牙周病是由宿主與口腔中的細菌交互作用所導致的疾病，會造成牙齒與周遭組織的附連喪失和齒槽骨的缺失，嚴重影響牙齒正常的功能。目前的研究已經指出人類牙周病與一些重要的牙周致病菌有關，其中又以Aggregatibacter actinomycetemcomitans (Aa)和Porphyromonas gingivalis (Pg) 最廣為研究。Aa和侵犯型牙周炎最為相關，而Pg則是慢性成人牙周炎發展過程中最主要的致病菌。目前有許多用來作為牙周病診斷的方法，像是牙周探針檢測法、全口X光和細菌培養等等，但是這些方法還是存在一些缺點，例如牙周探針可能會造成患者的不適，而全口X光無法做到早期診斷，因此我們想要藉由篩選出能夠與牙周病菌結合的適體，應用於牙周病診斷上。在本研究中，我們已經藉由cell-SELEX篩選出能分別和Pg以及Aa結合的適體，並且測試適體對於辨認菌的特性，利用傳統PCR和即時定量PCR觀察到適體對於目標菌株具有高度專一性和親和力。緊接著我們也將Pg適體應用於臨床檢體做測試，顯示在患者的檢體中Pg的含量比健康人高。這個結果顯示Pg適體可以用於偵測牙周致病菌，但要再藉由更多的檢體試驗來證明。我們將在未來的實驗中檢測Aa適體在臨床檢體上的效果。期望本研究成果在未來能夠有效改善牙周病診斷的方法。

In this study, we used two major periodontal bacteria, Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg), to select aptamer from random library by cell-SELEX and amplified the aptamer products by PCR. We obtained an Aa-binding aptamer (Aa-Ap 1) and a Pg-binding aptamer (Pg-AP). Then, we determined the binding specificity of Aa-AP1 and Pg-Ap and found that Aa-AP1 has better binding specificity for Aa. Furthermore, we analyzed the binding affinity of Aa-AP1 and Pg-Ap by real-time quantitative PCR. Both aptamers had high binding affinity to target periodontal bacteria. With the present experimental results, it will be important to evaluate the feasibility of periodontal disease diagnosis by Aa-AP1 and Pg-Ap in the future.

1 Hajishengallis, G. Periodontitis: from microbial immune subversion to systemic inflammation. Nature reviews. Immunology 15, 30-44, doi:10.1038/nri3785 (2015).
2 Vos, T. et al. Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the global burden of disease study 2010. Lancet 380, 2163-2196, doi:10.1016/S0140-6736(12)61729-2 (2012).
3 Eke, P. I. et al. Update on prevalence of periodontitis in adults in the United States: NHANES 2009 to 2012. Journal of Periodontology 86, 611-622, doi:10.1902/jop.2015.140520 (2015).
4 Lai, H. et al. A prediction model for periodontal disease: modelling and validation from a National Survey of 4061 Taiwanese adults. Journal of Clinical Periodontology 42, 413-421, doi:10.1111/jcpe.12389 (2015).
5 Wiebe, C. B. & Putnins, E. E. The periodontal disease classification system of the American Academy of Periodontology--an update. Journal of the Canadian Dental Association 66, 594-597 (2000).
6 Armitage, G. C. Development of a classification system for periodontal diseases and conditions. Annals of Periodontology 4, 1-6, doi:10.1902/annals.1999.4.1.1 (1999).
7 Tonetti, M. S. & Mombelli, A. Early-onset periodontitis. Annals of Periodontology 4, 39-53, doi:10.1902/annals.1999.4.1.39 (1999).
8 Grover, V., Kapoor, A., Malhotra, R. & Kaur, G. Clinical relevance of the advanced microbiologic and biochemical investigations in periodontal diagnosis: a critical analysis. Journal of Oral Diseases 2014, 11, doi:10.1155/2014/785615 (2014).
