SUPPLEMENT ARTICLE

DNA Probes and Primers in Dental Practice

Georg Conrads
R. M. Alden Research Laboratory and University of California—Los Angeles Medical Center, Santa Monica; University Hospital (Operative
Dentistry, Periodontology and Preventive Dentistry and the Institute of Medical Microbiology), Rhine-Westphalian Technical University, Aachen,
Germany

In clinical microbiology, molecular genetic techniques are increasingly being used to detect and/or differentiate
uncultivable, anaerobic, or fastidious microorganisms. During the past decade, DNA probe hybridization and
in vitro amplification by polymerase chain reaction have also been introduced to detect oral pathogens. The
present review describes experiences with methods and commercial test systems for the detection of pathogens
in periodontitis and caries.

Molecular microbiology has revolutionized modern
medicine. Especially in medical microbiology, a broad
spectrum of techniques are used to detect and/or dif-
ferentiate uncultivable, anaerobic, or fastidious micro-
organisms. The 2 basic methods are (1) hybridization
that uses either genomic or oligonucleotide probes and
(2) PCR, by which repeated cycles of oligonucleotide
(primer)–directed DNA synthesis of target “signature
sequences” are carried out in vitro.
Hybridization involves the denaturation of double-
stranded DNA into single strands and detection of
single-stranded DNA (ssDNA) with a labeled, com-
plementary ssDNA probe. For research, probe hybrid-
ization can be used to demonstrate genetic relatedness
in the DNA (e.g., ribosomal genes) of different organ-
isms to construct phylogenetic taxonomic schemes. In
the diagnostic laboratory, DNA probes are being used
for detection of uncultivable or fastidious organisms
directly in clinical specimen or for culture confirma-
tion. Commercially available probe tests include a num-
ber of bacterial pathogens, such as Campylobacter spe-
cies, Chlamydia trachomatis, Enterococcus species,
Gardnerella vaginalis, Haemophilus influenzae, Legi-
onella pneumophila, Listeria monocytogenes, Mycobac-
Reprints or correspondence: Dr. Georg Conrads, Institute of Medical
Microbiology, University Hospital, Pauwelsstraße 30, D-52057 Aachen, Germany
(gconradsphd@aol.com).
Clinical Infectious Diseases 2002; 35(Suppl 1):S72–7
 2002 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2002/3505S1-0014$15.00
terium species, Neisseria gonorrhoeae, Streptococcus aga-
lactiae, and Streptococcus pyogenes, and pathogenic
fungi, protozoa, and some viruses. Direct probe meth-
ods are much less sensitive (limit, 103–106 cells) when
used without DNA amplification procedures because
the number of target cells may fall below the sensitivity
of the assay.
However, by testing cell-rich oral specimens (e.g.,
samples from mixed anaerobic infected periodontal
pockets or plaque of initial carious lesions), this thresh-
old is met and probes can be used for most cases di-
rectly, without preamplification. Thus, additional tar-
gets for DNA probe detection can be found in the dental
practice. They are the periodontopathogens Actinoba-
cillus actinomycetemcomitans, Bacteroides forsythus, Por-
phyromonas gingivalis, Prevotella intermedia, Treponema
denticola, and caries agents—namely, Streptococcus mu-
tans–group species. In fact, because of the high prev-
alence of periodontal diseases and caries, oral infections
are a main indication for DNA probe application [1–5].
PCR is a highly sensitive technique by which minute
quantities of specific DNA (or RNA after reverse tran-
scription) can be enzymatically amplified. The tech-
nique can be used to detect very small amounts of
bacterial, fungal, or viral nucleic acid in clinical spec-
imens. PCR and other amplification techniques (es-
pecially nested and multiplex variants, transcription-
based amplification, strand displacement amplification,
and ligase-chain reaction) have a high variety of ap-
plications in the clinical laboratory. PCR is used for
direct detection or identification of microorganisms,

