International Dental Journal
ORIGINAL ARTICLE
doi: 10.1111/idj.12109
Evaluation of periodontal pathogens of the mandibular third molar pericoronitis by using real time PCR Metin Sencimen1, Isil Saygun2, Aydin Gulses3, Vehbi Bal1, Cengiz H. Acikel4 and Ayhan Kubar5 1
Department of Oral and Maxillofacial Surgery, G€ ulhane Military Medical Academy, Etlik, Ankara, Turkey; 2Department of Periodontology, G€ ulhane Military Medical Academy, Etlik, Ankara, Turkey; 3Centre for Oral & Dental Health, Ankara Mevki Military Hospital, Ankara, ulhane Military Medical Academy, Etlik, Ankara, Turkey; 5Department of Microbiology, G€ ulhane Turkey; 4Department of Epidemiology, G€ Military Medical Academy, Etlik, Ankara, Turkey.
Objectives: The aim of this study was to investigate the mandibular third molar pericoronitis flora by using real-time polymerase chain reaction (PCR). Materials and methods: The quantitative values of Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia (Tf) were evaluated in comparison with the healthy third molar flora by using real time PCR. Results: Aa, Cr, Pg, and Pi were not statistically significant but numerically higher than the pericoronitis group. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study revealed the strong relation between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times relative risk of pericoronitis as the individuals with an absence of Tf in their samples. Conclusion: Tf plays an important role in the development of clinical symptoms related to pericoronitis. Key words: Pericoronitis, real-time polymerase chain reaction, periodontal bacteria
INTRODUCTION Pericoronitis is a term used to describe the inflammation around the crown of a tooth, usually of an incompletely erupted mandibular third molar and refers to a polymicrobial mixed infectious periodontal pathology1,2. The microbiota that develops in the distally located pseudo-pocket is the major cause. The identification of the microbiota of the periodontal pocket-like crevice plays a key role in the medical treatment regimen3. However, clinical and population data focusing on the periodontal pathology affecting third molars remain limited4. It has been suggested that the microbiota predominantly responsible for pericoronitis is mainly anaerobically and contains Campylobacter gracilis, Capnocytophaga species, Fusobacterium species, Micromonas (Peptostreptococcus) micros, Prevotella intermedia, Prevotella nigrescens, Selenomonas noxia The current research has been conducted in full accordance with the World Medical Association Declaration of Helsinki. © 2014 FDI World Dental Federation
and Veillonella species4–9. Streptococci, staphylococci, actinomycetes, and enterobacteria as well as protozoa and fungi have also been described3. Treatment of the condition is limited to a local surgical procedure or involves empirical antibiotic treatment often of the b-lactam family5. Leung et al.10 showed that the multimicrobial and essentially anaerobic bacteria of pericoronitis resemble the species causing gingivitis or periodontitis. Detection of these putative pathogens in periodontal health and disease strongly depends on the techniques used, and the complexity of the microbiota, problems of sampling, transport and cultivation have to be taken into consideration for diagnostic procedures11. Cultivation and differentiation methods include dark-field microscopy, enzymatic and immunological assays, nucleic acid probes and polymerase chain reaction (PCR)12. Microbial diversity can be greatly underestimated by cultivation studies because many microorganisms cannot be cultivated by standard techniques and bacteria responsible for pericoronitis are mainly Gram-negative, requiring anaerobic growth conditions11. 1
Sencimen et al. The best model available at present for determining microbial diversity, without cultivation, is based on isolation of DNA from the target environment. Quantitative PCR has been reported to be highly specific and sensitive for detecting Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) in periodontitis samples in comparison to healthy subgingival samples13. Therefore, PCR has become an important tool for the rapid, sensitive, and specific detection of bacterial pathogens in third molar pericoronitis14. To the best of our knowledge, quantitative detection of periodontal bacteria in the mandibular third molar pericoronitis has not yet been investigated by real time PCR. The aim of this study was to investigate the mandibular third molar pericoronitis microbiota for the quantification of Aa, Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Pg, Prevotella intermedia (Pi) and Tannerella forsythia (Tf) in comparison with the healthy third molar microbiota in patients with no periodontal disease by using real-time PCR. MATERIALS AND METHODS
erupted. Seventy-eight per cent of the teeth were at most 50% visible (occlusal surface)and only 12% of the teeth were 70% or more visible (occlusal surface). There were no statistically significant differences between groups for the variables mentioned above. Clinical periodontal examination included the plaque index (PI),15 gingival index (GI),16 probing pocket depth (PD) and the percentage of sites that bled on probing (BOP). All measurements were taken using a Williams probe (23 W; Hu-Friedy, Chicago, IL, USA) with 0.5 mm units. Full-mouth periodontal examination at four sites per tooth, including third molars were conducted to detected periodontal condition, throughout the study, all measurements were made under standard conditions by a single clinician. Sampling procedure Before sampling, the sample site was gently cleaned of supragingival plaque and saliva with sterile cotton pellets, isolated with cotton rolls and air dried. A sterile periodontal curette was gently inserted to the bottom of the pocket and subgingival sample was removed by a simple stroke. The subgingival plaque samples were suspended in 500 lL of 10 mM Tris HCl containing 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8) (TE buffer) and homogenised by vigorous mixing on a vortex.
