https://doi.org/10.53453/ms.2024.1.5
Polymerase chain reaction significance in the diagnosis of periodontal
diseases
Karolina Budreikaitė
1
, Nomeda Basevičienė
2
1
Faculty of Odontology of Lithuanian University of Health Sciences, Kaunas, Lithuania
2
Faculty of Odontology of Lithuanian University of Health Sciences, Kaunas, Lithuania
Absrtract
Background. Polymerase chain reaction (PCR) is a revolutionary technique for rapidly amplifying millions of copies
of a specific segment of DNA, which can be used to make more accurate analysis. PCR is well-known for its sensitivity
and specificity to determine various microbiota and initially stood out as a novel molecular diagnostic method for
different fields of medicine, over time it became increasingly popular in the field of dentistry, particularly
periodontology.
Methods. The systematic review adhered to PRISMA guidelines and databases of PubMed, ScienceDirect, and The
Cochrane Library were used to perform the search.
Aim. To evaluate the importance of polymerase chain reaction in diagnosing periodontal diseases.
Results. 1356 adult patients with periodontitis and healthy group were evaluated microbiologically. All 7 articles
agreed that individual microbial species and total bacterial count in dental plaque samples may be accurately quantified
using Q-PCR or real-time PCR. Comparing Culture method and PCR, polymerase chain reaction showed better results
for the detection of F nucleatum (53 % and 73 % respectively), P. gingivalis (84 % and 94 % respectively) and T.
forsythensis (56 % and 93 % respectively).
Conclusions. PCR as a diagnostic tool upgrades the diagnostic field of periodontology and allows dentists to recognize
periopathology in the early stage and select the approprate treatment.
Keywords: polymerase chain reaction; PCR; periodontology; diagnostic tool; periodontal pathogens;
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Medical Sciences 2024 Vol. 12 (1), p. 32-40, https://doi.org/10.53453/ms.2024.1.5
32
1. Introduction
Periodontal diseases affect approximately 20-50 %
world’s population [1]. The onset of periodontitis is
triggered by the presence of periodontal pathogens,
particularly Gram-negative bacteria such as
Porphyromonas gingivalis, Treponema denticola, and
Tannerella forsythia, which are commonly detected
within subgingival dental plaque among patients with
periodontal disease [2]. According to recent literature,
there are three main classifications periodontal
diseases are specified into. The first one focuses on the
amount of bone loss around teeth (localized or
generalized). In the meantime, the second
classification is based on the severity of periodontal
disease (stage 1 –least severe; stage 4 – most severe).
The risk and rate of disease progression (3
rd
classification) have been divided into three grades
from the lowest risk of progression (grade A) to the
highest (grade C) [3]. Accurate diagnosis of the
disease is one of the most critical pre-treatment steps.
While the traditional clinical routine of diagnosing
periodontal disease (e.g. clinical attachment loss,
radiographic bone loss or bleeding on probing (BOP))
does not include the cause, progression, and prediction
of the disease, the scientists initiated a comparative
analysis of various microbial detection techniques and
came up with a new approach to identify perio-
pathogens – a polymerase chain reaction [4].
Polymerase chain reaction (PCR) is a revolutionary
technique for rapidly amplifying millions of copies of
a specific segment of DNA, which can be used to make
more accurate analysis. [5]. PCR involves the use of
short synthetic DNA fragments (primers) to select a
specific segment of the genome for amplification,
followed by multiple steps of DNA synthesis to further
amplify that segment [2, 4-8, 10, 20, 24]. PCR can
identify even one copy of the DNA targets from
clinical samples.
The aim of the article is to show the importance of
PCR in the detection of microbial dysbiosis of oral
microbiome.
2. Materials and methods
2.1 Methods
The systematic review adhered to PRISMA (Preferred
Reporting Items for Systematic Review) guidelines. A
focused question was formed according to the PICOS
model: how is polymerase chain reaction a significant
tool in the diagnostics of periodontal diseases?