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 detection of genes coding for virulence factors, determining the
presence of antimicrobial resistance genes, and typing of bac-
terial isolates in epidemiological studies. The list of organisms
for which PCR has been applied includes numerous bacterial
genera, such as Bordetella, Brucella, Campylobacter, Chlamydia,
Corynebacterium, Haemophilus, Helicobacter, Legionella, Lep-
tospira, Mycobacterium, Staphylococcus, and Streptococcus, as
well as fungi (Aspergillus, Candida, and Pneumocystis), parasites
(Enterocytozoon bieneusi, Encephalitozoon hellem, Plasmodium
spp., Toxoplasma gondii, and Trypanosoma cruzi), and almost
every virus known to be pathogenic in humans.
But again, oral microbiology is a very important field for
PCR application. PCR-based tests are for research and are com-
mercially available to detect the periodontopathogens as well
as cariogenic agents [6–9]; PCR is referred to as the new di-
agnostic standard for these common infections. The following
paragraphs describe some test systems available in the United
States and/or in Europe, especially those in daily use.
APPLICATION OF DNA PROBES AND PRIMERS
IN PERIODONTAL DISEASES
Periodontal diseases (gingivitis and periodontitis) are chronic
mixed anaerobic infections with a remarkably high prevalence
and morbidity. Whereas gingivitis, with some exceptions, is a
reversible polymicrobial infection with no single associated bac-
terial agent, periodontitis is moderately to rapidly progressive
and is associated with facultative or obligate anaerobic path-
ogens. The species A. actinomycetemcomitans, B. forsythus,
Campylobacter rectus, Eikenella corrodens, Fusobacterium nu-
cleatum, P. gingivalis, P. intermedia, and several forms of un-
cultivable spirochetes play major roles in the pathogenesis
[10–12]. Moderate forms of periodontitis are demonstrated by
50%–98% of adults, with regional and age-based differences.
Between 3% and 13% of the population may develop aggressive
forms of periodontitis, such as rapidly progressive, juvenile, or
refractory periodontitis. These individuals require prosthetic
treatment within a short period of time. Periodontal pathogens
not only destroy the integrity of the dentition or of oral mucous
membranes but can frequently be detected in the blood cultures
of patients, causing not only bacteremia but also, under some
circumstances, septicemia, organ abscesses, endocarditis, or car-
diovascular disorders [13–17].
For diagnosis of the activity of the different forms of per-
iodontitis, clinical symptoms alone may not be sufficient, be-
cause they provide a historical record only (pocket formation,
attachment loss, and alveolar bone loss) or have low predictive
value (bleeding during probing). But predictions of recurrence
of disease and prognosis for the patient can be significantly
improved when the presence or absence of periodontal path-
ogens is monitored as well [12].
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To monitor bacterial changes or shifts in the gingival sulcus
or the periodontal pocket, several molecular genetic test systems
have been introduced within the past decade, and millions of
samples (e.g., subgingival plaque) have been analyzed with
DNA probes and primers on a routine basis. In the next section,
some experiences from both the dentists and the laboratories
involved in these test procedures will be summarized.
Two different molecular genetic strategies have been intro-
duced for the detection of periodontal pathogens in subgingival
plaque, based on (1) genomic or oligonucleotide DNA probes
or (2) PCR. Murray and French [18] were the first investigators
who could detect P. intermedia and P. gingivalis with the aid
of purified DNA fragments labeled with 32P or biotin–11-dUTP
by nick translation. The sensitivity of these genomic probes was
increased, requiring only 102–103 bacteria. However, the same
procedure was found to be very difficult for the major perio-
dontal pathogen A. actinomycetemcomitans (A.a); thus, the au-
thors had to clone specific fragments of the genome into plas-
mids, and these recombinant DNA probes were then used
further for diagnosis. With this procedure, cross-hybridization
between the A.a. probe and other species of the genus Pasteu-
rella were reduced to the minimum of 1%. Other pathogens
were added into this system, including T. denticola (genomic
probe) and F. nucleatum, E. corrodens, C. rectus, and B. forsythus
(recombinant DNA probes). A similar testing system was mar-
keted by the US company OmniGene Laboratory Services and
is frequently used in some European countries (under the name
DMDx/PATHOTEK). The samples are centrally analyzed at
ANAWA Laboratories. In the year 2000, ∼16,000 of these tests
were performed in Europe, with 14,000 tests sent from German
dental practices, 600 from Switzerland, and the remaining tests
from practices in the rest of Europe.
In contrast to genomic probes, oligonucleotides are syn-
thetically produced, short, stable molecules and can easily be
introduced in automated systems, the future of diagnostics.
Chuba et al. [19] were the first to establish oligonucleotide
probes directed against species-specific sequences of the 16S
rRNA to detect the periodontopathogens P. gingivalis, P. inter-
media, and A. actinomycetemcomitans. By way of background,
the 16S rRNA is a part of the small subunit of bacterial ri-
bosomes. This amazing molecule consists of both highly con-
served regions, appropriate as primer targets for amplification,
and species-specific sequences, which are of major interest to
molecular taxonomists and, so far, an ideal target for deducing
specific oligonucleotide-probes able to distinguish between very
closely related species, as, for example, P. intermedia and P.
nigrescens are (figure 1). When pure cultures are used, the spec-
ificity of oligonucleotides can be noted as 100%, but this might
be reduced in detecting bacteria in complex samples such as
subgingival plaque. The sensitivity of oligonucleotide probes
depends on the labeling system but can be maximized to detect

