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Development of Quantitative Real-Time PCR Primers for the Detection of Aggregatibacter actinomycetemcomitans  

Park, Soon-Nang (Department of Oral Biochemistry, School of Dentistry, Chosun University)
Park, Jae-Yoon (Department of Biochemistry and Molecular Biology, Medical school, Chosun University)
Kook, Joong-Ki (Department of Oral Biochemistry, School of Dentistry, Chosun University)
Publication Information
International Journal of Oral Biology / v.36, no.1, 2011 , pp. 1-6 More about this Journal
Abstract
The purpose of this study was to develop species-specific real-time quantitative PCR (RT-qPCR) primers for use in the detection of Aggregatibacter actinomycetemcomitans. These primers were designed based on the nucleotide sequences of the RNA polymerase ${\beta}$-subunit gene (rpoB). We assessed the specificity of the primers against nine strains of A. actinomycetemcomitans, eight strains (three species) of the Haemophilus genus, and 40 strains of 40 other oral bacterial species. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC $33384^T$. Our data reveal that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 2 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these qRT-PCR primers are suitable for application in epidemiological studies.
Keywords
Aggregatibacter actinomycetemcomitans; rpoB; qRT- PCR primer;
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1 Lally, E.T., I.R. Kieba, D.R. Demuth, J. Rosenbloom, E.E. Golub, N.S. Taichman, and C.W. Gibson. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem Biophys Res Commun. 1989;159:256-262.   DOI   ScienceOn
2 Mandell, R.L. A longitudinal microbiological investigation of Actinobacillus actinomycetemcomitans and Eikenella corrodens in juvenile periodontitis. Infect Immun. 1984;45:778-780.
3 Robertson, P.B., M. Lantz, P.T. Marucha, K.S. Kornman, C.L. Trummel, and S.C. Holt. Collagenolytic activity associated with Bacteroides species and Actinobacillus actinomycetemcomitans. J Periodontal Res. 1982;17:275-283.   DOI
4 Kim SG, Kim SH, Kim MK, Kim HS, Kook JK. Identification of Actinobacillus actinomycetemcomitans using species-specific 16S rDNA primers. J Microbiol 2005;43:209-212
5 Ko KS, Lee HK, Park MY, Lee KH, Yun YJ, Woo SY, Miyamoto H, Kook YH. Application of RNA polymerase beta-subunit gene (rpoB) sequences for the molecular differentiation of Legionella species. J Clin Microbiol 2002;40:2653-2658.   DOI   ScienceOn
6 Kook, J.-K., M.-K. Kim, J.-H. Seong, D.-K. Kim, B.-O. Kim, J.-C. Park, K.-K. Kim, S.-J. Choe, and B.-M. Min. A new method for rapid screening of bacterial species- or subspecies-specific DNA probes. FEMS Microbiol Lett. 2003;219:121-127.   DOI   ScienceOn
7 Kim HS, Lee DS, Chang YH, Kim MJ, Koh S, Kim J, Seong JH, Song SK, Shin HS, Son JB, Jung MY, Park SN, Yoo SY, Cho KW, Kim DK, Moon S, Kim D, Choi Y, Kim BO, Jang HS, Kim CS, Kim C, Choe SJ, Kook JK. Application of rpoB and zinc protease gene for use in molecular discrimination of Fusobacterium nucleatum subspecies. J Clin Microbiol 2010;48:545-53.   DOI   ScienceOn
8 Krieg, N. R. Identification of Procaryotes. In D. R. Boone, R. W. Castenholz, and G. M. Garrity (ed.), Bergeys manual of systematic bacteriology, 2nd ed., vol. 1, p. 33-38. Springer, New York, NY. 2001.
9 Kiley, P. and S.C. Holt. Characterization of the lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27. Infect Immun. 1980;30:862-873.
10 Kim, B. J., S. H. Lee, M. A. Lyu, S. J. Kim, G. H. Bai, G. T. Chae, E. C. Kim, C. Y. Cha, and Y. H. Kook. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol. 1999;37:1714-1720.
11 Slots, J., A. Ashimoto, M.J. Flynn, G. Li, and C. Chen. Detection of putative periodontal pathogens in subgingival specimens by 16S ribosomal DNA amplification with the polymerase chain reaction. Clin Infect Dis. 1995;20(Suppl 2):S304-307.   DOI
12 Hyvarinen K, Laitinen S, Paju S, Hakala A, Suominen-Taipale L, Skurnik M, Kononen E, Pussinen PJ. Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR. Innate Immun. 2009;15:195-204.   DOI   ScienceOn
13 Khamis, A., D. Raoult, B. La Scola. rpoB gene sequencing for identification of Corynebacterium species. J Clin Microbiol. 2004;42:3925-3931.   DOI   ScienceOn
14 Genco, R.J., J.J. Zambon, and P.A. Murray. Serum and gingival fluid antibodies as adjuncts in the diagnosis of Actinobacillus actinomycetemcomitans-associated periodontal disease. J Periodontol. 1985:56;1-50.
15 Woese, C.R. Bacterial evolution. Microbiol Rev. 1987;51:221-271.
16 Drancourt, M. and D. Raoult. rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol. 2002;40:4333-4338.
17 Ashkenazi, M., R.R. White, and D.K. Dennison. Neutrophil modulation by Actinobacillus actinomycetemcomitans. I. Chemotaxis, surface receptor expression and F-actin polymerization. J Periodontal Res. 1992;27:264-273.   DOI
18 Asikainen, S. Occurrence of Actinobacillus actinomycetemcomitans and spirochetes in relation to age in localized juvenile periodontitis. J Periodontol. 1986;57:537-541.   DOI
19 Zambon, J.J. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol. 1985;12:1-20.   DOI
20 Conrads, G., T.F. Flemmig, I. Seyfarth, F. Lampert, and R. Lutticken. Simultaneous detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA gene-directed multiplex PCR. J Clin Microbiol. 1999;37:1621-1624.
21 Sugai, M., T. Kawamoto, S.Y. Peres, Y. Ueno, H. Komatsuzawa, T. Fujiwara, H. Kurihara, H. Suginaka, and E. Oswald. The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin. Infect Immun. 1998;66: 5008-5019.
22 Tran, S.D. and J.D. Rudney. Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis. J Clin Microbiol. 1999;37:3504-3508.
23 van Winkelhoff, A.J. and J. Slots. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in nonoral infections. Periodontol 2000. 1999;20:122-135.   DOI   ScienceOn
24 Shin HS, Kim MJ, Kim HS, Park SN, Kim do K, Baek DH, Kim C, Kook JK. Development of strain-specific PCR primers for the identification of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T and subsp. vincentii ATCC 49256T. Anaerobe 2010;16:43-46.   DOI   ScienceOn
25 Ashimoto, A., C. Chen, I. Bakker and J. Slots. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol. 1996;11: 266-273.   DOI   ScienceOn
26 Severinov K, Mustaev A, Kukarin A, Muzzin O, Bass I, Darst SA, Goldfarb A. Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase. J. Biol. Chem. 1996;271:27969-7974.   DOI   ScienceOn