Real-time quantitative polymerase chain reaction (QPCR) for the identification and quantification of Streptococcus Mutans in saliva and dental biofilm in children
Introduction: the objective of this study was to use real-time qPCR to identify and quantify the Streptococcus mutans species in samples of saliva and dental biofilm. Methods: 27 children were randomly chosen with the following criteria: 8 years of age, low socio-economic levels, residing in the northern metropolitan area of Santiago de Chile; they were asked to attend an appointment while fasting with no teeth brushing for at least 12 hours, in order to collect non-stimulated saliva and a pool of supragingival dental biofilm of all the mesio-vestibular sides of anterior and posterior teeth. The amount of S. mutans in the samples was quantified by qPCR using primers that amplify a fragment of the gtfB gene of S. mutans. Results: the amplification showed 98% efficiency with a fluorescence of 3.36 cycles. The melting curve presented a single maximum at the same temperature for all samples. Conclusion: the methodology allows the specific identification and quantification of gene gtfB of S. mutans in saliva and dental biofilm in a quick and reliable manner, contributing to the identification of individual cariogenic risk.
Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007; 369(9555): 51-59
Petersen PE. The World Oral Health Report 2003: continuous improvement of oral health in the 21st century—the approach of the WHO Global Oral Health Programme. Community Dent Oral Epidemiol 2003; 31 Suppl 1: 3-23.
Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C. The global burden of oral diseases and risks to oral health. Bull World Health Organ 2005; 83(9): 661-669.
Urzua I, Mendoza C, Arteaga O, Rodríguez G, Cabello R, Faleiros S et al. Dental caries prevalence and tooth loss in Chilean adult population: first national dental examination survey. Int J Dent 2012; 2012. http://dx.doi.org/10.1155/2012/810170.
Marsh P, Martin M, Lewis M, Williams D. Oral microbiology. 5 ed. Londres: Churchill Livingstone; 2009.
Simón-Soro A, Belda-Ferre P, Cabrera-Rubio R, Alcaraz LD, Mira A. A tissue-dependent hypothesis of dental caries. Caries Res 2013; 47(6): 591-600.
Burne RA. Oral streptococci... products of their environment. J Dent Res. 1998; 77(3): 445-452.
Rupf S, Merte K, Eschrich K, Kneist S. Streptococcus sobrinus in children and its influence on caries activity. Eur Arch Paediatr Dent 2006; 7(1): 17-22.
Gordan VV, Garvan CW, Ottenga ME, Schulte R, Harris PA, McEdward D et al. Could alkali production be considered an approach for caries control? Caries Res 2010; 44(6): 547-554.
Liu Y, Dong Y, Chen YY, Burne RA. Environmental and growth phase regulation of the Streptococcus gordonii arginine deiminase genes. Appl Environ Microbiol 2008; 74(16): 5023-5030.
Dong Y, Chen YY, Burne RA. Control of expression of the arginine deiminase operon of Streptococcus gordonii by CcpA and Flp. J Bacteriol 2004; 186(8): 2511-2514.
Fejerskov O. Changing paradigms in concepts on dental caries: consequences for oral health care. Caries Res 2004; 38(3): 182-191.
Nauntofte B, Tenovuo JO, Lagerlöf F. Secretion and composition of saliva. En: Fejerskov O, Kidd E (eds). 1 ed. Dental caries: the disease and its clinical management. Oxford: Blackwell; 2003. p. 7-27
Fejerskov O. Different concepts of dental caries and their implications. 2 ed. Copenhagen: Munksgaard; 1994.
Facklam R. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev 2002; 15(4): 613-630.
Nakano K, Nomura R, Nakagawa I, Hamada S, Ooshima T. Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype, k, in the human oral cavity. J Clin Microbiol 2004; 42(1): 198-202.
Nakano K, Inaba H, Nomura R, Nemoto H, Takeda M, Yoshioka H et al. Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens. J Clin Microbiol 2006; 44(9): 3313-3317.
Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res 2011; 45(1): 69-86.
Yano A, Konno N, Imai S, Kato H. Inhibitory effects of polysaccharides on the cariogenic activities of Streptococcus mutans. Biosci Biotechnol Biochem 2012; 76(12): 2313-2316.
Napimoga MH, Höfling JF, Klein MI, Kamiya RU, Gonçalves RB. Transmission, diversity and virulence factors of Streptococcus mutans genotypes. J Oral Sci 2005; 47(2): 59-64.
Senneby A, Mejàre I, Sahlin NE, Svensäter G, Rohlin M. Diagnostic accuracy of different caries risk assessment methods. A systematic review. J Dent 2015; 43(12): 1385-93.
Karjalainen S, Tolvanen M, Pienihäkkinen K, Söderling E, Lagström H, Simell O et al. High sucrose intake at 3 years of age is associated with increased salivary counts of mutans streptococci and lactobacilli, and with increased caries rate from 3 to 16 years of age. Caries Res 2015; 49(2): 125-132.
Twetman L, Twetman S. Comparison of two chair-side tests for enumeration of Mutans Streptococci in saliva. Oral Health Dent Manag 2014; 13(3): 580-583.
Childers NK, Osgood RC, Hsu KL, Manmontri C, Momeni SS, Mahtani HK et al. Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples. Eur J Oral Sci 2011; 119(6): 447-454.
Al-Robaiy S, Rupf S, Eschrich K. Rapid competitive PCR using melting curve analysis for DNA quantification. Biotechniques 2001; 31(6): 1382 1386, 1388.