Figure 1. Representative dot-blot hybridization for the detection of
Prevotella intermedia and Prevotella nigrescens. Lane A, ATCC 25611 (105,
104, and 103 cells); lane B, ATCC 33563 (105, 104, and 103 cells); lane C,
L1440, LR22, L583, and LR15; lane D, LR78, LR53, and LK71; lane E, LR35,
L621, LR100, LR119, and LK65; and lane F, LK 80, L610, LR20, and ACJ5.
The cell concentrations in lanes C–F are between 103 (strain L583) and
106 (strain LR119). In blot A, the Pi-probe was used, and in blot B, the
Pn-probe was used. No cross-reactions appeared when 65C was used
as the hybridization temperature.
factor in reaching therapeutic decisions (e.g., the most appro-
priate antibiotic therapy), is still the same when obtained by
in vitro amplification before hybridization. However, a testing
system distributed by the company HAIN Diagnostic is fre-
quently used in Germany. As an advantage, the samples are not
analyzed only in a central laboratory—the entire test system is
distributed to local medical laboratories, making specimen
transport easier.
In the future, mini-molecular laboratories will be available
for chair-side DNA probe testing in ⭐1 h (Periodontal Mi-
crobial Identification Test; Saigene Corp.) [21]. However, the
advice of a microbiologist might still sometimes be necessary
to avoid problems that may occur in the diagnostic process.
Some of these problems will be reviewed in the next section,
below.

102–103 cells per analysis. In the important publication by Dix

et al. [20], highly species-specific and 16S rRNA–directed DNA
probes of 24 bp length and 32P labeled were used for A. acti-
nomycetemcomitans, B. forsythus, P. gingivalis, P. intermedia, E.
corrodens, F. nucleatum, and C. rectus. The authors found that
the sensitivity and specificity of the probes was not reduced
when subgingival plaque was used for the samples, but this
might be true only under ideal conditions, which could be
problematic for routine testing. Meanwhile, oligonucleotide
probe–based test systems for periodontal pathogens are avail-
able in different formats on the European market, including
the IAI Pado Test 4.5 system (Institute for Applied Immunol-
ogy), which is available in some European countries, Switzer-
land, Germany, and The Netherlands. Whereas the IAI-Test
system uses radioactively labeled DNA probes, the new LCL
system (LCL Biokey) uses chemiluminescence-generating oli-
gonucleotide probes. Both companies perform ∼10,000 tests
per year in Europe. In the case of the LCL system, it is known
that the demand for such tests is rapidly growing among Figure 2. PCR analysis of subgingival plaque samples (1P–4P), tonsil
practitioners. swabs (1T–4T), buccal membrane swabs (1B–4B), and tongue mucosa
To further enhance specificity, PCR and combinatory mo- swabs (1Z–4Z). We used the molecular size marker AmplySize DNA
standard (50–2000-bp ladder, Bio-Rad Laboratories). Lane 1, Positive con-
lecular genetic techniques were also introduced for the routine
trol with both 100 ng of Bacteroides forsythus and Prevotella intermedia
diagnosis of periodontal pathogens. The extended sensitivity DNA isolated by a simple boiling method. Lane 2, The negative control
can be used to demonstrate P. intermedia or B. forsythus in missing all template DNA. Lanes 3–6, Whereas patient 1 harbors both
plaque and in oral mucous membranes (figure 2) [6]. species in the subgingival plaque sample (1P), the 3 other samples dem-
After amplification of the 16S rRNA gene, specific DNA onstrate P. intermedia (660 bp amplificon) exclusively. Lanes 8–11, All
samples isolated from patient 2 are negative for the 2 periodontal path-
probes used in a reversed hybridization procedure are able to
ogenic bacteria. Lanes 14–17, Patient 3 harbors both species in obviously
quantify the bacteria in the amplicon. This 2-step testing system high numbers in the subgingival plaque (3P) and on the buccal membrane
may also be very sensitive, but it seems questionable whether (3B). Lanes 19–22, Patient 4 shows B. forsythus on the buccal membrane
the ratio of different bacteria, a result that is an important only (4B).

