|Year : 2016 | Volume
| Issue : 2 | Page : 128-135
Polymerase chain reaction: A molecular diagnostic tool in periodontology
Rajendran Maheaswari, Jaishree Tukaram Kshirsagar, Nallasivam Lavanya
Department of Periodontics, Tamil Nadu Government Dental College and Hospital, Chennai, Tamil Nadu, India
|Date of Submission||30-Sep-2015|
|Date of Acceptance||16-Dec-2015|
|Date of Web Publication||11-Apr-2016|
Dr. Rajendran Maheaswari
No. 5, Poes 4th Street, Teynampet, Chennai - 600 018, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
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.
Keywords: Applications of polymerase chain reaction, identification of periodontal organisms, molecular techniques in periodontology, mRNA expression, polymerase chain reaction
|How to cite this article:|
Maheaswari R, Kshirsagar JT, Lavanya N. Polymerase chain reaction: A molecular diagnostic tool in periodontology. J Indian Soc Periodontol 2016;20:128-35
|How to cite this URL:|
Maheaswari R, Kshirsagar JT, Lavanya N. Polymerase chain reaction: A molecular diagnostic tool in periodontology. J Indian Soc Periodontol [serial online] 2016 [cited 2020 May 30];20:128-35. Available from: http://www.jisponline.com/text.asp?2016/20/2/128/176391
| 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). 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. 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.
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. 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. 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.
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. 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. Watson and Crick in 1953 described the double helix structure of DNA. In 1975, Southern blotting technology was used for genetic analysis. Its adaptation RFLP was developed in 1980 by Ray White.
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. 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.
PCR was utilized for the identification of periodontal pathogen Porphyromonas gingivalis (Pg) in oral plaque samples in 1993. 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.,,,,
In 2005, open-ended PCRs were used for genome mapping of the entire bacterial spectrum in the plaque sample. Later, the Human Oral Microbiome Database and CORE database to catalogue the entire bacterial species found in the oral cavity were developed. Recently, PCR was used in DNA microarray analysis for the rapid semiquantitative determination of about 10 periodontal pathogens.
| 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., 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. Primer pairs containing forward and reverse primer, each 16–20 base pairs in length are used. 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].
- 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. 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. 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.
| 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. 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.,
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.
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.
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.
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.
| 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. 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.
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. 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., 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. 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.
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. 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.
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., 28, ,,, The knowledge of the ecology of the oral cavity has been well-understood using PCR studies.
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. The microorganisms responsible for endodontic infections can be identified.,
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.
| 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.,,
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. 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., 20, ,,, Pg and Aa showed similar counts in aggressive periodontitis patients and controls, but only Aa was found to be related to the disease.
It is also used to identify Mt in gingival enlargement and osteomyelitis., Certain new microbial species like Methanobrevibacter oralis identified in periodontal diseases using PCR have not yet been cultivated. 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.
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.,, 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.
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.,,,, The negative influence of alcohol consumption on microbiological parameters were studied by real-time PCR.
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., 50, ,,,,,,,
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.
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. The fungal organisms including Candida species were identified at peri-implantitis and healthy implant sites, and they co-colonized with Pm and Tf. The uncultured phyla Chloroflexi, Synergistetes, Tenericutes and the organisms Pm, Pseudoramibacter alactolyticus, Peptostreptococcus stomatis, and Solobacterium moorei associated with peri-implantitis were also identified.
This technique also plays a role in detecting bacteria causing periimplantitis before implant placement to prevent the risk of periimplantitis.
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.
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.
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. 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.
Using semiquantitative PCR gene expression of receptor activator of NF-KB ligand (RANKL) to osteoprotegerin (OPG) ratio was found to be increased in periodontitis.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.
Smoking was associated with the mRNA expression of IL-1 β using real-time PCR in chronic periodontitis patients. 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. 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.,
Genetic polymorphism studies
The individual's susceptibility to periodontitis is attributed to genetic factors. 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.