Sixteen adults aged 18–34 years (nine men and seven women, mean age 27.62 years) without systemic disease, suffering from mandibular third molar pericoronitis with pain and without antibiotic treatment in the previous 6 months, were included in the study group. The clinical examination involved an investigation for regional and local manifestations: lymphadenopathy, oedema, trismus, presence of pus and pain, and bad taste in oral cavities. Radiographs were taken to assess the bony defects at the distal of third molars which confirms the presence of chronic pericoronitis. The control group consisted of 14 adults aged 19–34 years (seven men and seven women, mean age 30.07 years) without present and previous clinical signs and symptoms of mandibular third molar pericoronitis. Both pericoronitis and control groups consisted of subjects without periodontitis. The study was approved by the Institutional Internal Review and Ethics Board at the Gulhane Military Medical Academy, Sciences of Dentistry. The current research was conducted in full accordance with the World Medical Association Declaration of Helsinki. Verbal informed consent was obtained from all participants involved in the study.
Nucleic acid extraction DNA was extracted from the subgingival plaque sample using an alkali phenol– chloroform isoamyl alcohol procedure17. Briefly 100 lL of specimen was placed in 10 lL of protease solution (65 mg/mL) (Sigma-Aldrich Corp., St Louis, MO, USA) and 250 lL of potassium buffer for 60 minute at 42 °C. Following centrifugation at 10,000 g for 10 minute at 12 °C, DNA was extracted from the supernatant using a mixture of 250 lL of alkali phenol and 250 lL of chloroform-isoamyl alcohol (25:24:1) and then precipitated using 500 lL of isopropyl alcohol. DNA was washed in 75% ethyl alcohol at 10,000 g for 5 minutes at 4 °C, air dried at 37 °C and dissolved in 100 lL of distilled water.
Clinical examination
Real-time Taqman assay
The clinical examination was made after history-taking. The teeth were similarly erupted in comparison with the adjacent second molar. The level of eruption was 1.0 1.2 mm (mean and SD) under the occlusal surface of the adjacent second molar. In addition, the crowns of the lower third molars were similarly partly
The Taqman fluorogenic real-time PCR detection system was used to determine infectious agent counts18. The Taqman system uses species-specific primers and probes that are dually labelled with a fluorescent reporter and a quencher dye. The presence of Aa, Cr, Fn, Pg, Pi, and Tf in the strains isolated on media was
2
PCR analysis Nucleic acid extraction
© 2014 FDI World Dental Federation
Periodontal pathogens of pericoronitis evaluated. (Table 1) lists the nucleotide sequence of the PCR primers and probes, which were designed using the OLIGOWARE 1.0 software program (Integrated DNA Technologies, Coralville, IA, USA)19 (Table 1). Statistical analysis Statistical evaluation was performed using the Statistical Packages for the Social Sciences (SPSS, Chicago, IL, USA). Descriptive statistics were presented as median (min – max), mean standard deviation, frequency and percentages. Counts of the bacteria were normalised by conversion to logarithmic values. The values for each bacteria species were compared between the pericoronitis group and the control group. The comparisons of the categorical variables were analysed by the v2-test. We used Mann–Whitney U-test for comparison of continues variables which analysed the differences in the median of the groups. The analysis of the linear association between clinical parameters and periodontal pathogen values were made by using Spearman rho correlation test. Single variable logistic regression analysis was used for odds ratiobased risk calculation. Probability (P) values equal to or less than 0.05 were considered statistically significant.
