Background
Methods
Clinical specimens
Positive control strains
a) Aa strains
b) Other periodontal bacteria used as positive control
DNA extraction
Primers design
Name | Target | Strain | Sequence 5'--------------3' | GenBank Accession No | PCR product (bp) |
---|---|---|---|---|---|
OG 43 |
16S rRNA
|
Actinobacillus actimycetemcomitans
| CCAAGTGTGATTAGGTAGTT | M75036 | 226 |
OG 44 |
16S rRNA
|
Actinobacillus actimycetemcomitans
| ACCAACCTTCCTCAATAC | M75036 | |
OG53 |
16S rRNA
|
Prevotella intermedia
| CGTATCCAACCTTCCCTCC | X73965 | 389 |
OG54 |
16S rRNA
|
Prevotella intermedia
| ATTAGCCGGTCCTTATTCGAAG | X73965 | |
OG94 |
prtC
|
Porphyromonas gingivalis
| GAATCAAATACTTCAGCCGTCT | AB006973 | 143 |
OG95 |
prtC
|
Porpyromonas gingivalis
| TTGCAGTTCGTATCGGATCT | AB006973 | |
OG45 |
16S rRNA
|
Tannerella forsythensis
| GTCGGACTAATACCTCATAAAACA | L16495 | 222 |
OG46 |
16S rRNA
|
Tannerella forsythensis
| TCGCCCATTGACCAATATT | L16495 | |
OG348 |
16S rRNA
|
Treponema denticola
| AGAGAAAGGGTAATTTGAAG | AF179264 | 399 |
OG349 |
16S rRNA
|
Treponema denticola
| TATTATTGTCCCTTCTTTCTT | AF179264 |
Real time PCR
Differentiation between JP2 and 652 genotypes
Expression of the Aa concentration in dental plaque and saliva
Cultural method
Conventional PCR
Results
Reconstruction experiments: specificity and sensitivity of the method
Results with clinical samples
Patient | Disease type | Subgingival plaque | Saliva | |||
---|---|---|---|---|---|---|
Aa Real time PCR [cells/ml paper point] | Culture for Aa
| Conventional PCR (for 4 periodontal pathogens) | Real time PCR Aa cells/ml | Aa Culture CFU/ml vs Conventional PCR (Aa 16S rRNA only) | ||
C1 | aggressive periodontitis | 1,9*104 (652) | posit |
Aa
| 3,0*102 (652) | 1,5*101/pos |
C2 | aggressive periodontitis | 4,0*103 (JP2) | posit |
Aa, Pg
| 1,3*102 (JP2) | neg/pos |
C4 | gingivitis | 3,6*103 (652) | posit |
Aa, | Nd | neg/neg |
C5 | chronic periodontitis | 6,4*103 (JP2) | posit |
Aa,Tf
| Nd | neg/neg |
C6 | chronic periodontitis | 1,9*103 (JP2) | neg |
Aa,Tf
| Nd | neg/neg |
C7 | aggressive periodontitis | 2,8*104 (JP2) | posit |
Aa,Pi,Tf
| Nd | neg/neg |
C8 | aggressive periodontitis | 4,4*104 (JP2) | posit |
Aa, | Nd | neg/neg |
C25 | aggressive periodontitis | 2,0*103 (JP2) | posit |
Aa, | Nd | neg/neg |
C75 | aggressive periodontitis | 3,0 *104 (652) | posit |
Aa, Pg, Pi,Tf
| Nd | neg/neg |
C80 | no disease | 1,0*103 (652) | neg |
Aa, Pg, Pi,Tf
| Nd | neg/neg |