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.,,
TLR-4 gene polymorphism was found to be associated with chronic periodontitis, while TLR-9 was not associated.,,, 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., Various other polymorphisms including IL-10 gene, chemokine ligand (CCL5 and CCR5) gene, and OPG gene have been associated with periodontitis using PCR amplification.,,,
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. 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. 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., The 16 S rRNA PCR-based method is not able to distinguish between closely related and also highly recombinant species, for example, Neisseria More Details and certain Streptococci.
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.
Authors acknowledge the immense help received from the authors whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals, and books from where the literature for this article has been reviewed and discussed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Wolf DL, Lamster IB. Contemporary concepts in the diagnosis of periodontal disease. Dent Clin North Am 2011;55:47-61.
Armitage GC. Periodontal diseases: Diagnosis. Ann Periodontol 1996;1:37-215.
Sanz M, Newman MG, Quirynen M. Advanced diagnostic techniques. In: Newman MG, Takei HH, Klokkevold PR, Carranza FA, editors. Carranza's Clinical Periodontology. 11th
ed. New Delhi: Elsevier; 2012. p. 1353-9.
Sanz M, Lau L, Herrera D, Morillo JM, Silva A. Methods of detection of Actinobacillus actinomycetemcomitans
, Porphyromonas gingivalis
and Tannerella forsythensis
in periodontal microbiology, with special emphasis on advanced molecular techniques: A review. J Clin Periodontol 2004;31:1034-47.
Eick S, Pfister W. Comparison of microbial cultivation and a commercial PCR based method for detection of periodontopathogenic species in subgingival plaque samples. J Clin Periodontol 2002;29:638-44.
Dahm R. Friedrich Miescher and the discovery of DNA. Dev Biol 2005;278:274-88.
Tilstone WJ, Savage KA, Clark LA. Forensic Science: An Encyclopedia of History, Methods, and Techniques. 6th
ed. California: ABC-CLIO Publishers; 2006. p. 48.
Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1990;262:56-61, 64-5.
Pötsch L, Meyer U, Rothschild S, Schneider PM, Rittner C. Application of DNA techniques for identification using human dental pulp as a source of DNA. Int J Legal Med 1992;105:139-43.
Watanabe K, Frommel TO. Detection of Porphyromonas gingivalis
in oral plaque samples by use of the polymerase chain reaction. J Dent Res 1993;72:1040-4.
Leys EJ, Griffen AL, Strong SJ, Fuerst PA. Detection and strain identification of Actinobacillus actinomycetemcomitans
by nested PCR. J Clin Microbiol 1994;32:1288-94.
Riggio MP, Macfarlane TW, Mackenzie D, Lennon A, Smith AJ, Kinane D. Comparison of polymerase chain reaction and culture methods for detection of Actinobacillus actinomycetemcomitans
and Porphyromonas gingivalis
in subgingival plaque samples. J Periodontal Res 1996;31:496-501.
Saygun I, Sahin S, Ozdemir A, Kurtis B, Yapar M, Kubar A, et al.
Detection of human viruses in patients with chronic periodontitis and the relationship between viruses and clinical parameters. J Periodontol 2002;73:1437-43.
Kobayashi T, Sugita N, van der Pol WL, Nunokawa Y, Westerdaal NA, Yamamoto K, et al.
The Fcgamma receptor genotype as a risk factor for generalized early-onset periodontitis in Japanese patients. J Periodontol 2000;71:1425-32.
Lyons SR, Griffen AL, Leys EJ. Quantitative real-time PCR for Porphyromonas gingivalis
and total bacteria. J Clin Microbiol 2000;38:2362-5.
Kumar PS, Griffen AL, Moeschberger ML, Leys EJ. Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol 2005;43:3944-55.
Do T, Devine D, Marsh PD. Oral biofilms: Molecular analysis, challenges, and future prospects in dental diagnostics. Clin Cosmet Investig Dent 2013;5:11-9.
Topcuoglu N, Kulekci G. 16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis. Anaerobe 2015;35(Pt A):35-40.
Jordan RC, Daniels TE, Greenspan JS, Regezi JA. Advanced diagnostic methods in oral and maxillofacial pathology. Part I: Molecular methods. Oral Maxillofac Pathol 2001;92:650-69.
