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系統識別號 U0026-0507201317005100
論文名稱(中文) 以寡核苷酸晶片快速檢測結核菌群對第一及第二線藥物之抗藥性
論文名稱(英文) Rapid detection of antimicrobial resistance of Mycobacterium tuberculosis complex to the first- and second-line drugs by an oligonucleotide array
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 2
出版年 102
研究生(中文) 嚴文君
研究生(英文) Wen-Chun Yen
學號 t36004013
學位類別 碩士
語文別 中文
論文頁數 228頁
口試委員 口試委員-周如文
口試委員-吳俊忠
口試委員-盧柏樑
指導教授-張長泉
中文關鍵字 結核菌群  抗藥性結核病  寡核苷酸晶片  抗結核藥物 
英文關鍵字 Mycobacterium tuberculosis complex (MTBC)  Drug-resistant tuberculosis  Oligonucleotide array  Isoniazid (INH)  Rifampin (RIF)  Streptomycin (SM)  Ethambutol (EMB)  Ofloxacin (OFX)  Kanamycin (KM)  Amikacin (AM)  Capreomycin (CAP) 
學科別分類
中文摘要 依據世界衛生組織的統計,2011年全球約有870萬個結核病新案例,並有140萬人死於結核病。結核菌群之藥物感受性需耗時三週,造成延遲或不適當的治療。而第二線藥物之感受性試驗並非一般結核菌實驗室常規檢驗。結核菌群的抗藥性與基因發生點突變有關,rifampin (RIF), isoniazid (INH), ethambutol (EMB), fluoroquinolones [FQs, 如:ofloxacin (OFX)]及第二線注射藥物[streptomycin (SM), kanamycin (KM), amikacin (AM)及capreomycin (CAP)]的抗藥性分別和rpoB, katG及inhA regulatory region, embB, gyrA及gyrB, rrs, rpsL及eis上游區域發生突變有關。本研究目的為發展一寡核苷酸晶片,可同時檢測上述基因之突變。設計野生型及突變型探針包括embB (codons 306, 319, 406及497), rrs (nucleotides 491, 512, 513, 516, 906, 1401, 1402及1484), rpsL (codons 43及88), gyrA (codons 74, 88, 90, 91, 94及102), gyrB (codons 472及510), eis上游區域(nucleotides -10, -12, -14及-37),並整合先前已建構檢測RIF及INH抗藥性之探針。以多重聚合酶鏈反應,利用標誌毛地黃素的引子將待測基因同時放大,與尼龍膜上的探針雜合反應,以檢測是否有突變存在。
共測試546株(204位病人)結核菌群,晶片檢測抗藥性(以病人為計算基礎)的靈敏度分別為98.7% (RIF), 91.9% (INH), 84.6% (EMB), 85.3% (SM), 93.8% (OFX), 80% (KM), 60% (CAP)及75% (AM);特異性分別為 100% (RIF), 99.0% (INH), 99.3% (EMB), 98.9% (SM), 100% (OFX), 100% (KM), 97.9% (CAP)及100% (AM);陽性預測值分別為100% (RIF), 99.0% (INH), 97.8% (EMB), 98.9% (SM), 100% (OFX), 100% (KM), 60% (CAP)及100% (AM);陰性預測值分別為99.2% (RIF), 92.9% (INH), 95.0% (EMB), 85.4% (SM), 98.9% (OFX), 98.4% (KM), 97.9% (CAP)及 99.3% (AM)。檢測多重抗藥性(multi-drug resistance)及廣泛抗藥性(extensively drug resistance)菌株的靈敏度分別為91.5%及87.5%;特異性分別為98.5%及100%。晶片檢測時間可在一個工作天內完成。未來,將評估晶片檢測陽性液態培養檢體或是直接檢體之可行性。
英文摘要 Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC) and is one of the world’s most important infectious diseases. According to World Health Organization (WHO), there were 8.7 million new cases of TB and 1.4 million people died from TB in 2011. An alarming increase in the global incidence of drug-resistant TB has threatened the control and treatment of TB. Drug susceptibility of MTBC normally takes 3 weeks, resulting in deferred and inadequate treatment. Therefore, there is an urgent need to develop more rapid assays. Drug-resistant TB is associated with mutations in several genes, including rpoB for rifampin (RIF), katG and inhA regulatory region for isoniazid (INH), embB for ethambutol (EMB), gyrA and gyrB for fluoroquinolones [such as ofloxacin (OFX)], rrs, rpsL, and the promoter of eis for second-line injectable drugs [streptomycin (SM), kanamycin (KM), amikacin (AM), and capreomycin (CAP)]. This study aimed to develop an oligonucleotide array to detect mutations of the aformentioned genes.
Specific oligonucleotide probes were designed to detect mutations in embB (codons 306, 319, 406, and 497), rrs (nucleotides 491, 512, 513, 516, 906, 1401, 1402, and 1484), rpsL (codons 43 and 88), gyrA (codons 74, 88, 90, 91, 94, and 102), gyrB (codons 472 and 510), and the upstream of eis (nucleotides -10, -12, -14, and -37). Probes used to detect RIF and INH resistance were previously developed in our laboratory. The assay consisted of multiplex PCR amplification of 13 genes and the internal amplification control, followed by hybridization of the amplicons with probes on a nylon membrane.
