整合型微流體系統應用於胃幽門螺旋菌喹諾酮抗藥性檢測

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研究生：	趙芷郁 Chao, Chih Yu
論文名稱：	整合型微流體系統應用於胃幽門螺旋菌喹諾酮抗藥性檢測 An Integrated Microfluidic System for Diagnosis of the Resistance of Helicobacter pylori to Quinolone-Based Antibiotics
指導教授：	李國賓 Lee, Gwo Bin
口試委員:	楊瑞珍陳致真
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	78
中文關鍵詞：	微流體、抗藥性檢測、胃幽門螺旋菌、單核苷酸多態性的聚合酶鏈反應、光學檢測
外文關鍵詞：	microfluidic, antibiotic resistance detection, Helicobacter pylori, SNP-PCR, optical detection
相關次數：	點閱：1 下載：0
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胃幽門螺旋菌是一種感染胃黏膜之致病菌，胃幽門螺旋菌之感染被認為和許多腸胃道疾病的發生密切相關。臨床上使用三合一藥物組合做為胃幽門螺旋菌之治療方法，此藥物組合包含兩種不同之抗生素以及一質子幫浦抑制劑；近來有些菌株被發現對喹諾酮類抗生素具有抗藥性。常規之細菌抗藥性檢測方法為Epsilometer test 或Kirby-Bauer disk diffusion susceptibility test 此法通常在初次治療失敗時被用來確認感染菌株是否為抗藥性菌株。Epsilometer test 以及Kirby-Bauer disk diffusion susceptibility test 需較為冗長之培養時間，並且檢驗結果易受到採檢狀況與檢體運送過程所影響。因此，為減少此採檢流程對檢驗結果之影響與縮短檢驗時間，除了觀察抗藥性表現之外，分子診斷技術也被應用於檢測胃幽門螺旋菌之抗藥性。喹諾酮類抗生素之抗藥性的產生導因於促旋酶A 亞單位上的單個氨基酸被取代，影響藥物的結合能力，因而對喹諾酮類抗生素產生抗藥性，此單個氨基酸被取代是由於促旋酶A 亞單位基因上之點突變所導致。聚合酶鏈反應和隨後的基因定序檢測是常規的分子診斷技術來確認抗生素抗性。單核苷酸多態性的聚合酶鏈反應是一種新的分子診斷方法，由一對特異性引物，以區分正常基因與點突變之基因而不需後續的定序檢測。然而，此診斷過程需要昂貴、笨重的裝置以及專業訓練的人員。在這項研究中，我們開發一整合微流體系統，結合單核苷酸多態性的聚合酶鏈反應技術檢測胃幽門螺旋菌對於喹諾酮類抗生素之抗藥性。微流體系統可以統整整體實驗過程包括樣品前處理、單核苷酸多態性的聚合酶鏈反應、以及結果檢測。除此之外，一片晶片上包含一個以上的檢測反應槽，可同時檢測一個以上的樣本，整個檢測流程可在一個小時內完成。使用此開發的微流體檢測系統的檢測極限為100顆胃幽門螺旋菌，102顆單核苷酸多態性(突變)胃幽門螺旋菌。這個整合之檢測技術平台能快速地診斷胃幽門螺旋菌的感染以及其抗藥性，更加貼近臨床的使用需求。

Helicobacter pylori (H. pylori) is a kind of bacteria which can colonize on the stomach mucosa. Previous ntudies showed that it is closely associated with gastric diseases. H. pylori could be cured by using the triple therapy, which is composed of two antibiotics and a proton pump inhibitor and it has been comment used to eradicate H. pylori in routine practice. However, some strains of H. pylori were found which could express resistance to one kind of the antibiotics: quinolones. The Epsilometer test and Kirby-Bauer disk diffusion susceptibility test are the two presently used methods to confirm the resistance to antibiotics after treatment. However, these methods are relatively time-consuming and the culture result is strongly affected by the procedure of sample collection, transportation and storage. Therefore, a new method is in great need to prevent the artificial influence of sample collection, transportation and storage and shorten the experimental time as well. To detect the resistance of quinolones, besides phenotypic tests, some molecular diagnostic methods were reported recently. Some point mutations in the gyrase gene were found to express a resistance to quinolones. A mutation resulting in the change of a single amino acid substitution at any one or both of the two different sites in the gyrase A subunit affects the ability of the drug in binding to it. Polymerase chain reaction (PCR) and subsequent detection are conventional molecular diagnostic techniques for confirmation of the resistance to antibiotics. Single nucleotide polymorphism polymerase chain reaction (SNP-PCR) is a relatively new method to distinguish the single point mutations from a normal gene by applying a pair of specific primers without using complicated sequencing processes. Nevertheless, this diagnostic process requires relatively expensive and bulky apparatus and is labor-intensive. In this study, an integrated microfluidic system was developed to detect the resistance to quinolones in H. pylori by using the molecular diagnostic technique of SNP-PCR. The microfluidic system could combine the entire experimental procedure including sample pre-treatment, PCR/SNP-PCR, and optical detection on a single chip/system. Besides, more than one reaction chambers were designed on a single chip to detect multiple samples. The process could be performed within 1 hour with detection limits of 100, 102, and 102 bacterial cells for the detection of H. pylori and two different single nucleotide polymorphism site (mutation site) strains. Being accompanied by the advantages of microfluidic systems, the study fits clinical needs and may be promising for fast diagnosis of H. pylori and its drug-resistance.

Abstract    I
摘要    III
Table of contents    IV
List of figures    VI
List of tables    XIII 
Abbreviations     IV

Chapter 1  Introduction    1
1  Helicobacter pylori    1
2  Therapy and drug-resistance of H. pylori    3
3  Single nucleotide polymorphism polymerase chain reaction    5
4  MEMS and Bio-MEMS    8
5  Motivation and objectives    10

Chapter 2  Materials and methods……………………………………….14
1 Design and fabrication    14
1.1  Integrated microfluidic chip for H. pylori detection    14
1.2  Fabrication process    19
1.3  Custom-made control system    21
2  Preparation of the experimental materials    23
2.1  Design of specific primers    23
2.2  Preparation of bacterial samples    25
2.3  Preparation of magnetic beads conjugated specific probes    27
2.4.  Gel electrophoresis    30
3  Experimental process    31
4  Condition optimization    34
4.1 Pretreatment optimization    34
4.2 PCR optimization    36
5  Preparation of magnetic beads for capture rate test    37
5.1 Construction of H. pylori’s 16S rRNA DNA plasmid    37
5.2 Design of the experiment    40

Chapter 3  Results and discussion    42
1  Characterization of the pneumatic micropump    42
2  The experimental condition optimization test    44
2.1 Pre-treatment optimization    .44
2.2 PCR optimization    46
3  Capture rate of magnetic beads    49
4  Specificity of the microfluidic diagnostic assay    52 
5  Sensitivity of the microfluidic diagnostic assay    61
6  Multiple PCR using the integrated microfluidic system    66

Chapter 4  Conclusions and future perspectives    70
1  Conclusions    70
2  Future perspectives    72

References    73

                                

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