Jervøe-Storm PM, Koltzscher M, Falk W, Dörfler A, Jepsen S. Comparison of culture and real-time PCR for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples. J Clin Periodontol 2005;32:778-83.
Tille PM. Bailey and Scott's Diagnostic Microbiology. 13th
ed. Missouri: Mosby Elsevier Publishers; 2013. p. 112-22.
Turgeon ML. Linne's and Ringsrud's Clinical Laboratory Science: The Basics and Routine Techniques. 6th
ed. Missouri: Mosby Elsevier Publishers; 2011. p. 146-7.
Kuboniwa M, Amano A, Kimura KR, Sekine S, Kato S, Yamamoto Y, et al.
Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes. Oral Microbiol Immunol 2004;19:168-76.
Valones MA, Guimarães RL, Brandão LA, de Souza PR, de Albuquerque Tavares Carvalho A, Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: A review. Braz J Microbiol 2009;40:1-11.
Pimentel JD, Chan RC. Desulfovibrio fairfieldensis bacteremia associated with choledocholithiasis and endoscopic retrograde cholangiopancreatography. J Clin Microbiol 2007;45:2747-50.
Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ. New bacterial species associated with chronic periodontitis. J Dent Res 2003;82:338-44.
Ashimoto A, Chen C, Bakker I, Slots J. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 1996;11:266-73.
Kumar V, Abbas AK, Fausto N, Astra JC. Robbins and Cotran Pathologic Basis of Disease. 8th
ed. Philadelphia: Saunders Elsevier Publishers; 2010. p. 173.
LaGier MJ, Bilokopytov I, Cockerill B, Threadgill DS. Identification and characterization of a putative chemotaxis protein, CheY, from the oral pathogen Campylobacter rectus
. Internet J Microbiol 2014;12. pii: 21300.
Ashworth M, Horan KL, Freeman R, Oren E, Narita M, Cangelosi GA. Use of PCR-based Mycobacterium tuberculosis
genotyping to prioritize tuberculosis outbreak control activities. J Clin Microbiol 2008;46:856-62.
Lynas C, Hurlock NJ, Copplestone JA, Prentice AG, McGonigle RJ. HLA-DR typing for kidney transplants: Advantage of polymerase chain reaction with sequence specific primers in a routine hospital laboratory. J Clin Pathol 1994;47:609-12.
Boutaga K, Savelkoul PH, Winkel EG, van Winkelhoff AJ. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction. J Periodontol 2007;78:79-86.
Garlet GP, Cardoso CR, Silva TA, Ferreira BR, Avila-Campos MJ, Cunha FQ, et al.
Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans
through the modulation of MMPs, RANKL, and their physiological inhibitors. Oral Microbiol Immunol 2006;21:12-20.
Figuero E, Lindahl C, Marín MJ, Renvert S, Herrera D, Ohlsson O, et al.
Quantification of periodontal pathogens in vascular, blood, and subgingival samples from patients with peripheral arterial disease or abdominal aortic aneurysms. J Periodontol 2014;85:1182-93.
García-Delaney C, Sánchez-Garcés MÁ, Figueiredo R, Sánchez-Torres A, Gay-Escoda C. Clinical significance of interleukin-1 genotype in smoking patients as a predictor of peri-implantitis: A case-control study. Med Oral Patol Oral Cir Bucal 2015;20:e737-43.
Bizzarro S, Loos BG, Laine ML, Crielaard W, Zaura E. Subgingival microbiome in smokers and non-smokers in periodontitis: An exploratory study using traditional targeted techniques and a next-generation sequencing. J Clin Periodontol 2013;40:483-92.
Okada M, Soda Y, Hayashi F, Doi T, Suzuki J, Miura K, et al.
PCR detection of Streptococcus mutans
and S. sobrinus
in dental plaque samples from Japanese pre-school children. J Med Microbiol 2002;51:443-7.
Bogen G, Slots J. Black-pigmented anaerobic rods in closed periapical lesions. Int Endod J 1999;32:204-10.
Kim Y, Flynn TR, Donoff RB, Wong DT, Todd R. The gene: The polymerase chain reaction and its clinical application. J Oral Maxillofac Surg 2002;60:808-15.
