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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇剛毅(Kang-Yi Su) | |
| dc.contributor.author | Hsiao-Yi Hung | en |
| dc.contributor.author | 洪孝儀 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:43:56Z | - |
| dc.date.available | 2015-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-20 | |
| dc.identifier.citation | 1. Salton, M.R.J. and K.S. Kim, Structure, in Medical Microbiology, S. Baron,
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Schiller, Resistance mechanisms in an in vitro-selected amoxicillin-resistant strain of Helicobacter pylori. Antimicrob Agents Chemother, 2006. 50(12): p. 4174-6. 38. Rimbara, E., et al., Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori. Microbiol Immunol, 2007. 51(10): p. 939-44. 39. Rimbara, E., et al., Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori. J Antimicrob Chemother, 2008. 61(5): p. 995-8. 40. Paul, R., et al., Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin. Antimicrob Agents Chemother, 2001. 45(3): p. 962-5. 41. Kim, J.J., et al., Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J Antimicrob Chemother, 2001. 47(4): p. 459-61. 42. Brodersen, D.E., et al., The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell, 2000. 103(7): p. 1143-54. 43. Chopra, I. and M. Roberts, Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev, 2001. 65(2): p. 232-60 ; second page, table of contents. 44. Gerrits, M.M., et al., 16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori. Antimicrob Agents Chemother, 2002. 46(9): p. 2996-3000. 45. Trieber, C.A. and D.E. Taylor, Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline. J Bacteriol, 2002. 184(8): p. 2131-40. 46. Gerrits, M.M., et al., Effects of 16S rRNA gene mutations on tetracycline resistance in Helicobacter pylori. Antimicrob Agents Chemother, 2003. 47(9): p. 2984-6. 47. Cattoir, V., et al., Update on fluoroquinolone resistance in Helicobacter pylori: new mutations leading to resistance and first description of a gyrA polymorphism associated with hypersusceptibility. Int J Antimicrob Agents, 2007. 29(4): p. 389-96. 48. Glupczynski, Y., et al., European multicentre survey of in vitro antimicrobial resistance in Helicobacter pylori. Eur J Clin Microbiol Infect Dis, 2001. 20(11): p. 820-3. 49. Glocker, E. and M. Kist, Rapid detection of point mutations in the gyrA gene of Helicobacter pylori conferring resistance to ciprofloxacin by a fluorescence resonance energy transfer-based real-time PCR approach. J Clin Microbiol, 2004. 42(5): p. 2241-6. 50. Fujimura, S., et al., In vitro activity of fluoroquinolone and the gyrA gene mutation in Helicobacter pylori strains isolated from children. J Med Microbiol, 2004. 53(Pt 10): p. 1019-22. 51. Bogaerts, P., et al., Prevalence and mechanisms of resistance to fluoroquinolones in Helicobacter pylori strains from patients living in Belgium.Helicobacter, 2006. 11(5): p. 441-5. 52. Infection with Helicobacter pylori. IARC Monogr Eval Carcinog Risks Hum, 1994. 61: p. 177-240. 53. Bessede, E., et al., Helicobacter pylori generates cells with cancer stem cell properties via epithelial-mesenchymal transition-like changes. Oncogene, 2014. 33(32): p. 4123-31. 54. Whittal, R.M. and L. Li, High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal Chem, 1995. 67(13): p. 1950-4. 55. Braun, A., D.P. Little, and H. Koster, Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry. Clin Chem, 1997. 43(7): p. 1151-8. 56. Haff, L.A. and I.P. Smirnov, Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res, 1997. 