Polymerase chain reaction: A molecular diagnostic tool in periodontology.

This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

INTRODUCTION

A clinical diagnosis of periodontal disease is made by measuring the loss of connective tissue attachment on the root surface (clinical attachment loss) and loss of alveolar bone (radiographic bone loss).[1] But the clinical diagnosis does not indicate the cause, pathogenesis, clinical course, progress, and prognosis of the disease. In addition to the conventional examination, various diagnostic methods play a vital role in the confirmation of the clinical diagnosis.

The traditional culture methods have inherent advantages, but have shortcomings, including the need to preserve bacterial vitality, the inability to detect low numbers of microorganisms with a detection limit averaging 103–104 bacterial cells, labor intensiveness, need for experienced personnel, strict sampling, transport conditions, and a prolonged period of time before results.[2] Other microbiological tests such as dark field microscopy are not able to detect the nonmotile periodontal pathogen, and immunodiagnostic methods like flow cytometry, immunofluorescence assay, etc., and enzymatic assays can lead to false positive results and cross-reactions.[3]

The molecular biological techniques analyze deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. Restriction fragment length polymorphism (RFLP) was the DNA profiling genetic technique that exploited variations in homologous DNA sequences; it was laborious, time-consuming, expensive, and needed large sample. The nucleic acid probe, a nucleic acid molecule artificially synthesized and labelled for detection of a specific organism has the limitation of cross-reactivity.[3] Hybridization refers to the pairing of complementary DNA strands to produce double stranded nucleic acid and the checkerboard DNA-DNA hybridization technology used for epidemiological research, and ecologic studies require sophisticated laboratory equipment and expertise.[3] Polymerase chain reaction (PCR) overcomes the above limitations and is capable of detecting even one copy of the searched DNA targets from clinical microbiologic samples.[4]

The subgingival microbiota in patients with periodontitis is complex and the difference in plaque composition serves as the basis for the clinical application of microbiological techniques in the diagnosis and therapy control of progressive and refractory forms of periodontitis.[5] The development of PCR has generated vast benefits in genetic analysis for the study of gene expression and diagnosis of genetic diseases. Genetic analysis using PCR for identification of susceptibility of an individual to periodontitis will help in the determination of the type and frequency of treatment. Studies based on PCR for the determination of mRNA expression of various immune and inflammatory markers are useful in understanding the pathogenesis of periodontitis.

The MEDLINE and PubMed databases were searched manually and electronically in English language by typing PCR, applications of PCR, PCR in Periodontics, Polymorphism studies in Periodontitis, and molecular techniques in periodontology. Out of 248 searched articles, 88 articles describing PCR process and its relation to periodontology were used to prepare a concise review. The aim of this review is to discuss the principles, advantages, applications, and limitations of PCR in the field of periodontology with their future perspectives.

HISTORY OF POLYMERASE CHAIN REACTION

The field of human genetics started on when DNA was first isolated by Johann Friedrich Miescher in 1869.[6] Watson and Crick in 1953 described the double helix structure of DNA.[6] In 1975, Southern blotting technology was used for genetic analysis. Its adaptation RFLP was developed in 1980 by Ray White.[7]

One of the most important revolutionary techniques in molecular biology, the PCR, was introduced by Mullis in the 1983, and he won the Noble prize in Chemistry in 1993 for its discovery. They developed it as a rapid and 2 times sensitive procedure than standard Southern blotting for the detection of the sickle cell mutation which is the first application of PCR in the field of medicine.[8] This molecular technique, invented three decades ago, now has revolutionized various fields. In dentistry, as early as 1992, PCR was used to identify DNA from human tooth pulp tissue for use in forensic dentistry.[9]

PCR was utilized for the identification of periodontal pathogen Porphyromonas gingivalis (Pg) in oral plaque samples in 1993.[10] Various derivatives of conventional PCR including nested PCR, multiplex-PCR, reverse transcriptase PCR (RT-PCR), allele-specific PCR, and quantitative PCR (Q-PCR) or real-time PCR subsequently evolved playing a significant role in the field of periodontology.[1112131415]

In 2005, open-ended PCRs were used for genome mapping of the entire bacterial spectrum in the plaque sample.[16] Later, the Human Oral Microbiome Database and CORE database to catalogue the entire bacterial species found in the oral cavity were developed.[17] Recently, PCR was used in DNA microarray analysis for the rapid semiquantitative determination of about 10 periodontal pathogens.[18]

PRINCIPLES OF POLYMERASE CHAIN REACTION

PCR, an in vitro technique, allows amplification and study of genes and their RNA transcripts obtained from various tissue sources including peripheral blood, skin, saliva, gingival crevicular fluid, semen, and hair.[1920] Each assay requires the presence of template DNA, primers, nucleotides, and DNA polymerase.