odontal parameters (PI, GI, BOP and PD) of the third molar regions are presented in (Table 2). The clinical examination parameters (GI, BOP and PD) in the third molar region were statistically significantly different for the subjects with pericoronitis compared with subjects enrolled with no pericoronitis. (PI P = 0.068, GI P < 0.001, BOP P < 0.001 and PD P = 0.01). Analysis of microbial data Samples obtained from 16 subjects with pericoronitis and 14 control subjects were evaluated by real-time PCR assays and the levels of periodontopathogens were determined (Table 3 and Figure 1). It was found that Aa, Cr, Pg, and Pi were numerically higher than in the pericoronitis group but this was not statistically significant. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study noted the strong association between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times greater relative risk of pericoronitis as the individuals with an absence of Tf in their samples (OR 7.500, 95% CI 1.484–37.905, P = 0.011).
RESULTS Analysis of the periodontal clinical parameters
Analysis of relations between the periodontal pathogens and clinical parameters
Males and females were almost equally presented. Frequencies of subjects according to the clinical peri-
The analysis of non-parametric correlations between the periodontal pathogens and clinical parameters
Table 1 Polymerase chain reaction primers and Taqman probes for detecting bacteria Infectious agents (Genbank accession number) Porphyromonas gingivalis (AB 261608.1) Forward 50 -TGGGACTTGCTGCTCTTGCTATG-30 Reverse 50 -GATGGCTTCCTGCTGTTCTCCA-30 Probe FAM-50 -CAAAGACAACGAGGCAGAACCCGTTA-TAMRA-30 Tannerella forsythia Forward 50 GCGTATGTAACCTGCCCGCA-30 Reverse 50 CCGTTACCTCACCAACTACCTAATG-30 Probe FAM-50 -AGGGATAACCCGGCGAAAGTCGGA-TAMRA-30 Prevotella intermedia (AY689226.1) Forward 50 -AGACGGCCTAATACCCGATGTTG-30 Reverse 50 -TTACCCGCACCAACAAGCTAATCAG-30 Probe JOE-50 -TGGCATCTGACGTGGACCAAAGATTC-TAMRA-30 Aggregatibacter actinomycetemcomitans (AF359451.1) Forward 50 -CGGTTACCGTTATGACCGTGTGA-30 Reverse 50 -GCCCGGAATGCTTTGCTATATTTC-30 Probe FAM-50 -AGGCAAGACGGGAAGCTAACGCAAA-TAMRA-30 Fusobacterium nucleatum (EF089177.1) Forward 50 -GCGGAACTACAAGTGTAGAGGTG-30 Reverse 50 -GTTCGACCCCCAACACCTAGTA-30 Probe JOE-50 -AATGCCGATGGGGAAGCCAGCTTA-TAMRA-30 Campylobacter rectus (AF035193.1) Forward 50 -CACCCGATAACCCTACTCCTCCTA-30 Reverse 50 -GATCCGTTCCATCAGTACCCACTA-30 Probe FAM-50 -CCGGTACCGAATCCTGAGGAACCA-TAMRA-30
Amplicon size in bp (reference) 194 (this study)
149 (this study)
105 (this study)
288 (this study)
175 (this study)
132 (this study)
FAM, 6-carboxyfluorescein labelled reporter dye; JOE, 6-carboxy-40 ,50 -dichloro20 , 70 -dimethoxyfluorescein labelled reporter dye; TAMRA, 6-carboxytetramethyl-rhodamine labelled quencher dye. © 2014 FDI World Dental Federation
3
Sencimen et al. Table 2 Comparisions of subjects according to the clinical periodontal parameters (PI, GI, BOP and PD) of the third molar regions Group Control (n = 14) Median (min–max) Pericoronitis (n = 16) Median (min–max) P-value
Age
PD
GI
BOP (%)
31.50 22.00–37.00
2 1.00–4.00
1.125 1.00–2.00
25 0–100
25.50 18–38 0.33
3.33 1.75–5.00 0.01
2 1.25–2.00
ORIGINAL ARTICLE
doi: 10.1111/idj.12109
Evaluation of periodontal pathogens of the mandibular third molar pericoronitis by using real time PCR Metin Sencimen1, Isil Saygun2, Aydin Gulses3, Vehbi Bal1, Cengiz H. Acikel4 and Ayhan Kubar5 1
Department of Oral and Maxillofacial Surgery, G€ ulhane Military Medical Academy, Etlik, Ankara, Turkey; 2Department of Periodontology, G€ ulhane Military Medical Academy, Etlik, Ankara, Turkey; 3Centre for Oral & Dental Health, Ankara Mevki Military Hospital, Ankara, ulhane Military Medical Academy, Etlik, Ankara, Turkey; 5Department of Microbiology, G€ ulhane Turkey; 4Department of Epidemiology, G€ Military Medical Academy, Etlik, Ankara, Turkey.