7(4): p. 378-88. 57. Taranenko, N.I., et al., Laser desorption mass spectrometry for point mutation detection. Genet Anal, 1996. 13(4): p. 87-94. 58. Fu, D.J., et al., Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry. Nat Biotechnol, 1998. 16(4): p. 381-4. 59. Hurst, G.B., et al., Detection of bacterial DNA polymerase chain reaction products by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom, 1996. 10(3): p. 377-82. 60. Jurinke, C., et al., Detection of hepatitis B virus DNA in serum samples via nested PCR and MALDI-TOF mass spectrometry. Genet Anal, 1996. 13(3): p. 67-71. 61. Borromeo, M., J.R. Lambert, and K.J. Pinkard, Evaluation of 'CLO-test' todetect Campylobacter pyloridis in gastric mucosa. J Clin Pathol, 1987. 40(4): p. 462-3. 62. Das, S.S., et al., Rapid diagnosis of Campylobacter pyloridis infection. J Clin Pathol, 1987. 40(6): p. 701-2. 63. Clayton, C.L., et al., Sensitive detection of Helicobacter pylori by using polymerase chain reaction. J Clin Microbiol, 1992. 30(1): p. 192-200. 64. Espinoza, M.G., et al., Detection of the glmM gene in Helicobacter pylori isolates with a novel primer by PCR. J Clin Microbiol, 2011. 49(4): p. 1650-2. 65. Liou, J.M., et al., The Primary Resistance of Helicobacter pylori in Taiwan after the National Policy to Restrict Antibiotic Consumption and Its Relation to Virulence Factors-A Nationwide Study. PLoS One, 2015. 10(5): p. e0124199. 66. Burucoa, C., et al., T2182C mutation is not associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother, 2005. 49(2): p. 868; author reply 868-70. 67. Khan, R., et al., T2182C mutation in 23S rRNA is associated with clarithromycin resistance in Helicobacter pylori isolates obtained in Bangladesh. Antimicrob Agents Chemother, 2004. 48(9): p. 3567-9. 68. Agudo, S., et al., High prevalence of clarithromycin-resistant Helicobacter pylori strains and risk factors associated with resistance in Madrid, Spain. J Clin Microbiol, 2010. 48(10): p. 3703-7. 69. Tanih, N.F. and R.N. Ndip, Molecular Detection of Antibiotic Resistance in South African Isolates of Helicobacter pylori. Gastroenterol Res Pract, 2013. 2013: p. 259457. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54190 | - |
| dc.description.abstract | 胃癌目前是全球排名第四常見的癌症，亦是與癌症死亡率高的癌症。世界衛生組織也證實了幽門螺旋桿菌與胃癌的相關性，因此降低感染率或能有效根除此菌，便能降低胃癌或胃潰瘍的發生率。幽門螺旋桿菌屬於螺旋微嗜氧菌，為一革蘭氏陰性菌，具鞭毛，會導致胃潰瘍、十二指腸潰瘍及胃炎，嚴重甚至會導致胃上皮細胞癌。目前在治療幽門螺旋桿菌的方法選擇，包括抗生素療法和質子幫浦抑制劑（proton pump inhibitor, PPI）療法。儘管一開始對於無論良性或惡性的幽門螺旋桿菌相關之胃十二指腸疾病，抗生素確實有其作用，但近幾年對抗生素產生抗藥性的比率在全世界都日益升高。所以本篇論文主要目的是要建立一個具高度敏感性的檢測平台，利用核酸質譜儀（MALDI-TOF MS）偵測幽門螺旋桿菌的感染與否，以及預測其藥物耐受性。
有關其抗藥基因包括23S rRNA, 16S rRNA, PBP-1, rdxA, gyrA以及frxA六個基因，這些基因都將會設計成為偵測平台探針。其中23S rRNA和gyrA這兩個基因突變位點較明確，先將這兩個基因所知突變位點建立於核酸質譜儀的偵測平台；其他四個基因包括PBP-1, rdxA, gyrA以及frxA則因為其突變位點仍在研究，尚未明確，所以先將由臨床檢體所分離出的菌株利用Sanger method做定序，欲找出和抗藥基因相關突變頻率較高的突變位點，再陸續將突變熱點建立於核酸質譜儀的偵測平台。截至目前，我們建立了一個可以偵測兩種基因（23S rRNA和gyrA）15個突變位點的偵測平台，剩下的四種基因將來也會一併加入偵測平台。針對核酸質譜儀的偵測平台，其檢測極值根據序列稀釋的結果看可達1copy。在未來我們將致力於1. 將偵測敏感度以及專一性最佳化 2. 找出16S rRNA, PBP-1, rdxA和frxA這四個基因的突變熱點 3.將此偵測平台應用於臨床，檢驗來自患有胃潰瘍或胃癌病患之檢體。待此偵測平台建立完成，利用微量檢體偵測幽門螺旋桿菌的存在，以及預測其藥物耐受性，以非侵入性的檢驗指日可待。 | zh_TW |