Template DNA is the known target sequence that needs to be amplified, and it ranges from 100 to 1000 base pairs in length. Primers are short, single-stranded sequences of nucleic acid (oligonucleotides) selected to specifically anneal to a particular nucleic acid target.[21] Primer pairs containing forward and reverse primer, each 16–20 base pairs in length are used.[22] DNA polymerase is the DNA replicating key enzyme that links individual nucleotides together to form the PCR product and hence to amplify target sequences of DNA.

The nucleic acid is first extracted from the clinical sample by heat, chemical, or enzymatic methods. Once extracted, target nucleic acid is added to the reaction mix containing primers, components to optimize polymerase activity (i.e., buffer, cation [MgCl2], salts, and deoxynucleotides) and enzymes in a test tube or 96-well plate and then placed in a thermal cycler that allows repeated cycles of DNA amplification to occur in the following three basic steps [Figure 1].

Figure 1

Principle of polymerase chain reaction

DNA denaturation – Separation of the double DNA strands into two single strands is accomplished by heating to 94°C

Primary annealing – At 50–58°C, when the primer pair is mixed with the denatured target DNA, forward primer anneals to a specific site at one end of the target sequence of one target strand, and the reverse primer anneals to a specific site at the opposite end of the other complementary target strand

Extension of the primed DNA sequence – The enzyme DNA polymerase synthesizes new complementary strands by the extension of primers at 72°C.[22] Taq polymerase is commonly used because of its ability to function efficiently at elevated temperatures.

Automated programmable thermal cyclers carry the PCR mixture through each reaction step at the precise temperature and for an optimal duration.

In general, the process is repeated 30 times. At the end of 30 cycles, the reaction mixture contains about 230 molecules of the desired product.[22] Once amplification reaction has occurred, a variety of manual and automated methods are available to detect the amplified product known as amplicon, of which the simplest is to identify the product by size after migration through electrophoresis on an agarose gel or polyacrylamide gel stained with ethidium bromide. Products appear as a single band matching to the size of the amplified sequence and fluoresces when illuminated by ultraviolet light.[21]

TYPES OF POLYMERASE CHAIN REACTION

Quantitative polymerase chain reaction

Is an approach where the accumulation of amplicon is monitored, as it is generated by the labeling of primers, oligonucleotide probes, or amplicons with molecules capable of fluorosing.[21] The fluorescent probes can be those that involve the nonspecific binding of a fluorescent dye to double stranded DNA (e.g., SYBER® Green I) or that bind specifically to the target of interest (e.g., TaqMan®). These probes produce a change in fluorescent signal following their direct interaction with or hybridization to the amplicon which is measured by the optical system to capture fluorescence and computer software capable of receiving and processing the data. Fluorescence values are recorded during each cycle and represent the amount of amplified product.[2324]

Nested polymerase chain reaction

Involves the sequential use of two primer sets; the first set is used to amplify a target sequence and the amplicon obtained is then used as the target sequence for a second amplification using primers internal to those of the first amplicon.[21]

Real-time polymerase chain reaction

Amplifies an RNA target. It involves two steps, first RNA is reverse transcribed into cDNA using an RT and then the resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes.

Multiplex polymerase chain reaction

Uses multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. Thus, it has the ability to search for different targets, organisms or genes using one reaction, avoiding the use of multiple reaction vessels and minimizing the volume of the specimen required.[21]

Allele-specific polymerase chain reaction

Is a diagnostic or cloning technique to identify or utilize single-nucleotide polymorphisms (SNPs). It uses primers whose 3' ends encompass the SNP and will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions.