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 EXPERIENCES IN DENTAL PRACTICE
As was mentioned above, German dentists are confronted with
various genetic test systems for periodontal pathogens. With
this in mind, we perormed a survey among practitioners.
Among the ∼50,000 dentists practicing in Germany, 3%–6%
(only) specialize in the diagnosis and treatment of periodontitis
and were thus chosen to report their experiences. The 100 most
experienced practices (according to data from LCL Biokey)
were contacted for a statement, and 32 responses were received.
More details about this survey, especially representative answers
and categories, have been published elsewhere [22].
In general, these dentists seemed to be very satisfied with
the current test systems. However, the information I got from
numerous lengthy discussions with dentists in a more private
setting is somewhat different. The problems “under the surface”
are interesting and are as follows. (1) Periodontal diseases are
chronic infections and “healing” is almost impossible, even with
the most sophisticated diagnostic and treatment strategies. The
prolonged treatment is definitely very time consuming and
costly (health insurance covers only a portion of the expenses)
and can be frustrating for both dentist and patient. As a
consequence, some practitioners might give up on these
cases—unfortunately, sometimes too early. (2) Some practi-
tioners, highly encouraged, use different test formats over time
(sometimes for 1 patient) to select a favorite. To a certain extent,
they get different results from the different tests. Some of the
discrepancies are minor (e.g., different cell counts of an indi-
vidual species) but still confusing, and others are major (dif-
ferent species detected because of different formats, e.g., some
P. intermedia–directed tests do still include P. nigrescens, and
others do or do not include T. denticola. As a further problem
for comparison, the detection limits vary among the test sys-
tems, and some laboratories report real cell counts, whereas
others report percentages of the total flora). More validation
is needed for microbial test systems in periodontal diseases
before one can improve the acceptance of these useful tests.
EXPERIENCES FROM LABORATORIES
In our survey, we also asked the managers of laboratories about
their experiences with dentists (the 6 “major players” were
contacted, with 4 responding). Most of the laboratories are very
satisfied with the collaboration and are very optimistic about
the future of their testing systems for periodontal pathogens.
However, the present review article tries to concentrate on
problems in the communication that can occur between den-
tists and microbiologists, some of which are as follows.
Some dentists are overenthusiastic at the beginning and send
samples from almost every patient. Testing periodontal path-
ogens should, for several reasons, be concentrated on a small
spectrum of diseases and patients. Patients for whom microbial
testing is indicated include
1. patients with advanced attachment and bone loss be-
fore age 25 years (juvenile periodontitis or prepubertal peri-
odontitis);
2. patients, usually aged 25–35 years, with rapid destruc-
tion of attachment and bone in a relatively short period of time
(rapidly progressive periodontitis); and
3. patients who continue to lose attachment despite ade-
quate treatment (refractory periodontitis).
To receive useful information, an appropriate mechanical
treatment and an optimized oral hygiene regimen are essential
preconditions for subsequent testing. Reducing the biofilm is
necessary both for access to the important, base-located, sub-
gingival plaque for testing and for supporting the diffusion of
antimicrobial agents.
A fraction of the dentists seem to accept only “positive”
results from the tests to support their (predetermined) anti-
biotic therapy. But even in a patient with serious clinical symp-
toms, the periodontal disease has nonactive periods, when mi-
crobiological testing is “truly negative.” Therefore, diagnosis
needs both clinical and microbiological data [23–25].
A diagnostic result can only be as representative as the sample
that was analyzed. This is true for medical microbiology in
general but is especially true for periodontal diseases. According
to Mombelli et al. [26], the deepest periodontal pocket with
the highest tendency for bleeding must be chosen from each
quadrant for sampling. The dentist must get an overview of
the destructive processes in 1 session before sampling in a
second session, to avoid provocation of bleeding. It is extremely
important that sampling is not performed in a bleeding peri-
odontal pocket because the paper points, the sampling device
in most cases, can absorb only ∼20 mL (1 drop) of fluid, and
if this volume is mainly blood or saliva or supragingival plaque,
not much is left for the pathogen-containing deep fluid. Several
paper points should be used for sampling and either tested
separately or “pooled” (more cost-efficient) [27].
To get the highest benefit from testing marker bacteria in
periodontal diseases, the education of the staff involved must be
improved. It is very important that clinical microbiologists learn
more about oral and anaerobic microbiology, dentistry, and per-
iodontology and that periodontologists keep in touch with mi-
crobiology, antibiotics, and the principles of modern diagnostic
systems, especially their predictive values and shortcomings.
APPLICATION OF DNA PROBES AND PRIMERS
IN CARIES PREVENTION
Dental caries is a slow decomposition of teeth that results from
the loss of hydroxyapatite crystals. The mineralized matrix dis-