Objectives: The aim of this study was to investigate the mandibular third molar pericoronitis flora by using real-time polymerase chain reaction (PCR). Materials and methods: The quantitative values of Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia (Tf) were evaluated in comparison with the healthy third molar flora by using real time PCR. Results: Aa, Cr, Pg, and Pi were not statistically significant but numerically higher than the pericoronitis group. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study revealed the strong relation between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times relative risk of pericoronitis as the individuals with an absence of Tf in their samples. Conclusion: Tf plays an important role in the development of clinical symptoms related to pericoronitis. Key words: Pericoronitis, real-time polymerase chain reaction, periodontal bacteria
INTRODUCTION Pericoronitis is a term used to describe the inflammation around the crown of a tooth, usually of an incompletely erupted mandibular third molar and refers to a polymicrobial mixed infectious periodontal pathology1,2. The microbiota that develops in the distally located pseudo-pocket is the major cause. The identification of the microbiota of the periodontal pocket-like crevice plays a key role in the medical treatment regimen3. However, clinical and population data focusing on the periodontal pathology affecting third molars remain limited4. It has been suggested that the microbiota predominantly responsible for pericoronitis is mainly anaerobically and contains Campylobacter gracilis, Capnocytophaga species, Fusobacterium species, Micromonas (Peptostreptococcus) micros, Prevotella intermedia, Prevotella nigrescens, Selenomonas noxia The current research has been conducted in full accordance with the World Medical Association Declaration of Helsinki. © 2014 FDI World Dental Federation
and Veillonella species4–9. Streptococci, staphylococci, actinomycetes, and enterobacteria as well as protozoa and fungi have also been described3. Treatment of the condition is limited to a local surgical procedure or involves empirical antibiotic treatment often of the b-lactam family5. Leung et al.10 showed that the multimicrobial and essentially anaerobic bacteria of pericoronitis resemble the species causing gingivitis or periodontitis. Detection of these putative pathogens in periodontal health and disease strongly depends on the techniques used, and the complexity of the microbiota, problems of sampling, transport and cultivation have to be taken into consideration for diagnostic procedures11. Cultivation and differentiation methods include dark-field microscopy, enzymatic and immunological assays, nucleic acid probes and polymerase chain reaction (PCR)12. Microbial diversity can be greatly underestimated by cultivation studies because many microorganisms cannot be cultivated by standard techniques and bacteria responsible for pericoronitis are mainly Gram-negative, requiring anaerobic growth conditions11. 1
Sencimen et al. The best model available at present for determining microbial diversity, without cultivation, is based on isolation of DNA from the target environment. Quantitative PCR has been reported to be highly specific and sensitive for detecting Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) in periodontitis samples in comparison to healthy subgingival samples13. Therefore, PCR has become an important tool for the rapid, sensitive, and specific detection of bacterial pathogens in third molar pericoronitis14. To the best of our knowledge, quantitative detection of periodontal bacteria in the mandibular third molar pericoronitis has not yet been investigated by real time PCR. The aim of this study was to investigate the mandibular third molar pericoronitis microbiota for the quantification of Aa, Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), Pg, Prevotella intermedia (Pi) and Tannerella forsythia (Tf) in comparison with the healthy third molar microbiota in patients with no periodontal disease by using real-time PCR. MATERIALS AND METHODS
erupted. Seventy-eight per cent of the teeth were at most 50% visible (occlusal surface)and only 12% of the teeth were 70% or more visible (occlusal surface). There were no statistically significant differences between groups for the variables mentioned above. Clinical periodontal examination included the plaque index (PI),15 gingival index (GI),16 probing pocket depth (PD) and the percentage of sites that bled on probing (BOP). All measurements were taken using a Williams probe (23 W; Hu-Friedy, Chicago, IL, USA) with 0.5 mm units. Full-mouth periodontal examination at four sites per tooth, including third molars were conducted to detected periodontal condition, throughout the study, all measurements were made under standard conditions by a single clinician. Sampling procedure Before sampling, the sample site was gently cleaned of supragingival plaque and saliva with sterile cotton pellets, isolated with cotton rolls and air dried. A sterile periodontal curette was gently inserted to the bottom of the pocket and subgingival sample was removed by a simple stroke. The subgingival plaque samples were suspended in 500 lL of 10 mM Tris HCl containing 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8) (TE buffer) and homogenised by vigorous mixing on a vortex.