| dc.description.abstract | Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death in the world. Among all risk factor, the infection of H. pylori is presented in approximately 63% of patients. H. Pylori is a spiral-shaped, microaerophilic gram-negative flagellate bacterium contributes to duodenal/gastric ulcer, gastritis and gastric adenocarcinoma. Eradication therapy of H. pylori including antibiotics, PPI (Proton Pump Inhibitor) has emerged as the treatment of choice. Although antibiotics have been useful in the treatment of H. pylori-related benign and malignant gastroduodenal disease, the rate of resistance to standard therapies has increased a result of the widespread use of antibiotics in recent year. Our specific aim is to establish a multiplexed high sensitive nucleotide MALDI-TOF MS to detect H. pylori infection and predict its world. Among all risk factor, the infection of H. pylori 23S rRNA, 16S rRNA, PBP-1, rdxA, gyrA and frxA genes were selected for multiplex panel design. 23S rRNA and gyrA that drug resistant related mutation sites were well known was pioneer for MALDI-TOF MS establishment. Other four genes were sequenced by Sanger method in clinical isolates for identifying high prevalence mutation sites correlated drug resistance for MALDI-TOF MS platform in the future. Up to date, we had established two reaction multiplex gene testing panel for 15 mutations within 2 genes.The following mutations important for testing will be recruited in the panel in the future. The detection limitation of our platform is around 1 copy according to serial dilution of H. pylori genome DNA. Future direction will focus on: 1. Optimization of detection sensitivity and specificity; 2. Identification of drug resistant mutation hot spot among 16S rRNA, PBP-1, rdxAand frxA genes; 3. Feasibility of H. pylori detection in clinical specimens such as biopsy from ulcer lesion or gastric cancer patients. With this platform set up, we can develop non-invasive detection strategy of H. pylori and predict its resistance in clinical specimens. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:43:56Z (GMT). No. of bitstreams: 1 ntu-104-R02424021-1.pdf: 7619928 bytes, checksum: 749516f1dbda93f4b37a0eac4c3cb8e6 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 英文摘要
中文摘要 壹、緒論 1.革蘭氏陰性菌p.1 2.幽門螺旋桿菌 2.1.簡介.p.1 2.2.流行病學p.2 2.3.致病機轉p.3 2.4.診斷p.4 2.5.預防及治療p.5 2.6.抗藥性p.7 3.胃潰瘍p.9 4.胃癌p.10 5.核酸質譜儀p.11 貳、研究動機 p.12 參、材料方法 a.實驗材料 p.13 b.實驗方法p.14 1.幽門螺旋桿菌菌株p.14 2.聚合酶鏈連鎖反應p.15 3.聚核酶鏈連鎖反應引子p.15 4.電泳p.16 5.DNA 定序p.16 6. 序列稀釋p.16 7. 核酸質譜儀p.16 肆、結果 1.引子測試p.20 2.基因定序p.20 3.核酸質譜儀準確性測試p.21 4.核酸質譜儀敏感度測試p.23 伍、討論 p.24 陸、圖表p.30 柒、參考資料p.67 表目錄 表ㄧ、臨床檢體列表p.30 表二、基因定序實驗所用引子p.33 表三、質譜儀實驗所用引子p.34 表四、質譜儀所用探針p.34 表五、質譜圖判讀表p.35 表六、所有檢體偵測結果p.36 表七、對levofloxacin 具抗藥性之臨床菌株與基因型之統計表p.40 表八、對clarithromycin 具抗藥性之臨床菌株與基因型之統計表p.40 表九、對levofloxacin 具抗藥性之胃組織黏膜切片菌株與基因型之 統計表p.40 表十、對clarithromycin 具抗藥性之胃組織黏膜切片菌株與基因型 之統計表p.40 圖目錄 圖ㄧ、實驗流程簡圖p.41 圖二、引子設計示意圖p.42 圖三、核酸質譜儀實驗中，探針及引子設計序列位置圖p.43 圖四、各引子增幅之產物p.44 圖五、基因定序結果比較圖p.45 圖六、標準菌株於核酸質譜儀第一次測試質譜圖p.46 圖七、核酸質譜儀偵測各基因突變位點探針位置示意圖p.47 圖八、核酸質譜儀偵測各基因突變位點探針位置示意圖p.48 圖九、質譜圖判讀p.49 圖十、標準菌株於核酸質譜儀第一個反應槽測試質譜圖p.50 圖十一、標準菌株於核酸質譜儀第二個反應槽測試質譜圖p.51 圖十二、臨床菌株檢體於核酸質譜儀檢測結果之質譜圖p.52 圖十三、臨床菌株檢體與胃黏膜組織切片檢體於核酸質譜儀檢測 之結果圖p.56 圖十四、臨床菌株檢體、胃黏膜組織切片檢體與糞便檢體於核酸 質譜儀檢測之結果圖p.62 圖十五、序列稀釋THHP3706 於核酸質譜儀的檢測結果p.66 | |
| dc.language.iso | zh-TW | |
| dc.title | 利用核酸質譜儀建立幽門螺旋桿菌抗藥突變基因高敏感度偵測平台 | zh_TW |
| dc.title | Development of Highly Sensitive Drug Resistant Mutation Gene Detection in Helicobacter pylori by MALDI-TOF Mass Spectrometry | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉志銘(Chi-Ming Liu),楊雅倩(Ya-Chien Yang),林亮音(Liang-Yin Lin) | |
| dc.subject.keyword | 幽門螺旋桿菌,核酸質譜儀,抗藥基因, | zh_TW |
| dc.subject.keyword | Helicobacter pylori,MALDI-TOF Mass Spectrometry,drug resistant gene, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-21 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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