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 solution reduces the structural integrity of the teeth. The bac-
terial nature of this process may result in a chronic infection
of the tooth and eventual loss of teeth and supporting alveolar
bone. Current understanding supports the “plaque-host–
substrate” theory, whereby caries has a bacterial etiology in-
terdependent on (1) the host defense system (e.g., tooth brush-
ing and buffer capacity of saliva), (2) dietary factors, and (3)
time. Caries occurs when all these factors are operating together.
Depending on the bacterial composition, plaque can be ag-
gressive or not toward the enamel surface. The most consis-
tently found bacterial organisms in (human) initial dental le-
sions are Streptococcus mutans–group species (S. mutans, S.
sobrinus, S. cricetus, and S. rattus) [28–32]. They ferment sugars
(especially sucrose) to lactic acid, and, in parallel, they poly-
merize insoluble extracellular dextran (called mutan), which
allows bacteria to stick to the tooth surface. As a consequence,
the tooth surface–located acid production derives the demi-
neralization of enamel in vivo. A single enzyme, glucosyltran-
ferase, is responsible for the first step in acid and dextran pro-
duction by S. mutans [33, 34].
For more than a decade, culture strips have been available
to monitor S. mutans cell count in saliva and plaque to predict
the individual patients (especially children’s) risk for future
demineralization [35]. However, this procedure is sometimes
inaccurate, time-consuming, and laborious. DNA probes and
PCR primers could probably overcome these limitations. Tar-
gets chosen for DNA probe hybridization and/or in vitro am-
plification are the genes encoding for glucosyltransferase (gtfB),
fructosyltransferase (ftf), surface proteins (wapA and spaP),
dextranase (dex) [2, 36, 37], and specific regions of the 16S
rRNA.
In addition, typing of S. mutans and S. sobrinus by arbitrarily
primed PCR fingerprinting has been introduced [8]. Routine
screening for S. mutans by DNA probe hybridization and glu-
cosyltransferase-directed PCR in saliva and plaque is available
for private practices (LCL Biokey).
Of course, the use of DNA probes and PCR reaction for risk
assessment in caries is “high-end” medicine and might be re-
served for countries with high standards in dentistry. However,
because the costs for molecular techniques are constantly drop-
ping, this could be a future standard worldwide in industrialized
countries.
CONCLUSION
Microbiological testing that uses DNA probes and primers in
advanced forms of periodontitis is a very promising tool to
determine active disease and predict future attachment loss,
ultimately improving treatment prognosis. In addition, molec-
ular screening for S. mutans could help prevent caries, the most
frequently found and cost-intensive infectious disease.
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