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標題: | 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 Development of Highly Sensitive Drug Resistant Mutation Gene Detection in Helicobacter pylori by MALDI-TOF Mass Spectrometry |
作者: | Hsiao-Yi Hung 洪孝儀 |
指導教授: | 蘇剛毅(Kang-Yi Su) |
關鍵字: | 幽門螺旋桿菌,核酸質譜儀,抗藥基因, Helicobacter pylori,MALDI-TOF Mass Spectrometry,drug resistant gene, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54190 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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