Colony polymerase chain reaction

Is a technique in which the samples for PCR are taken using a sterile pipette from the bacterial colonies for bacterial identification by 16S rRNA gene sequencing.[25]

Hot start polymerase chain reaction

Improves specificity and DNA yield of PCR technique by reducing nonspecific amplification in the initial set up stage of the PCR and inhibiting the activity polymerase at ambient temperature.

Digital polymerase chain reaction

Limits dilution of template targets. It separates individual nucleic acid samples within a single specimen into separate regions or droplets and quantifies rare initial nucleic acid targets, important in areas such as cancer and prenatal diagnostics.

Polymerase chain reaction-cloning-sequencing method

Is a type of open-ended PCR in which 16S rRNA genes are amplified directly from samples and amplicons obtained are then cloned and sequenced by means of the traditional Sanger method.[26]

ADVANTAGES OF POLYMERASE CHAIN REACTION

The ease of quantification, greater sensitivity, rapid analysis, precision, reproducibility, quality control, and least contamination are the main advantages of PCR.[27] It also allows precise identification of bacterial strains with divergent phenotype. A large number of samples can be measured at 1 time. As cell viability is not a deterrent to PCR technique, it is advantageous in the study of strictly anaerobic infections, in which cell death could occur during sampling and transportation.

As PCR allows several million times amplifications of DNA or RNA, it is possible to use as few as 1–100 cells, and 0.1 µl of blood or cells scraped from buccal mucosa for analysis; exhibits excellent detection limits.[28]

Q-PCR has the ability to quantify the actual number of targets present in the clinical specimen. Multiplex PCR has the ability to search for different organisms or genes in one reaction.[21] Nested PCR facilitates the detection of bacterial DNA present at very low levels. RT-PCR is a sensitive method for detection of viruses and mRNA expression levels.[2129] Colony PCR was used for bacterial identification from bacterial colonies.

APPLICATIONS OF POLYMERASE CHAIN REACTION

Detection and characterization of microorganisms in the various medical fields of bacteriology, mycology, parasitology, virology, and dentistry has been revolutionized by the PCR technique.[24] The clinician and the researcher use the PCR technique for detecting microorganisms, diagnosing diseases, cloning and sequencing genes, and carrying out quantitative and genomic studies in a very rapid and sensitive manner. PCR has widespread application in various areas including genetic analysis, medical applications, and in research.

The ability to quantify “infectious burden” using Q-PCR has tremendous implications for studying and understanding the disease state (e.g. AIDS), prognosis of certain infections, and effectiveness of antimicrobial therapy. Genotyping allows the study of bacteria such as Mycobacterium tuberculosis (Mt). Thus, early recognition and optimized treatment are favored for public health.[30]

As clinically important viruses have genomes composed of RNA rather than DNA (e.g., human immunodeficiency virus [HIV], hepatitis B virus), the ability to amplify RNA using RT-PCR facilitates laboratory-based diagnostic testing to these infectious agents to a greater extent.[21] Identification of criminals has been made possible by PCR assay in forensic medicine. It is a sensitive test for tissue typing and plays an essential role in organ transplantation.[31]

The presence of genetic disease mutation can be detected in samples by PCR. Mutations in oncogenes and tumor suppressor genes are studied by PCR-based tests, and the results can be used to customize the therapy.

IN DENTISTRY

PCR plays an important role in various fields of dentistry. The subgingival plaque, saliva, mouthwash, blood, gingival tissue, and buccal mucosa scraping are used in the PCR to identify microorganisms, genetic polymorphisms, and mRNA gene expression of various inflammatory mediators in dentistry.[202832333435] The knowledge of the ecology of the oral cavity has been well-understood using PCR studies.[36]

Epidemiological studies based on the microbiology of dental diseases, genetic polymorphisms, and their relation to systemic diseases can be established.

Dental caries pathogens can be identified by PCR, and it also explains the progress of dental caries.[37] The microorganisms responsible for endodontic infections can be identified.[3839]

Genetic markers for oral cancers are identified by the PCR technique, and they are used in diagnosing and predicting the outcome and response to treatment.[40]

APPLICATIONS IN PERIODONTOLOGY

Identification of microbial pathogens

The PCR technique is a more accurate, sensitive, and rapid technique for the detection, identification, and quantification of periodontal bacteria.[51241]

Q-PCR or real-time PCR with species-specific primers provide accurate quantification of individual microbial species and total bacterial count in dental plaque samples.[15] This precise and sensitive method serves as a useful tool for studies on etiology of periodontal diseases.