2.2 Search strategy
A comprehensive literature search using advanced
features of Pubmed, Science Direct, Cochrane Library,
and Scopus databases was carried out. The following
search terms were used: “polymerase chain reaction”
OR “PCR” AND “periodontology” AND “diagnostic
tool” OR “ periodontal pathogens”. The search was
supplemented using additional articles in references
and lists of similar studies.
2.3 Eligibility criteria
The inclusion criteria for the studies were: studies in
English language, studying periodontal patients’
microbiota with PCR. The articles describing PCR
process and its relation to periodontology were
collected and used to prepare a concise review. Case
reports, systematic reviews, meta-analyses, and
animal studies were excluded from the search. There
was no limitation for publication time.
2.4 Study selection and data collection process
Firstly, the possible studies from the initial search
were selected for further screening based on the title
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33
and the abstract by the authors. Secondly, the selected
studies were analyzed and the ones that did not match
the inclusion criteria were discarded. Randomized
controlled trials, as
well as comparative studies, double-blinded,
controlled clinical trials were included in the article.
3. Results
3.1 Study selection
Primary database search yielded 397 results out of
which 11 were duplicates and were excluded. Titles
and abstracts of 386 articles were screened and after
the process, 28 studies were used for further full-text
analysis. After checking the content and relevance of
the articles, 7 articles were used. 1356 adult patients
with periodontitis and healthy individuals (the control
group) were evaluated microbiologically.
3.2 Risk of bias assessment
All 7 included studies were evaluated qualitatively by
the tools of Cohraine Collaboration for the risk of bias
(Figure 1). Two studies [6, 9] had a high risk of bias in
allocation concealment. The highest proportion of low
risk of bias included other bias allocation
concealment, blinding of outcomes, selective
reporting and incomplete outcome data. Meanwhile
blinding of participants and personnel, selective
reporting, and allocation concealment were noted as
the highest proportion of unclear risk bias.
Figure 1. Cohraine Collaboration for the risk of bias
Random sequence
generation (selection
bias)
Allocation
concealment
(selection bias)
Blinding of
participants and
personnel
(performance
) =bias)
Blinding of outcomes
assessment
(detection
bias)
Incomplete outcome
data
(attrition bias)
Selective reporting
(reporting bias)
Other bias
Hee Sam Na et al.
[2]
P.-M. Jervøe-Storm et al
[7]
Jin Uk Choi et al
[8]
Khalil Boutaga et al
[6]
Nicole B. Arweiler et al.
[9]
Preeti Ingalagi et al
[10]
Braga RR et al
[11]
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Random sequence generation (selection bias)
Allocation concealment (selection bias)
Blinding of participants and personnel
(performance bias)
Blinding of outcome assessment (detection
bias)
Incomplete outcome data (attrition bias)
Selective reporting (reporting bias)
Other bias
3.3 Characteristics of included studies
2 of the studies were cross-sectional, 2- comparative
studies and 3 studies were controllend clinical trials. A
total of 1356 patients were included in the studies.
Despite the control group, only patients with
periodontal diseases were included in the study.
Participants were enrolled in either the
healthy/gingivitis group or the periodontitis group (in
most of the studies). Patients in the healthy/gingivitis
group had < 3 mm attachment loss, >4 mm periodontal
probe depth (PD), and no radiographic alveolar bone
loss. Patients with periodontitis showed at least 4 sites
with radiographic bone loss, 4 sites with more than 3
mm attachment loss, and at least 4 sites with more than
4 mm PD.
3.4 Principles of polymerase chain reaction
The polymerase chain reaction (PCR) amplifies a
single or a few copies of a piece of DNA through
multiple orders of magnitude, producing thousands to
millions of copies of a specific DNA sequence [5].
The PCR process consists of three essential steps:
denaturation, annealing, and extension [4].
In the first step, the DNA is denatured at high
temperatures (90-97 degrees Celsius). Primers anneal
to the DNA template strands in step two to prime
extension . In step three, the annealed primers are
extended to form a complementary copy strand of
DNA. [4,5,8].
There are a lot of types of PCR (such as Quantitative
polymerase chain reaction, Nested polymerase chain
reaction, Real-time polymerase chain reaction,
Multiplex polymerase chain reaction, and etc.) but all of
them have identical benefits and drawbacks (Figure 2).