Murdoch-Kinch CA. Oral medicine: Advances in diagnostic procedures. J Calif Dent Assoc 1999;27:773-80, 782-4.
Morillo JM, Lau L, Sanz M, Herrera D, Silva A. Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans
and Porphyromonas gingivalis
. J Periodontal Res 2003;38:518-24.
Hayashi F, Okada M, Zhong X, Miura K. PCR detection of Capnocytophaga
species in dental plaque samples from children aged 2 to 12 years. Microbiol Immunol 2001;45:17-22
Sencimen M, Saygun I, Gulses A, Bal V, Acikel CH, Kubar A. Evaluation of periodontal pathogens of the mandibular third molar pericoronitis by using real time PCR. Int Dent J 2014;64:200-5.
Oettinger-Barak O, Sela MN, Sprecher H, Machtei EE. Clinical and microbiological characterization of localized aggressive periodontitis: A cohort study. Aust Dent J 2014;59:165-71.
Bringuier A, Khelaifia S, Richet H, Aboudharam G, Drancourt M. Real-time PCR quantification of Methanobrevibacter oralis
in periodontitis. J Clin Microbiol 2013;51:993-4.
Sánchez GA, Acquier AB, De Couto A, Busch L, Mendez CF. Association between Aggregatibacter actinomycetemcomitans
and Porphyromonas gingivalis
in subgingival plaque and clinical parameters, in Argentine patients with aggressive periodontitis. Microb Pathog 2015;82:31-6.
Jan SM, Khan FY, Bhat MA, Behal R. Primary tuberculous gingival enlargement – A rare clinical entity: Case report and brief review of the literature. J Indian Soc Periodontol 2014;18:632-6.
Bakutra G, Manohar B, Mathur L. Tuberculous osteomyelitis affecting periodontium: A rare case report. J Indian Soc Periodontol 2015;19:578-81.
Parra B, Slots J. Detection of human viruses in periodontal pockets using polymerase chain reaction. Oral Microbiol Immunol 1996;11:289-93.
Pucar A, Milasin J, Lekovic V, Vukadinovic M, Ristic M, Putnik S, et al.
Correlation between atherosclerosis and periodontal putative pathogenic bacterial infections in coronary and internal mammary arteries. J Periodontol 2007;78:677-82.
Sharma S, Tapashetti RP, Patil SR, Kalra SM, Bhat GK, Guvva S. Revelation of viral – Bacterial interrelationship in aggressive periodontitis via polymerase chain reaction: A microbiological study. J Int Oral Health 2015;7:101-7.
Aimetti M, Romano F, Guzzi N, Carnevale G. Full-mouth disinfection and systemic antimicrobial therapy in generalized aggressive periodontitis: A randomized, placebo-controlled trial. J Clin Periodontol 2012;39:284-94.
Hakki SS, Bozkurt SB. Effects of different setting of diode laser on the mRNA expression of growth factors and type I collagen of human gingival fibroblasts. Lasers Med Sci 2012;27:325-31.
Milne TJ, Coates DE, Leichter JW, Soo L, Williams SM, Seymour GJ, et al.
Periodontopathogen levels following the use of an Er: YAG laser in the treatment of chronic periodontitis. Aust Dent J 2015.
Ardila CM, Martelo-Cadavid JF, Boderth-Acosta G, Ariza-Garcés AA, Guzmán IC. Adjunctive moxifloxacin in the treatment of generalized aggressive periodontitis patients: Clinical and microbiological results of a randomized, triple-blind and placebo-controlled clinical trial. J Clin Periodontol 2015;42:160-8.
Rodrigues AS, Lourenção DS, Lima Neto LG, Pannuti CM, Hirata RD, Hirata MH, et al.
Clinical and microbiologic evaluation, by real-time polymerase chain reaction, of non-surgical treatment of aggressive periodontitis associated with amoxicillin and metronidazole. J Periodontol 2012;83:744-52.
Lages EJ, Costa FO, Cortelli SC, Cortelli JR, Cota LO, Cyrino RM, et al.
Alcohol consumption and periodontitis: Quantification of periodontal pathogens and cytokines. J Periodontol 2015;86:1058-68.
