SUPPLEMENTARY
Isolation of DNA
Isolation of DNA involved samples first being lyophilized. This was followed by a
sequence of enzymatic lysis and addition of Proteinase-K (Qiagen, Manchester, UK).
Following this, total DNA was isolated from the plaque sample using a DNeasy
Blood and Tissue kit (Qiagen, Manchester, UK) according to manufacturer’s
instructions. The quality and quantity of DNA was determined using a Nanodrop
Spectrophotometer (Thermo Fischer Scientific, Rockford, IL, USA). DNA samples
were stored at -80°C until required for further analysis.
qPCR
Amplification primers were designed using Primer-Express software (Applied
Biosystems, Foster City, CA, USA). Sequences derived from the 16S rRNA genes
(Ashimoto et al. 1996), of the organisms of interest were aligned and inspected for
regions of conserved and variable sequences (GenBank,
http://www.ncbi.nlm.nih.gov/GenBank/index). Regions specific for target bacteria
were selected and evaluated for Tm and several other characteristics using the
PrimerExpress software. Sequences with appropriate specificity were designated as
probes and appropriate amplification primers were designed, again using the PrimerExpress software (Table S1). Quantified bacterial DNA from the four representative
strains were used to prepare serial tenfold dilutions from 100 to 105 cells as the
positive control standards to be used.
A master mix of all the components for the PCR amplification reaction except the
target DNA was prepared (PCR SYBR Master Mix, Qiagen Inc.). The components
were combined and 19 μl of Master Mix dispensed into the wells of a 96 well assay
plate (MicroAmp™ Optical 96 Well Reaction Plate, PE/ABI). Triplicate 1ul samples
of target DNA from plaque samples or dilutions of bacterial genomic DNA standards
were added to the wells and cycled in an ABI 7700 Sequence Detector (Applied
Biosystems) 40 times (95°C 30 seconds, 60°C 60seconds), after an initial
denaturation at 95°C for 10 minutes. A disassociation curve was included at the end
of the cycles to ensure one specific PCR product was formed in each reaction.
Bacterial quantification
Based on the positive control value, standard curves for quantification were thus
established. Numbers of bacteria in each sample were determined by comparing the
amplification curve of each sample to those of the standards, and results were
normalized to the concentration of DNA used. The sensitivity of each assay was
determined to be the lowest dilution of DNA that produced statistically significant
(p<0.001) average signal higher than the no template control using a t-test; this meant
the threshold for positivity of a pathogen being detected was the lowest standard used
(i.e. >100 cells). Subjects were thus classified as having the pathogen present or not;
this was in fact an aggregate of all their plaque samples, meaning that if least one
sample per subject was positive the subject was reported as positive for that organism.
Reference
Ashimoto, A., Chen, C., Bakker, I. & Slots, J. (1996) Polymerase chain reaction
detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis
and advanced periodontitis lesions. Oral microbiology and immunology 11, 266273.
Table S1. qPCR primer information
Primer
Direction
Sequence (5’ → 3’)
Tm (°C)
Porphyromonas gingivalis
Forward
CATAGATATCACGAGGAACTCCGATT
63.02
Reverse
AAACTGTTAGCAACTACCGATG
58.94
Forward
GGCACGTAGGCGGACCTT
64.46
Reverse
ACCAGGGCTAAAGCCCAATC
62.45
Forward
CTTCCGCAATGGACGAAAGT
60.4
Reverse
CAACCTTTCGGCCTTCTTCA
60.4
Forward
GGGTGAGTAACGCGTATGTAAC
62.67
Reverse
ACCCATCCGCAACCAATAAA
58.35
Aggregatibacter
actinomycetemcomitans
Treponema denticola
Tannerella forsythia
Table S2. Baseline characteristics for the participants.
Study cohort
(n=518)
Age (years), mean (SD)
63.6 (3.0)
Number of teeth, mean (SD)
19.6 (5.8)
Periodontal Disease, n (%)
None / Mild
328 (63.3%)
Moderate
98 (18.9%)
Severe
92 (17.8%)
BMI (kg/m2), mean (SD)
27.3 (3.5)
Cholesterol (mmol/L), mean (SD)
5.6 (0.9)
Diabetes reported, n (%)
22 (4.2%)
Hypertension reported, n (%)
146 (28.2%)
Current smoker, n (%)
81 (15.6%)
Men living alone, n (%)
58 (11.2%)
Material conditions, n (%)
Low
149 (28.8%)
Medium
130 (25.1%)
High
238 (45.9%)