A total of 546 MTBC isolates (204 patients) were analyzed. The performance of the array (based on patient) were: sensitivities, 98.7% (RIF), 91.9% (INH), 84.6% (EMB), 85.3% (SM), 93.8% (OFX), 80% (KM), 60% (CAP), and 75% (AM), respectively; specificities, 100% (RIF), 99.0% (INH), 99.3% (EMB), 98.9% (SM), 100% (OFX), 100% (KM), 97.9% (CAP), and 100% (AM), respectively; positive predictive values, 100% (RIF), 99.0% (INH), 97.8 % (EMB), 98.9 % (SM), 100% (OFX), 100 % (KM), 60% (CAP), and 100% (AM), respectively; negative predictive values, 99.2% (RIF), 92.9% (INH), 95.0% (EMB), 85.4% (SM), 98.9% (OFX), 98.4% (KM), 97.9% (CAP), and 99.3% (AM), respectively. For multidrug-resistant TB and extensively drug-resistant TB, the sensitivities were 91.5% and 87.5%, respectively, while the specificities were respectively 98.5% and 100%. In conclusion, the array can effectively detect gene mutations causing drug resistance to the first- and second-line anti-tuberculosis antibiotics, except CAP, in a working day.
論文目次 中文摘要 I
Abstract III
致謝 V
目錄 VI
表目錄 XII
圖目錄 XIV
緒論 1
結核菌群 (Mycobacterium tuberculosis complex, MTBC) 1
結核病之流行病學 1
臨床實驗室診斷 4
抗藥性結核菌之分生機制 5
1. Rifampin (RIF) 5
2. Isoniazid (INH) 6
3. Ethambutol (EMB) 7
4. Fluoroquinolones (FQs) 8
5. Streptomycin (SM) 9
6. Kanamycin (KM), amikacin (AM)及capreomycin(CAP) 9
抗藥性結核菌之分子檢測方法 10
研究目的 12
研究架構 12
材料與方法 14
參考菌株 14
臨床結核菌群菌株之收集 14
DNA萃取 15
分子選殖(molecular cloning) 15
質體DNA萃取 18
以定點突變(site-directed mutation)建構含特定單一核苷酸變異(SNP)之DNA片段 19
抗藥性基因之增幅及定序 20
特異性探針(specific probes)之設計及修改 21
內部增幅對照組(internal amplification control, IC) DNA 25
結核菌群抗藥性晶片製備 26
多重聚合酶鏈反應(multiplex PCR) 27
晶片雜合反應(array hybridization) 28
晶片雜合反應之結果判讀 30
臨床結核菌群菌株抗藥性基因定序 31
Sensititre® MYCOTB plates套組 32
靈敏度(sensitivity)、特異性(specificity)、陽性預測值(positive predictive value, PPV)、陰性預測值(negative predictive value, NPV)、一致性(agreement)之定義 33
結果 35
抗藥性基因之增幅 35
具定點突變之DNA建構 35
探針篩選 36
以多重聚合酶鏈反應進行晶片一及晶片二之測試 36
以多重聚合酶鏈反應進行晶片三之測試 37
結核菌群(Mycobacterium tuberculosis complex)探針 38
非結核分枝桿菌(nontuberculous mycobacteria, NTM)之晶片測試 39
臨床結核菌群(MTBC)菌株抗藥性檢測 40
1. RIF抗藥性之檢測 40
2. INH抗藥性之檢測 41
3. EMB抗藥性之檢測 42
4. SM抗藥性之檢測 42
5. OFX抗藥性之檢測 43
6. KM抗藥性之檢測 43
7. CAP及AM抗藥性之檢測 43
8. MDR-TB及XDR-TB之檢測 43
偽陽性及偽陰性菌株之基因定序 44
1. RIF抗藥性菌株 44
2. INH抗藥性菌株 44
3. EMB抗藥性菌株 44
4. SM抗藥性菌株 45
5. OFX抗藥性菌株 45
6. KM抗藥性菌株 45
7. CAP抗藥性菌株 46
8. AM抗藥性菌株 46
偽陽性菌株之最低抑菌濃度試驗 46
晶片靈敏度(sensitivity)、特異性(specificity)、陽性預測值(positive predictive value, PPV)與陰性預測值(negative predictive value, NPV) 47
1. RIF之結果 47
2. INH之結果 47
3. EMB之結果 47
4. SM之結果 48
5. OFX之結果 48
6. KM之結果 48
7. CAP之結果 49
8. AM之結果 49
9. 晶片檢測MDR-TB之結果 49
10. 晶片檢測XDR-TB之結果 49
11. 晶片之表現性(以病人為計算基礎) 50
討論 51
RIF抗藥性和rpoB突變之相關性 51
INH抗藥性與katG及inhA regulatory region突變之相關性 52
EMB抗藥性與embB突變之相關性 53
SM抗藥性與rrs及rpsL突變之相關性 54
OFX抗藥性與gyrA及gyrB突變之相關性 55
二線注射型藥物抗藥性與rrs及eis上游區域突變之相關性 55
其他突變對晶片雜合反應之影響 57
晶片對結核菌群異質性抗藥性(hetero-resistance)之檢測 58
抗藥性晶片與商業化套組之比較 59
非結核分枝桿菌與探針交叉反應 60
結論 62
參考文獻 63
附錄 157
附錄一、結核病治療 157
附錄二、臨床菌株清單 159
附錄三、臨床菌株之抗藥相關基因定序 191
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