3.5 Qualitative synthesis of results
All 7 articles agreed that individual microbial species
and total bacterial count in dental plaque samples may
be accurately quantified using Q-PCR or real-time
PCR. [2, 7-11].
Low risk of bias Unclear risk of bias High risk of bias
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Figure 2. Advantages and disadvantages of PCR
Benefits of PCR
Limitations of PCR
Exact identification of bacterial strains with
disparate phenotypes
Expensive cost
The simplicity of quantifying
High technical capabilities are required.
Precision
Changing the specificity of PCR product produced
Amplification of DNA or RNA millions of times
Results that are either positive or falsely negative
Rapid examination
Creating a high sterile atmosphere has constraints.
Least contamination
Contamination of DNA
Increased sensitivity
Low detection ability between closely related
and highly recombinant species
Reproducibility
Multiplex PCR using various primers has limits.
The capacity to measure several targets in a
clinical specimen
The ability to contaminate other reaction vials
Quality assurance
The capacity to look for several organisms or genes in
a single response.
Identification of microorganisms from bacterial colonies
Detection of very tiny quantities of samples
The study of strictly anaerobic infections
Virus detection and mRNA expression levels
Porphyromonas gingivalis (Pg), Aggregatibacter
actinomycetemcomitans (Aa), Tannerella forsythia
(Tf), Prevotella intermedia (Pi), Prevotella
nigrescens, Parvimonas micra (Pm), Eubacteria,
Campylobacter rectus (Cr), Capnocytophaga
sputigena, Capnocytophaga ochracea, and
Capnocytophaga gingivalis have all been found in
subgingival plaque samples [2, 4, 6, 7, 10, 11]. Pg and
Aa levels were comparable in aggressive periodontitis
patients and controls, but according to Shahriar Shahi
et al only Aa was linked to periodontal disease [4].
Some of the articles compared bacterial cultivation
(the golden standard) and real-time PCR for the
detection of the most common periodontal microbes
[6, 7, 10].
P.-M. Jervøe-Storm et al found that polymerase chain
reaction showed better results for the detection of F.
nucleatum (53 % and 73 % respectively), P. gingivalis
(84 % and 94 % respectively) and T. forsythensis
(56 % and 93 % respectively). The culture method was
better for the detection of A. actinomycetemcomitans
and P. intermedia [7].
Other article used both methods for detection of P.
gingivalis in subgingival plaque samples. The results
revealed that P. gingivalis was detected in 111 (43 %)
of the 259 subgingival plaque samples by culture and
in 138 (53 %) samples by PCR. The sensitivity,
specificity, and positive and negative predictive values
of the real-time PCR were 100, 94, 94, and 100 %,
respectively [6].
In the meantime, Jin Uk Choi et al demonstrated that
the amount of salivary Pg was more prominent in
patients with periodontitis than that in healthy people
[8]. On account of PCR, it may be suggested that Pg in
saliva has the potential to be utilized as a diagnostic
marker of periodontitis [8, 11].
Jin UK Choi et al demonstrated that the majority of
target bacteria exhibited increased counts as the
severity of periodontitis increased. Pg, Tf, Td, Pm, Cr,
and En were significantly correlated with the severity
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of periodontal disease (ρ = 0.530, 0.438, 0.209, 0.276,
0.283, 0.311, respectively) [8].
Some articles compared the prevalence of
periopathogens in healthy individuals and patients
with periodontitis [10, 11]
Renato R. R. Braga et al [11] compared the
quantification of five putative periodontal pathogens
(A. actinomycetemcomitans, E. corrodens, F. nuclea-
tum, P. gingivalis and P. intermedia). Oral specimens
from all individuals tested positive for A. actino-
mycetemcomitans, E. corrodens, and F. nucleatum.
Besides, P. gingivalis was detected in 70.0 % and
46.6% and P. intermedia in 90.0 % and 80.0 % of
periodontal patients and healthy subjects, respectively.
However, only P. gingivalis, which was found in
greater quantities in specimens from individuals with
chronic periodontitis, showed a statistically significant
difference (p = 5.2 × 10
−3
) [11].
