Real-time quantitative polymerase chain reaction (QPCR) for the identification and quantification of Streptococcus Mutans in saliva and dental biofilm in children
DOI:
https://doi.org/10.17533/udea.rfo.v28n1a4Keywords:
S. muntans, qPCR, gtfB gen, Dental biofilm, SalivaAbstract
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.
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References
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.
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