Sixteen adults aged 18–34 years (nine men and seven women, mean age 27.62 years) without systemic disease, suffering from mandibular third molar pericoronitis with pain and without antibiotic treatment in the previous 6 months, were included in the study group. The clinical examination involved an investigation for regional and local manifestations: lymphadenopathy, oedema, trismus, presence of pus and pain, and bad taste in oral cavities. Radiographs were taken to assess the bony defects at the distal of third molars which confirms the presence of chronic pericoronitis. The control group consisted of 14 adults aged 19–34 years (seven men and seven women, mean age 30.07 years) without present and previous clinical signs and symptoms of mandibular third molar pericoronitis. Both pericoronitis and control groups consisted of subjects without periodontitis. The study was approved by the Institutional Internal Review and Ethics Board at the Gulhane Military Medical Academy, Sciences of Dentistry. The current research was conducted in full accordance with the World Medical Association Declaration of Helsinki. Verbal informed consent was obtained from all participants involved in the study.
Nucleic acid extraction DNA was extracted from the subgingival plaque sample using an alkali phenol– chloroform isoamyl alcohol procedure17. Briefly 100 lL of specimen was placed in 10 lL of protease solution (65 mg/mL) (Sigma-Aldrich Corp., St Louis, MO, USA) and 250 lL of potassium buffer for 60 minute at 42 °C. Following centrifugation at 10,000 g for 10 minute at 12 °C, DNA was extracted from the supernatant using a mixture of 250 lL of alkali phenol and 250 lL of chloroform-isoamyl alcohol (25:24:1) and then precipitated using 500 lL of isopropyl alcohol. DNA was washed in 75% ethyl alcohol at 10,000 g for 5 minutes at 4 °C, air dried at 37 °C and dissolved in 100 lL of distilled water.
Clinical examination
Real-time Taqman assay
The clinical examination was made after history-taking. The teeth were similarly erupted in comparison with the adjacent second molar. The level of eruption was 1.0 1.2 mm (mean and SD) under the occlusal surface of the adjacent second molar. In addition, the crowns of the lower third molars were similarly partly
The Taqman fluorogenic real-time PCR detection system was used to determine infectious agent counts18. The Taqman system uses species-specific primers and probes that are dually labelled with a fluorescent reporter and a quencher dye. The presence of Aa, Cr, Fn, Pg, Pi, and Tf in the strains isolated on media was
2
PCR analysis Nucleic acid extraction
© 2014 FDI World Dental Federation
Periodontal pathogens of pericoronitis evaluated. (Table 1) lists the nucleotide sequence of the PCR primers and probes, which were designed using the OLIGOWARE 1.0 software program (Integrated DNA Technologies, Coralville, IA, USA)19 (Table 1). Statistical analysis Statistical evaluation was performed using the Statistical Packages for the Social Sciences (SPSS, Chicago, IL, USA). Descriptive statistics were presented as median (min – max), mean standard deviation, frequency and percentages. Counts of the bacteria were normalised by conversion to logarithmic values. The values for each bacteria species were compared between the pericoronitis group and the control group. The comparisons of the categorical variables were analysed by the v2-test. We used Mann–Whitney U-test for comparison of continues variables which analysed the differences in the median of the groups. The analysis of the linear association between clinical parameters and periodontal pathogen values were made by using Spearman rho correlation test. Single variable logistic regression analysis was used for odds ratiobased risk calculation. Probability (P) values equal to or less than 0.05 were considered statistically significant.
