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

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

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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.
Figure 1. Representative dot-blot hybridization for the detection of Some of these problems will be reviewed in the next section,
Prevotella intermedia and Prevotella nigrescens. Lane A, ATCC 25611 (105, below.
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.

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

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