9 American Academy of Periodontology task force report on the update to the 1999 classification of periodontal diseases and conditions. Journal of Periodontology 86, 835-838, doi:10.1902/jop.2015.157001 (2015).
10 Tugnait, A., Clerehugh, V. & Hirschmann, P. N. The usefulness of radiographs in diagnosis and management of periodontal diseases: a review. Journal of Dentistry 28, 219-226 (2000).
11 Armitage, G. C. The complete periodontal examination. Periodontology 2000 34, 22-33 (2004).
12 AlJehani, Y. A. Risk factors of periodontal disease: review of the literature. International Journal of Dentistry 2014, 182513, doi:10.1155/2014/182513 (2014).
13 Kubota, M., Tanno-Nakanishi, M., Yamada, S., Okuda, K. & Ishihara, K. Effect of smoking on subgingival microflora of patients with periodontitis in Japan. BMC Oral Health 11, 1, doi:10.1186/1472-6831-11-1 (2011).
14 Zini, A., Sgan-Cohen, H. D. & Marcenes, W. Socio-economic position, smoking, and plaque: a pathway to severe chronic periodontitis. Journal of Clinical Periodontology 38, 229-235, doi:10.1111/j.1600-051X.2010.01689.x (2011).
15 Saremi, A. et al. Periodontal disease and mortality in type 2 diabetes. Diabetes Care 28, 27-32 (2005).
16 Bartova, J. et al. Periodontitis as a risk factor of atherosclerosis. Journal of Immunology Research 2014, 636893, doi:10.1155/2014/636893 (2014).
17 Kim, J. & Amar, S. Periodontal disease and systemic conditions: a bidirectional relationship. Odontology 94, 10-21, doi:10.1007/s10266-006-0060-6 (2006).
18 Darveau, R. P. Periodontitis: a polymicrobial disruption of host homeostasis. Nature Reviews Microbiology 8, 481-490, doi:10.1038/nrmicro2337 (2010).
19 Paster, B. J., Olsen, I., Aas, J. A. & Dewhirst, F. E. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontology 2000 42, 80-87, doi:10.1111/j.1600-0757.2006.00174.x (2006).
20 Berezow, A. B. & Darveau, R. P. Microbial shift and periodontitis. Periodontology 2000 55, 36-47, doi:10.1111/j.1600-0757.2010.00350.x (2011).
21 Dogan, B., Kipalev, A. S., Okte, E., Sultan, N. & Asikainen, S. E. Consistent intrafamilial transmission of Actinobacillus actinomycetemcomitans despite clonal diversity. Journal of Periodontology 79, 307-315, doi:10.1902/jop.2008.070270 (2008).
22 Kou, Y. et al. Inflammatory responses of gingival epithelial cells stimulated with Porphyromonas gingivalis vesicles are inhibited by hop-associated polyphenols. Journal of Periodontology 79, 174-180, doi:10.1902/jop.2008.070364 (2008).
23 Chahboun, H., Arnau, M. M., Herrera, D., Sanz, M. & Ennibi, O. K. Bacterial profile of aggressive periodontitis in Morocco: a cross-sectional study. BMC Oral Health 15, 25, doi:10.1186/s12903-015-0006-x (2015).
24 Rylev, M., Bek-Thomsen, M., Reinholdt, J., Ennibi, O. K. & Kilian, M. Microbiological and immunological characteristics of young Moroccan patients with aggressive periodontitis with and without detectable Aggregatibacter actinomycetemcomitans JP2 infection. Molecular Oral Microbiology 26, 35-51, doi:10.1111/j.2041-1014.2010.00593.x (2011).
25 Aberg, C. H., Kelk, P. & Johansson, A. Aggregatibacter actinomycetemcomitans: virulence of its leukotoxin and association with aggressive periodontitis. Virulence 6, 188-195, doi:10.4161/21505594.2014.982428 (2015).
26 Kachlany, S. C. Aggregatibacter actinomycetemcomitans leukotoxin: from threat to therapy. Journal of Dental Research 89, 561-570, doi:10.1177/0022034510363682 (2010).