The other study investigated that the prevalence of P.
gingivalis was low in healthy people (9.9 % by RT-
PCR) but rose to 45.5 % in periodontitis patients. T.
forsythia was detected in 33.2 % of healthy people and
had a prevalence of 89.2 % in patients when tested
with RT-PCR. P. intermedia exhibited a significant
difference in healthy individuals: 23.2 %, whereas it
increased to 83 % in periodontitis patients [10].
Also, all of the studies (that compared people with
periodontitis and healthy humans) indicate higher rate
of the A. actinomycetemcomitans and P. gingivalis in
periodontal patients than healthy individuals
[2,4,10,11].
None of the articles found a specific amount of
pathogens that determines the onset of periopathology.
4. Discussion
PCR was first developed in the mid-80s and is well-
known for its sensitivity and specificity to determine
various microbiome [5]. While the polymerase chain
reaction initially stood out as a novel molecular
diagnostic method for different fields of medicine,
over time it became increasingly popular in the field
of dentistry, particularly periodontology.
Although the cost of using PCR for diagnosing
periodontal disease is high and not yet widely
available in many dental clinics, a PCR as a diagnostic
tool will eventually become the golden standard for
minimally invasive periodontal treatment [17].
Exploring the pathological pathways that lead to the
development, progression, and management of
periodontal diseases could be highly beneficial and has
some potential to provide proactive strategies for
prevention and treatment, as well as reducing the risk
for relevant systemic conditions [4, 8, 10, 11, 15-23].
The upcoming publication of the PCR will be
beneficial in developing a more comprehensive
understanding, from the identification of disease-
promoting agents in the periodontium to effective
treatment strategies.
The high sensitivity and specificity of PCR allow it to
be a precise, efficient, and rapid method for the
detection, identification, and quantification of
microorganisms [4, 5, 26-28]. Several potential
periodontal pathogens such as Porphyromonas
gingivalis (Pg), Aggregatibacter actinomycetecomi-
tans (Aa), Tannerella forsythia (Tf), Prevotella
intermedia (Pi), Prevotella nigrescens, Parvimonas
micra (Pm), Eubacteria, Campylobacter rectus (Cr),
Capnocytophaga sputigena, Capnocytophaga
ochracea, and Capnocytophaga gingivalis have been
identified in samples of subgingival plaque (as
referenced in the sources) [16].
This method also plays a crucial role in identifying
bacteria responsible for periimplantitis before implant
placement, thereby mitigating the risk of
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37
periimplantitis [12, 13, 18, 25]. Quantitative PCR (Q-
PCR) has identified opportunistic pathogens like E.
faecalis within the peri-implant environment of
diseased implants. This discovery suggests that
removing the prosthesis and regularly
decontaminating the implant surface and implant
abutment connection may be necessary [12, 25].
Moreover, PCR is used for the detection of
Mycobacterium tuberculosis in cases of osteomyelitis
and hypertrophic gingivitis [4, 16]. Open-ended
PCR/sequencing techniques are utilized to identify
Gram-positive bacteria, including
Peptostreptococcus, Filifactor, Desulfobulbus,
Deferribacteres, Atopobium, Treponema,
Megasphaera, Dialister, Eubacterium, Selenomonas,
Catonella, Streptococcus, Tannerella, and
Campylobacter in such situations [16].
Unfortunately, the use of PCR has some limitations.
The most prominent of these is the cost of equipment
and tests, as well as the potential for false positives and
false negatives [4, 6-11, 24, 26, 28]. Additionally,
PCR has a limited capacity to detect closely related
and highly recombinant species [4, 5].
5. Conclusions
Investigating the pathological processes that
contribute to the formation, progression, and
management of periodontal diseases offers the
potential to give proactive preventive and treatment
techniques, as well as reduce the risk for associated
systemic disorders. PCR as a diagnostic tool upgrades
the diagnostic field of periodontology and allows
dentists to recognize periopathology in the early stage
and select the approprate treatment.
Declaration of interests
There are no conflicts of interest to declare.
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