Mahendra J, Mahendra L, Kurian VM, Jaishankar K, Mythilli R. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque. Indian J Dent Res 2010;21:248-52.
Swati P, Thomas B, Vahab SA, Kapaettu S, Kushtagi P. Simultaneous detection of periodontal pathogens in subgingival plaque and placenta of women with hypertension in pregnancy. Arch Gynecol Obstet 2012;285:613-9.
Aemaimanan P, Amimanan P, Taweechaisupapong S. Quantification of key periodontal pathogens in insulin-dependent type 2 diabetic and non-diabetic patients with generalized chronic periodontitis. Anaerobe 2013;22:64-8.
Bastos JA, Diniz CG, Bastos MG, Vilela EM, Silva VL, Chaoubah A, et al.
Identification of periodontal pathogens and severity of periodontitis in patients with and without chronic kidney disease. Arch Oral Biol 2011;56:804-11.
Hernández-Vigueras S, Martínez-Garriga B, Sánchez MC, Sanz M, Estrugo-Devesa A, Vinuesa TT, et al.
Oral microbiota, periodontal status and osteoporosis in postmenopausal women. J Periodontol 2015:1-15. [Epub ahead of print].
Paju S, Bernstein JM, Haase EM, Scannapieco FA. Molecular analysis of bacterial flora associated with chronically inflamed maxillary sinuses. J Med Microbiol 2003;52(Pt 7):591-7.
Nakano K, Nemoto H, Nomura R, Inaba H, Yoshioka H, Taniguchi K, et al.
Detection of oral bacteria in cardiovascular specimens. Oral Microbiol Immunol 2009;24:64-8.
Ohki T, Itabashi Y, Kohno T, Yoshizawa A, Nishikubo S, Watanabe S, et al.
Detection of periodontal bacteria in thrombi of patients with acute myocardial infarction by polymerase chain reaction. Am Heart J 2012;163:164-7.
Casado PL, Otazu IB, Balduino A, de Mello W, Barboza EP, Duarte ME. Identification of periodontal pathogens in healthy periimplant sites. Implant Dent 2011;20:226-35.
Schwarz F, Becker K, Rahn S, Hegewald A, Pfeffer K, Henrich B. Real-time PCR analysis of fungal organisms and bacterial species at peri-implantitis sites. Int J Implant Dent 2015;1:9.
Koyanagi T, Sakamoto M, Takeuchi Y, Ohkuma M, Izumi Y. Analysis of microbiota associated with peri-implantitis using 16S rRNA gene clone library. J Oral Microbiol 2010;2:10.
Ito T, Yasuda M, Kaneko H, Sasaki H, Kato T, Yajima Y. Clinical evaluation of salivary periodontal pathogen levels by real-time polymerase chain reaction in patients before dental implant treatment. Clin Oral Implants Res 2014;25:977-82.
Canullo L, Rossetti PH, Penarrocha D. Identification of Enterococcus faecalis
and Pseudomonas aeruginosa
on and in implants in individuals with peri-implant disease: A cross-sectional study. Int J Oral Maxillofac Implants 2015;30:583-7.
Vaz P, Gallas MM, Braga AC, Sampaio-Fernandes JC, Felino A, Tavares P. IL1 gene polymorphisms and unsuccessful dental implants. Clin Oral Implants Res 2012;23:1404-13.
Shelburne CE, Gleason RM, Germaine GR, Wolff LF, Mullally BH, Coulter WA, et al.
Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis
gene expression in vivo
. J Microbiol Methods 2002;49:147-56.
Yamada M, Nakae H, Yumoto H, Shinohara C, Ebisu S, Matsuo T. N-acetyl-D-galactosamine specific lectin of Eikenella corrodens
induces intercellular adhesion molecule-1 (ICAM-1) production by human oral epithelial cells. J Med Microbiol 2002;51:1080-9.
Liu D, Xu JK, Figliomeni L, Huang L, Pavlos NJ, Rogers M, et al.
Expression of RANKL and OPG mRNA in periodontal disease: Possible involvement in bone destruction. Int J Mol Med 2003;11:17-21.