27 Castillo-Ruiz, M. et al. Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm. Applied and Environmental Microbiology 77, 3157-3159, doi:10.1128/AEM.02115-10 (2011).
28 Brigido, J. A., da Silveira, V. R., Rego, R. O. & Nogueira, N. A. Serotypes of Aggregatibacter actinomycetemcomitans in relation to periodontal status and geographic origin of individuals-a review of the literature. Medicina Oral Patologia Oral y Cirugia Bucal 19, e184-191 (2014).
29 Kawamoto, D. et al. Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates. Oral Microbiology and Immunology 24, 493-501, doi:10.1111/j.1399-302X.2009.00547.x (2009).
30 Kim, T. S., Frank, P., Eickholz, P., Eick, S. & Kim, C. K. Serotypes of Aggregatibacter actinomycetemcomitans in patients with different ethnic backgrounds. Journal of Periodontology 80, 2020-2027, doi:10.1902/jop.2009.090241 (2009).
31 Mysak, J. et al. Porphyromonas gingivalis: major periodontopathic pathogen overview. Journal of Immunology Research 2014, 476068, doi:10.1155/2014/476068 (2014).
32 Al Batran, R., Al-Bayaty, F. H. & Al-Obaidi, M. M. In-vivo effect of andrographolide on alveolar bone resorption induced by Porphyromonas gingivalis and its relation with antioxidant enzymes. BioMed Research International 2013, 276329, doi:10.1155/2013/276329 (2013).
33 Hajishengallis, G. Immune evasion strategies of Porphyromonas gingivalis. Journal of Oral Biosciences 53, 233-240, doi:10.2330/joralbiosci.53.233 (2011).
34 Bodet, C., Chandad, F. & Grenier, D. [Pathogenic potential of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, the red bacterial complex associated with periodontitis]. Pathologie Biologie (Paris) 55, 154-162, doi:10.1016/j.patbio.2006.07.045 (2007).
35 Holt, S. C. & Ebersole, J. L. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the "red complex", a prototype polybacterial pathogenic consortium in periodontitis. Periodontology 2000 38, 72-122, doi:10.1111/j.1600-0757.2005.00113.x (2005).
36 Laine, M. L. & van Winkelhoff, A. J. Virulence of six capsular serotypes of Porphyromonas gingivalis in a mouse model. Oral Microbiology and Immunology 13, 322-325 (1998).
37 Laine, M. L., Appelmelk, B. J. & van Winkelhoff, A. J. Novel polysaccharide capsular serotypes in Porphyromonas gingivalis. Journal of Periodontal Research 31, 278-284 (1996).
38 Brunner, J. et al. The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis. BMC Microbiology 10, 252, doi:10.1186/1471-2180-10-252 (2010).
39 Olsen, I. & Yilmaz, O. Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases. Journal of oral microbiology 8, 30385, doi:10.3402/jom.v8.30385 (2016).
40 Yamaguchi, Y., Kurita-Ochiai, T., Kobayashi, R., Suzuki, T. & Ando, T. Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated atherosclerosis. Pathogens and Disease 73, doi:10.1093/femspd/ftv011 (2015).
41 Olsen, I. & Singhrao, S. K. Can oral infection be a risk factor for Alzheimer's disease? Journal of oral microbiology 7, 29143, doi:10.3402/jom.v7.29143 (2015).
42 Furusho, H. et al. Dental infection of Porphyromonas gingivalis exacerbates high fat diet-induced steatohepatitis in mice. Journal of Gastroenterology 48, 1259-1270, doi:10.1007/s00535-012-0738-1 (2013).
43 Seror, R. et al. Association of anti-porphyromonas gingivalis antibody titers with nonsmoking status in early rheumatoid arthritis: results from the prospective French cohort of patients with early rheumatoid arthritis. Arthritis & Rheumatology 67, 1729-1737, doi:10.1002/art.39118 (2015).