Various putative perio-pathogens such as Pg, Aggregatibacter actinomycetecomitans (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 detected in subgingival plaque samples.[152042434445] Pg and Aa showed similar counts in aggressive periodontitis patients and controls, but only Aa was found to be related to the disease.[46]

It is also used to identify Mt in gingival enlargement and osteomyelitis.[4748] Certain new microbial species like Methanobrevibacter oralis identified in periodontal diseases using PCR have not yet been cultivated.[45] Recently, open-ended PCR/sequencing techniques are used to detect Gram-positive organisms Peptostreptococcus and Filifactor, genera Megasphaera and Desulfobulbus, species or phylotypes of Atopobium, Campylobacter, Catonella, Deferribacteres, Dialister, Eubacterium, Selenomonas, Streptococcus, Tannerella, and Treponema which are elevated in periodontal disease.[16]

PCR is used for research purposes to determine the prevalence of herpes simplex virus, human papillomavirus, HIV, human cytomegalovirus, and Epstein-Barr virus Type I and II (1 and 2) in the gingival crevicular fluid of the individuals with various forms of periodontal disease.[134950] It was found using hot start PCR that herpes virus might cause direct damage or impair the resistance of the periodontium to permit subgingival overgrowth of pathogenic bacteria in aggressive periodontitis.[51]

The microbial levels can be assessed following various treatment modalities and thus, can be an indicator for efficacy of treatment in chronic and aggressive periodontitis.[5253545556] The negative influence of alcohol consumption on microbiological parameters were studied by real-time PCR.[57]

It is also used to study the association of the systemic diseases such as coronary heart disease, pregnancy complications, diabetes, chronic kidney disease, osteoporosis, and respiratory disease with periodontitis by identifying the periodontal pathogen levels in various tissue samples such as subgingival plaque, thrombi, carotid endarterectomy, coronary artherosclerotic plaque, aortic valves, placenta, maxillary sinus tissue/wash samples.[34505859606162636465]

Diagnostic tests such as the MicroDent® Test, ParoCheck® kits, MyPerioPath® Test and oral DNA® using multiplex PCR scheme are commercially available to evaluate the microbiota in subgingival plaque samples and they give crucial information for a prevention strategy for healthy patients and treatment plans for “at risk” patients.[17]

Polymerase chain reaction as a diagnostic tool in peri-implantitis

Periodontal pathogens Aa, Pg, Pi, Td, and Tf have been detected in the foci of peri-implantitis using PCR.[66] The fungal organisms including Candida species were identified at peri-implantitis and healthy implant sites, and they co-colonized with Pm and Tf.[67] The uncultured phyla Chloroflexi, Synergistetes, Tenericutes and the organisms Pm, Pseudoramibacter alactolyticus, Peptostreptococcus stomatis, and Solobacterium moorei associated with peri-implantitis were also identified.[68]

This technique also plays a role in detecting bacteria causing periimplantitis before implant placement to prevent the risk of periimplantitis.[69]

Q-PCR has detected opportunistic pathogens such as E. faecalis in peri-implant environment of diseased implants suggesting removal of prosthesis and routine decontamination of implant surface and implant abutment connection.[70]

The success of dental implants was mainly associated with a negative TGP (Genetic Test for Periodontitis) which determines polymorphisms of − 889 IL1A gene and + 3953 IL1B gene using PCR whereas no success was related to a positive result.[71]

Immune and inflammatory markers identification

Amidst a deregulated oral environment, the subgingival biofilm triggers the release of pro-inflammatory cytokines and host-derived enzymes causing tissue breakdown. PCR has become the mainstay in protein detection in Periodontics, with special importance to microbial antigens, extracellular matrix proteins, and cytokines detection. The genetic expression of Pg virulence factors was studied using Q-PCR.[72] mRNA expression of adhesion molecule (ICAM-1) in periodontopathogen Eikenella corrodens (Ec) infected epithelial cells were determined using real-time PCR, and it was found to increase after exposure to N-acetyl-D-galactosamine adherence lectin of Ec.[73]