Meenawat A, Govila V, Goel S, Verma S, Punn K, Srivastava V, et al.
Evaluation of the effect of nicotine and metabolites on the periodontal status and the mRNA expression of interleukin-1ß in smokers with chronic periodontitis. J Indian Soc Periodontol 2015;19:381-7.
Perri R, Nares S, Zhang S, Barros SP, Offenbacher S. MicroRNA modulation in obesity and periodontitis. J Dent Res 2012;91:33-8.
Ghaderi H, Kiany F, Razmkhah M, Dadras S, Chenari N, Hosseini A, et al.
mRNA expression of pattern recognition receptors and their signaling mediators in healthy and diseased gingival tissues. J Indian Soc Periodontol 2014;18:150-4.
Michalowicz BS, Aeppli D, Virag JG, Klump DG, Hinrichs JE, Segal NL, et al.
Periodontal findings in adult twins. J Periodontol 1991;62:293-9.
Hart TC, Hart PS, Bowden DW, Michalec MD, Callison SA, Walker SJ, et al.
Mutations of the cathepsin C gene are responsible for Papillon-Lefèvre syndrome. J Med Genet 1999;36:881-7.
Sakellari D, Koukoudetsos S, Arsenakis M, Konstantinidis A. Prevalence of IL-1A and IL-1B polymorphisms in a Greek population. J Clin Periodontol 2003;30:35-41.
Li QY, Zhao HS, Meng HX, Zhang L, Xu L, Chen ZB, et al.
Association analysis between interleukin-1 family polymorphisms and generalized aggressive periodontitis in a Chinese population. J Periodontol 2004;75:1627-35.
Quappe L, Jara L, López NJ. Association of interleukin-1 polymorphisms with aggressive periodontitis. J Periodontol 2004;75:1509-15.
Schröder NW, Meister D, Wolff V, Christan C, Kaner D, Haban V, et al.
Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene. Genes Immun 2005;6:448-51.
Fukusaki T, Ohara N, Hara Y, Yoshimura A, Yoshiura K. Evidence for association between a toll-like receptor 4 gene polymorphism and moderate/severe periodontitis in the Japanese population. J Periodontal Res 2007;42:541-5.
Reddy BH, Jayakumar ND, Akula SR, Sharma R, Kaarthikeyan G, Sankari. Analysis of association between TLR-4 Asp299Gly and Thr399Ile gene polymorphisms and chronic periodontitis in a sample of south Indian population. J Indian Soc Periodontol 2011;15:366-70.
Ashok N, Warad S, Kalburgi NB, Bilichodmath S, Prabhakaran PS, Tarakji B. Toll-like receptor 9 gene polymorphism in chronic and aggressive periodontitis patients. J Indian Soc Periodontol 2014;18:723-7.
Hans VM, Mehta DS. Genetic polymorphism of Fcγ-receptors IIa, IIIa and IIIb in South Indian patients with generalized aggressive periodontitis. J Oral Sci 2011;53:467-74.
Debabrata K, Prasanta B, Vineet N, Anshul G, Arindam S, Satadal D. Aggressive periodontitis: An appraisal of systemic effects on its etiology-genetic aspect. J Indian Soc Periodontol 2015;19:169-73.
Armingohar Z, Jørgensen JJ, Kristoffersen AK, Schenck K, Dembic Z. Polymorphisms in the interleukin-10 gene and chronic periodontitis in patients with atherosclerotic and aortic aneurysmal vascular diseases. J Oral Microbiol 2015;7:26051.
Shih YS, Fu E, Fu MM, Lin FG, Chiu HC, Shen EC, et al.
Association of CCL5 and CCR5 gene polymorphisms with periodontitis in Taiwanese. J Periodontol 2014;85:1596-602.
Park OJ, Shin SY, Choi Y, Kim MH, Chung CP, Ku Y, et al.
The association of osteoprotegerin gene polymorphisms with periodontitis. Oral Dis 2008;14:440-4.
Chai L, Song YQ, Leung WK. Genetic polymorphism studies in periodontitis and Fcγ receptors. J Periodontal Res 2012;47:273-85.