odontal parameters (PI, GI, BOP and PD) of the third molar regions are presented in (Table 2). The clinical examination parameters (GI, BOP and PD) in the third molar region were statistically significantly different for the subjects with pericoronitis compared with subjects enrolled with no pericoronitis. (PI P = 0.068, GI P < 0.001, BOP P < 0.001 and PD P = 0.01). Analysis of microbial data Samples obtained from 16 subjects with pericoronitis and 14 control subjects were evaluated by real-time PCR assays and the levels of periodontopathogens were determined (Table 3 and Figure 1). It was found that Aa, Cr, Pg, and Pi were numerically higher than in the pericoronitis group but this was not statistically significant. In contrast to samples from control subjects, statistically significant higher numbers of Tf were detected in samples from pericoronitis patients. The study noted the strong association between risk of pericoronitis and the presence of Tf. Individuals who have Tf in their samples present with an almost eight times greater relative risk of pericoronitis as the individuals with an absence of Tf in their samples (OR 7.500, 95% CI 1.484–37.905, P = 0.011).
RESULTS Analysis of the periodontal clinical parameters
Analysis of relations between the periodontal pathogens and clinical parameters
Males and females were almost equally presented. Frequencies of subjects according to the clinical peri-
The analysis of non-parametric correlations between the periodontal pathogens and clinical parameters
Table 1 Polymerase chain reaction primers and Taqman probes for detecting bacteria Infectious agents (Genbank accession number) Porphyromonas gingivalis (AB 261608.1) Forward 50 -TGGGACTTGCTGCTCTTGCTATG-30 Reverse 50 -GATGGCTTCCTGCTGTTCTCCA-30 Probe FAM-50 -CAAAGACAACGAGGCAGAACCCGTTA-TAMRA-30 Tannerella forsythia Forward 50 GCGTATGTAACCTGCCCGCA-30 Reverse 50 CCGTTACCTCACCAACTACCTAATG-30 Probe FAM-50 -AGGGATAACCCGGCGAAAGTCGGA-TAMRA-30 Prevotella intermedia (AY689226.1) Forward 50 -AGACGGCCTAATACCCGATGTTG-30 Reverse 50 -TTACCCGCACCAACAAGCTAATCAG-30 Probe JOE-50 -TGGCATCTGACGTGGACCAAAGATTC-TAMRA-30 Aggregatibacter actinomycetemcomitans (AF359451.1) Forward 50 -CGGTTACCGTTATGACCGTGTGA-30 Reverse 50 -GCCCGGAATGCTTTGCTATATTTC-30 Probe FAM-50 -AGGCAAGACGGGAAGCTAACGCAAA-TAMRA-30 Fusobacterium nucleatum (EF089177.1) Forward 50 -GCGGAACTACAAGTGTAGAGGTG-30 Reverse 50 -GTTCGACCCCCAACACCTAGTA-30 Probe JOE-50 -AATGCCGATGGGGAAGCCAGCTTA-TAMRA-30 Campylobacter rectus (AF035193.1) Forward 50 -CACCCGATAACCCTACTCCTCCTA-30 Reverse 50 -GATCCGTTCCATCAGTACCCACTA-30 Probe FAM-50 -CCGGTACCGAATCCTGAGGAACCA-TAMRA-30
Amplicon size in bp (reference) 194 (this study)
149 (this study)
105 (this study)
288 (this study)
175 (this study)
132 (this study)
FAM, 6-carboxyfluorescein labelled reporter dye; JOE, 6-carboxy-40 ,50 -dichloro20 , 70 -dimethoxyfluorescein labelled reporter dye; TAMRA, 6-carboxytetramethyl-rhodamine labelled quencher dye. © 2014 FDI World Dental Federation
3
Sencimen et al. Table 2 Comparisions of subjects according to the clinical periodontal parameters (PI, GI, BOP and PD) of the third molar regions Group Control (n = 14) Median (min–max) Pericoronitis (n = 16) Median (min–max) P-value
Age
PD
GI
BOP (%)
31.50 22.00–37.00
2 1.00–4.00
1.125 1.00–2.00
25 0–100
25.50 18–38 0.33
3.33 1.75–5.00 0.01
2 1.25–2.00