Using semiquantitative PCR gene expression of receptor activator of NF-KB ligand (RANKL) to osteoprotegerin (OPG) ratio was found to be increased in periodontitis.[74] Using Q-PCR expression of matrix metalloproteinases and RANKL was found to be correlated with expression of interleukin-1β, TNF-α, IF-gamma, intense inflammatory reaction and alveolar bone loss but IL-4, IL-10, TIMPs, and OPG expression reduced the cellular infiltration and alveolar bone loss.[33]

Smoking was associated with the mRNA expression of IL-1 β using real-time PCR in chronic periodontitis patients.[75] The expression of specific micro RNA species in periodontitis targeting and modulating cytokine mRNA using quantitative micro RNA PCR assay which provides insights to modify periodontal inflammation.[76] Quantitative mRNA expression of various growth factors; receptors toll-like receptors (TLRs), NOD2 and NALP3; signaling mediators CD14, MYD88, and TIR-domain-containing adapter-inducing interferon-beta were also determined by RT-PCR.[5377]

Genetic polymorphism studies

The individual's susceptibility to periodontitis is attributed to genetic factors.[78] The correlation of known genetic polymorphisms with phenotypes for certain patient groups currently appear to provide the most promising application of genetic determinants in treating periodontitis.

Genetic polymorphisms affect periodontal disease with a number of SNPs occurring in the gene coding for cytokines, receptors, and immune cells are associated with severity and susceptibility of periodontitis. PCR was used to identify a modified gene on chromosome 11 which caused a decrease in cathepsin C activity and resulted in Papillon–Lefevre syndrome.[79]

PCR has been used in linkage and segregation analysis of genetic studies in periodontal disease. Several studies have been conducted using PCR exploring the role of IL-1 gene polymorphism as a severity factor in periodontal diseases in various population and ethnic groups.[808182]

TLR-4 gene polymorphism was found to be associated with chronic periodontitis, while TLR-9 was not associated.[83848586] Polymorphisms in Fc gamma receptor gene and MPO-463G/A gene were studied using allele-specific and RT-PCR, respectively, which were found to be related with aggressive periodontitis.[8788] Various other polymorphisms including IL-10 gene, chemokine ligand (CCL5 and CCR5) gene, and OPG gene have been associated with periodontitis using PCR amplification.[89909192]

Limitations of polymerase chain reaction

The enormous cost of the very accurate PCR technique is a deterrent to its widespread application in routine diagnostic procedures.[24] High level of expertise is required to process samples and to carry out data analysis. The specificity of amplified PCR product could be altered by nonspecific binding of primers to the similar sequences of the template DNA. The DNA polymerase used in the PCR reaction is prone to errors which can lead to mutations in the fragment generated.

In the quantitative real-time PCR, the fluorescent signal cannot discriminate specific versus nonspecific amplified products. In multiplex PCR mixing different primers can cause some interference in amplification.[21] A high number of false negative results has been reported when nested PCR was used for identifying DNA of periodontal pathogens, and it is also capable of contaminating other reaction vials.[2136] The 16 S rRNA PCR-based method is not able to distinguish between closely related and also highly recombinant species, for example, Neisseria and certain Streptococci.

Future perspectives

Identification of microbial pathogens associated with the etiology of periodontitis is the first step toward the development of effective periodontal therapeutic approach. PCR may soon become the ideal detection method for periodontal pathogens owing to its inherent capacity of sensitivity and specificity. Test for diseases of the oral cavity may likely combine inherited polymorphisms with oral microbial profiles and might also include assays of gene expression and proteomic data measured in saliva or other oral tissues.

CONCLUSION

The PCR is a revolutionary watershed in scientific and medical fields and has now become a standard diagnostic and research tool in periodontology. Understanding the pathogenesis involved in the onset, progression and resolution of the periodontal diseases could greatly help in establishing the effective ways for prevention and treatment besides decreasing the risk factor for relevant systemic conditions. The future of PCR is promising in producing greater insight starting from identification of periodontal pathogens to effective therapeutic approach.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest