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頁籤選單縮合
題 名 | Determination of the Sensitivity and Specificity of PCR Assays Using Different Target DNAs for the Detection of Mycobacterium Tuberculosis=以不同標的核酸進行結核桿菌聚合酵素連鎖反應分子診斷的敏感性與專一性分析 |
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作 者 | 魏誠佑; 李俊年; 朱清華; 黃娟娟; 李展平; | 書刊名 | The Kaohsiung Journal of Medical Sciences |
卷 期 | 15:7 1999.07[民88.07] |
頁 次 | 頁396-405 |
分類號 | 415.2773 |
關鍵詞 | 核酸; 結核桿菌; 聚合酵素連鎖反應; Molecular diagnosis; Mycobacterium tuberculosis; Polymerase chain reaction; |
語 文 | 英文(English) |
中文摘要 | 為了建立一套敏感、專一與重覆性高的聚合酵素連鎖反應方法,作為結核分枝桿菌的分子診斷,我們評估三種不同標的核酸;IS6110、65kDa、及mtp40。我們培養15種結核分枝桿菌(包括人型結核分枝桿菌,牛型結核分枝桿菌及其他相關分枝桿菌菌種)並純化其基因體核酸。研究中各有三對引子是針對標的核酸IS6110及mtp40,另有二對引子是針對標的核酸65kDa。聚合酵素連鎖反應方法包括了單一聚合酵素連鎖反應(single-step PCR)與巢式聚合酵素連鎖反應(nested PCR)。使用標的核酸65 kDa,能偵測所有研究中的分枝桿菌。使用標的核酸IS6110,能偵測人型與牛型的結核分枝桿菌,而使用標的核酸mtp40能區分人型與牛型的結核分枝桿菌。使用巢式聚合酵素連鎖反應方法,能夠偵測到1至10個結核分枝桿菌的基因體核酸。此方法証明為一敏感性高與專一性良好的分子診斷方法,可以應用到通常含結核分枝桿菌稀少的各種臨床檢體,例如腦膜炎病患的腦脊髓液。 |
英文摘要 | To establish a sensitive, specific and reproducible PCR assay for the detection of Mycobacterium tuberculosis, we evaluated three target DNAs: IS6110, 65 kDa heat shock protein gene; and mtp40 genomic fragment. We purified genomic DNA from 15 mycobacterial strains including four M. tuberculosis isolates, four M. bovis BCG isolates, and one of each for M. fortuitum, M. avium, M. intracelluare, M.szulgai, M. scrofulaceum, M. chelonei, and M. gordonae from the culture and used them as the template DNA. We studied 3 primer sets for IS6110, 2 primer sets for 65 kDa, and 3 primer sets for mtp40. Depending on the assay, these primer sets were used in the single-step PCR and/or nested PCR. The PCR assay targeting the 65 kDa protein gene could detect all of the tested mycobacterial strains, whereas targeting the IS6110 sequence resulted in detection of only M. tuberculosis and M. bovis BCG. Furthermore, targeting the mtp40 genomic fragment could be used to distinguish M. tuberculosis from M. bovis BCG.. Using a nested PCR assay with primer sets specifically targeting the IS6110 or 65 kDa, we have been able to detect single copy M. tuberculosis genomic DNA. When the mtp40 genomic fragment was used as the target DNA, the sensitivity of detection was 10 copies of M. tuberculosis genomic DNA. This assay was demonstrated to have good sensitivity and specificity for detection and discrimination of mycobacterial species, and could be used in analyzing the clinical samples with low copy number infections such as the cerebrospinal fluid from the patient with twberculous meningitis. |
本系統中英文摘要資訊取自各篇刊載內容。