Rupf S, Merte K, Eschrich K. Quantification of bacteria in oral samples by competitive polymerase chain reaction. J Dent Res 1999; 78(4): 850-856.
Rupf S, Merte K, Kneist S, Al-Robaiy S, Eschrich K. Comparison of different techniques of quantitative PCR for determination of Streptococcus mutans counts in saliva samples. Oral Microbiol Immunol 2003; 18(1): 50-53.
Chen Z, Saxena D, Caufield PW, Ge Y, Wang M, Li Y. Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples. FEMS Microbiol Lett 2007; 272(2): 154-162.
Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K et al. The real-time polymerase chain reaction. Mol Aspects Med 2006; 27(2-3): 95-125.
Sloots TP, Nissen MD, Ginn AN, Iredell JR. Rapid identification of pathogens using molecular techniques. Pathology 2015; 47(3): 191-198.
Atieh MA. Accuracy of real-time polymerase chain reaction versus anaerobic culture in detection of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: a meta-analysis. J Periodontol 2008; 79(9): 1620-1629.
Karsai A, Müller S, Platz S, Hauser MT. Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. Biotechniques 2002; 32(4): 790-792, 794-796.
Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002; 29(1): 23-39.
Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 2002; 309(2): 293-300.
Taylor SC, Mrkusich EM. The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE). J Mol Microbiol Biotechnol 2014; 24(1): 46-52.
Yoshida A, Suzuki N, Nakano Y, Kawada M, Oho T, Koga T. Development of a 5’ nuclease-based real-time PCR assay for quantitative detection of cariogenic dental pathogens Streptococcus mutans and Streptococcus sobrinus. J Clin Microbiol 2003; 41(9): 4438-4441.
Boutaga K, van Winkelhoff AJ, Vandenbroucke-Grauls CM, Savelkoul PH. Comparison of real-time PCR and culture for detection of Porphyromonas gingivalis in subgingival plaque samples. J Clin Microbiol 2003; 41(11): 4950-4954.
Weng T, Jin N, Liu L. Differentiation between amplicon polymerization and nonspecific products in SYBR green I real-time polymerase chain reaction. Anal Biochem 2005; 342(1): 167-169.
Morgan CA, Herman N, White PA, Vesey G. Preservation of micro-organisms by drying; a review. J Microbiol Methods 2006; 66(2): 183-193.
Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19(1): 165-256.
Ono T, Hirota K, Nemoto K, Fernandez EJ, Ota F, Fukui K. Detection of Streptococcus mutans by PCR amplificaction of spaP gene. J Med Microbiol 1994; 41(4): 231-235.
Hata S, Hata H, Miyasawa-Hori H, Kudo A, Mayanagi H. Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR. Lett Appl Microbiol 2006; 42(2): 127-131.
Choi EJ, Lee SH, Kim YJ. Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries. Int J Paediatr Dent 2009; 19(2): 141-147.
Durán-Contreras GL, Torre-Martínez HH, de la Rosa EI, Hernández RM, de la Garza Ramos M. spaP gene of Streptococcus mutans in dental plaque and its relationship with early childhood caries. Eur J Paediatr Dent 2011; 12(4): 220-224.
Vásquez S, Lobos O, Padilla C. Presencia de genes de virulencia gtfB y spaP en Streptococcus mutans aislados desde saliva y su relación con el índice COPD y ceod. Rev Clin Periodoncia Implantol Rehabil Oral 2014; 7(2): 65-71.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Copyright comprises moral and patrimonial rights.
1. Moral rights: are born at the moment of the creation of the work, without the need to register it. They belong to the author in a personal and unrelinquishable manner; also, they are imprescriptible, unalienable and non negotiable. Moral rights are the right to paternity of the work, the right to integrity of the work, the right to maintain the work unedited or to publish it under a pseudonym or anonymously, the right to modify the work, the right to repent and, the right to be mentioned, in accordance with the definitions established in article 40 of Intellectual property bylaws of the Universidad (RECTORAL RESOLUTION 21231 of 2005).
2. Patrimonial rights: they consist of the capacity of financially dispose and benefit from the work trough any mean. Also, the patrimonial rights are relinquishable, attachable, prescriptive, temporary and transmissible, and they are caused with the publication or divulgation of the work. To the effect of publication of articles in the journal Revista de la Facultad de Odontología, it is understood that Universidad de Antioquia is the owner of the patrimonial rights of the contents of the publication.
The content of the publications is the exclusive responsibility of the authors. Neither the printing press, nor the editors, nor the Editorial Board will be responsible for the use of the information contained in the articles.
I, we, the author(s), and through me (us), the Entity for which I, am (are) working, hereby transfer in a total and definitive manner and without any limitation, to the Revista Facultad de Odontología Universidad de Antioquia, the patrimonial rights corresponding to the article presented for physical and digital publication. I also declare that neither this article, nor part of it has been published in another journal.
Open Access Policy
The articles published in our Journal are fully open access, as we consider that providing the public with free access to research contributes to a greater global exchange of knowledge.
Creative Commons License
The Journal offers its content to third parties without any kind of economic compensation or embargo on the articles. Articles are published under the terms of a Creative Commons license, known as Attribution – NonCommercial – Share Alike (BY-NC-SA), which permits use, distribution and reproduction in any medium, provided that the original work is properly cited and that the new productions are licensed under the same conditions.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.