Efficacy of DNA Extraction Methods Using Simple Boiling and Commercial Kit in Extracting Streptococcus Mutans in Caries
Keywords:
DNA Extraction, Dental Caries, Genetics, Streptococcus mutansAbstract
The rapid progression of molecular-based technology now enables us to analyse huge number of samples. Nonetheless, DNA (Deoxyrebonucleic acid) extraction is always a limiting factor. In this study, we analysed human microbes to compare the performance of DNA extraction methods: simple boiling method and MasterPureTM Complete DNA and RNA Purification Kit. Dental plaque was initially collected from 12 subjects, 6 of which were from individuals with caries active lesions with International Caries Detection and Assessment System (ICDAS) score five and six, and 6 samples from non-caries subjects, were collected in deionised water. The bacterial samples were extracted by the two aforementioned methods and examined using gel electrophoresis, followed by polymerase chain reaction (PCR). Streptococcus mutans was detected using conventional polymerase chain reaction (PCR). Our results demonstrated that boiling method produced higher DNA concentration (100 – 350 ng/μl), however, the commercial kit yield superior in DNA quality with single and specific PCR products. Based on the findings, the commercial kit is the better choice and practicable method for DNA extraction considering the quality of the DNA yield.
Downloads
References
Dani, S., Prabhu, A., Chaitra, K.R., Desai, N.C., Patil, S.R. and Rajeev, R., 2016. Assessment of Streptococcus mutans in healthy versus gingivitis and chronic periodontitis: A clinico-microbiological study. Contemporary clinical dentistry, 7(4), p.529.
WHO (2003). The World Oral Health Report 2003, Community dentistry and oral epidemiology, 31 Suppl 1, pp. 3-23. doi: 10.1046/j..2003.com122.x.
Razak, I. (2018). Tooth Decay: Malaysian Dental Association. [online] Mda.org.my. Available at: https://www.mda.org.my/05-03-healthcare-04.html [Accessed 14 Aug. 2019].
Daboor, S.M., Masood, F.S.S., Al‐Azab, M.S. and Nori, E.E., 2015. A review on streptococcus mutans with its diseases dental caries, dental plaque and endocarditis. Indian Journal of Microbiology Research, 2(2), pp.76-82.
Krzyściak, W., Jurczak, A., Kościelniak, D., Bystrowska, B. and Skalniak, A., 2014. The virulence of Streptococcus mutans and the ability to form biofilms. European Journal of Clinical Microbiology & Infectious Diseases, 33(4), pp.499-515.
Forssten, S.D., Björklund, M. and Ouwehand, A.C., 2010. Streptococcus mutans, caries and simulation models. Nutrients, 2(3), pp.290-298.
Hartmann, M. and Widmer, F., 2008. Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling. FEMS Microbiology Ecology, 63(2), pp.249-260.
Dashti, A.A., Jadaon, M.M., Abdulsamad, A.M. and Dashti, H.M., 2009. Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques. Kuwait Med J, 41(2), pp.117-122.
Merk, S., Meyer, H., Greiser‐Wilke, I., Sprague, L.D. and Neubauer, H., 2006. Detection of Burkholderia cepacia DNA from Artificially Infected EDTA‐blood and Lung Tissue Comparing Different DNA Isolation Methods. Journal of Veterinary Medicine, Series B, 53(6), pp.281-285.
Morono, Y., Terada, T., Hoshino, T. and Inagaki, F., 2014. Hot-alkaline DNA extraction method for deep-subseafloor archaeal communities. Appl. Environ. Microbiol., 80(6), pp.1985-1994.
Jose, J.J.M. and Brahmadathan, K.N., 2006. Evaluation of simplified DNA extraction methods for emm typing of group A streptococci. Indian journal of medical microbiology, 24(2), p.127.
Wang, X., Lim, H.J. and Son, A., 2014. Characterization of denaturation and renaturation of DNA for DNA hybridization. Environmental health and toxicology, 29.
Warner, P., Karakousis, A., Schiemann, A., Eglinton, J., Langridge, P. and Barr, A., 2001. An investigation of a rapid DNA extraction method for routine MAS in the SA Barley Improvement Program. In Proceedings of the 10th Barley Technical Symposium’. Canberra, ACT. (2001 Australian Barley Technical Symposium Inc.).
Kim, J., Kim, M., Lee, D., Baik, B., Yang, Y. and Kim, J., 2014. Rapid detection of pathogens associated with dental caries and periodontitis by PCR using a modified DNA extraction method. Journal of The Korean Academy of Pediatric Dentistry, 41(4), pp.292-297.
Dilhari, A., Sampath, A., Gunasekara, C., Fernando, N., Weerasekara, D., Sissons, C., McBain, A. and Weerasekera, M., 2017. Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method. Amb Express, 7(1), p.179.
Sergeant, M.J., Constantinidou, C., Cogan, T., Penn, C.W. and Pallen, M.J., 2012. High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature. PloS one, 7(5), p.e38094.
Natarajan, V.P., Zhang, X., Morono, Y., Inagaki, F. and Wang, F., 2016. A modified SDS-based DNA extraction method for high quality environmental DNA from seafloor environments. Frontiers in microbiology, 7, p.986.
Chen, Z., Saxena, D., Caufield, P.W., Ge, Y., Wang, M. and Li, Y., 2007. Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples. FEMS microbiology letters, 272(2), pp.154-162.
Cruaud, P., Vigneron, A., Lucchetti-Miganeh, C., Ciron, P.E., Godfroy, A. and Cambon-Bonavita, M.A., 2014. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems. Appl. Environ. Microbiol., 80(15), pp.4626-4639.
Luo, A.H., Yang, D.Q., Xin, B.C., Paster, B.J. and Qin, J., 2012. Microbial profiles in saliva from children with and without caries in mixed dentition. Oral diseases, 18(6), pp.595-601.
Islam, B., Khan, S.N. and Khan, A.U., 2007. Dental caries: from infection to prevention. Medical Science Monitor, 13(11), pp.RA196-RA203.
Listl, S., Galloway, J., Mossey, P.A. and Marcenes, W., 2015. Global economic impact of dental diseases. Journal of dental research, 94(10), pp.1355-1361.
Sudan, J., Raina, M., Singh, R., Mustafiz, A. and Kumari, S., 2017. A modified protocol for high-quality DNA extraction from seeds rich in secondary compounds. Journal of crop improvement, 31(5), pp.637-647.
Branquinho, M.R., Ferreira, R.T.B. and Cardarelli-Leite, P., 2012. Use of real-time PCR to evaluate two DNA extraction methods from food. Food Science and Technology, 32(1), pp.112-118.
Published
Issue
Section
License
Copyright (c) 2020 Malaysian Journal of Human Genetics
This work is licensed under a Creative Commons Attribution 4.0 International License.