table 2). In the PCR results four samples showed Aa DNA only (samples C1, C4, C8, C25) and these results were in accordance with the PCR real time and cultural result s(Table 2).