44 Mikuls, T. R. et al. Periodontitis and Porphyromonas gingivalis in patients with rheumatoid arthritis. Arthritis & Rheumatology 66, 1090-1100, doi:10.1002/art.38348 (2014).
45 Katz, J., Onate, M. D., Pauley, K. M., Bhattacharyya, I. & Cha, S. Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma. International Journal of Oral Science 3, 209-215, doi:10.4248/IJOS11075 (2011).
46 Anil, S. & Baiju, F. M. Current research in diagnostic methods for assessing periodontal disease. Indian Journal of Dental Research 9, 120-123 (1998).
47 Paster, B. J. & Dewhirst, F. E. Molecular microbial diagnosis. Periodontology 2000 51, 38-44, doi:10.1111/j.1600-0757.2009.00316.x (2009).
48 IviÊ-Kardum, M., Beader, N. & Štaudt-Škaljac, G. Diagnostic methods for evaluation of microbial flora in periodontitis. Acta stomatologica Croatica 35, 137-140 (2001).
49 Liu, J., Cao, Z. & Lu, Y. Functional nucleic acid sensors. Chemical Reviews 109, 1948-1998, doi:10.1021/cr030183i (2009).
50 Tan, W., Donovan, M. J. & Jiang, J. Aptamers from cell-based selection for bioanalytical applications. Chemical Reviews 113, 2842-2862, doi:10.1021/cr300468w (2013).
51 Santosh, B. & Yadava, P. K. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics. BioMed Research International 2014, 540451, doi:10.1155/2014/540451 (2014).
52 Hamaguchi, N., Ellington, A. & Stanton, M. Aptamer beacons for the direct detection of proteins. Analytical Biochemistry 294, 126-131, doi:10.1006/abio.2001.5169 (2001).
53 Germer, K., Leonard, M. & Zhang, X. RNA aptamers and their therapeutic and diagnostic applications. International Journal of Biochemistry and Molecular Biology 4, 27-40 (2013).
54 Hamula, C. L., Zhang, H., Guan, L. L., Li, X. F. & Le, X. C. Selection of aptamers against live bacterial cells. Analytical Chemistry 80, 7812-7819, doi:10.1021/ac801272s (2008).
55 Zimbres, F. M., Tarnok, A., Ulrich, H. & Wrenger, C. Aptamers: novel molecules as diagnostic markers in bacterial and viral infections? BioMed Research International 2013, 731516, doi:10.1155/2013/731516 (2013).
56 Song, K.-M., Lee, S. & Ban, C. Aptamers and their biological applications. Sensors (Basel, Switzerland) 12, 612-631, doi:10.3390/s120100612 (2012).
57 Stoltenburg, R., Reinemann, C. & Strehlitz, B. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular Engineering 24, 381-403, doi:10.1016/j.bioeng.2007.06.001 (2007).
58 Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822 (1990).
59 Lee, J. H., Kim, H., Ko, J. & Lee, Y. Interaction of C5 protein with RNA aptamers selected by SELEX. Nucleic Acids Research 30, 5360-5368 (2002).
60 Ohuchi, S. Cell-SELEX Technology. BioResearch Open Access 1, 265-272, doi:10.1089/biores.2012.0253 (2012).
61 Cao, X. et al. Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus. Nucleic Acids Research 37, 4621-4628, doi:10.1093/nar/gkp489 (2009).
62 Yang, M. et al. Highly specific and cost-efficient detection of Salmonella paratyphi a combining aptamers with single-walled carbon nanotubes. Sensors (Basel, Switzerland) 13, 6865-6881, doi:10.3390/s130506865 (2013).
63 Duan, N. et al. Selection and characterization of aptamers against Salmonella typhimurium using whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX). Journal of Agricultural and Food Chemistry 61, 3229-3234, doi:10.1021/jf400767d (2013).
64 Chen, F., Hu, Y., Li, D., Chen, H. & Zhang, X.-L. CS-SELEX generates high-affinity ssDNA aptamers as molecular probes for hepatitis C virus envelope glycoprotein E2. PLoS ONE 4, e8142, doi:10.1371/journal.pone.0008142 (2009).
65 Hung, L.-Y., Wang, C.-H., Hsu, K.-F., Chou, C.-Y. & Lee, G.-B. An on-chip Cell-SELEX process for automatic selection of high-affinity aptamers specific to different histologically classified ovarian cancer cells. Lab on a Chip 14, 4017-4028, doi:10.1039/C4LC00587B (2014).
66 Zhang, C. et al. Whole-cell based aptamer selection for selective capture of microorganisms using microfluidic devices. Analytical Methods 7, 6339-6345, doi:10.1039/C5AY01016K (2015).
67 Keefe, A. D., Pai, S. & Ellington, A. Aptamers as therapeutics. Nature Reviews Drug Discovery 9, 537-550 (2010).
68 Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564-566 (1992).
69 Lebruska, L. L. & Maher, L. J., 3rd. Selection and characterization of an RNA decoy for transcription factor NF-kappa B. Biochemistry 38, 3168-3174, doi:10.1021/bi982515x (1999).
70 Ng, E. W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. ature Reviews Drug Discovery 5, 123-132, doi:10.1038/nrd1955 (2006).
71 Group, V. I. S. i. O. N. C. T. et al. Year 2 efficacy results of 2 randomized controlled clinical trials of pegaptanib for neovascular age-related macular degeneration. Ophthalmology 113, 1508 e1501-1525, doi:10.1016/j.ophtha.2006.02.064 (2006).
72 Kuo, T.-C., Tsai, C.-W., Lee, P.-C. & Chen, W.-Y. Revisiting the streptavidin–biotin binding by using an aptamer and displacement isothermal calorimetry titration. Journal of Molecular Recognition 28, 125-128, doi:10.1002/jmr.2366 (2015).
73 Wang, H.-Q., Wu, Z., Tang, L.-J., Yu, R.-Q. & Jiang, J.-H. Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection. Nucleic Acids Research 39, e122, doi:10.1093/nar/gkr559 (2011).
74 Highfield, J. Diagnosis and classification of periodontal disease. Australian Dental Journal 54, S11-S26, doi:10.1111/j.1834-7819.2009.01140.x (2009).
75 Park, J. P. et al. Screening and development of DNA aptamers specific to several oral pathogens. Journal of Microbiology and Biotechnology 25, 393-398 (2015).
76 de Josselin de Jong, E., Higham, S. M., Smith, P. W., van Daelen, C. J. & van der Veen, M. H. Quantified light-induced fluorescence, review of a diagnostic tool in prevention of oral disease. Journal of Applied Physics 105, 102031, doi:doi:http://dx.doi.org/10.1063/1.3116138 (2009).
77 Amaechi, B. T. & Higham, S. M. Quantitative light-induced fluorescence: a potential tool for general dental assessment. Journal of Biomedical Optics 7, 7-13, doi:10.1117/1.1427044 (2002).
78 van der Veen, M. H., Thomas, R. Z., Huysmans, M. C. & de Soet, J. J. Red autofluorescence of dental plaque bacteria. Caries Research 40, 542-545, doi:10.1159/000095655 (2006).
79 Koenig, K., Hibst, R., Meyer, H., Flemming, G. & Schneckenburger, H. Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria. SPIE 170-180, doi:10.1117/12.166180 (1993).
80 Han, S.-Y., Kim, B.-R., Ko, H.-Y., Kwon, H.-K. & Kim, B.-I. Assessing the use of quantitative light-induced fluorescence-digital as a clinical plaque assessment. Photodiagnosis and Photodynamic Therapy 13, 34-39, doi:http://dx.doi.org/10.1016/j.pdpdt.2015.12.002 (2016).