Use of Polymerase Chain Reaction-Restriction Fragment Analysis for Detection and Identification of Mycobacterium tuberculosis

目的：我們結合核酸聚合酵素連鎖反應與限制酵素分解片段分析的技術進行結核分枝桿菌的檢測與鑑定分析。病人及方法：核酸聚合酵素連鎖反應本身所利用的標的核酸為65kDa、IS6110、mtp40 三種。其中65 kDa為一蛋白質基因用以檢測分枝桿菌屬，IS6110序列用來檢測結核分枝桿複合群；主要包含了結核分枝桿菌、牛型分枝桿菌(含卡介苗菌種)，而mtp40則是用來區分結核分枝桿菌和牛型分技桿菌。另一方面，限制酵素分解片段分析不需使用核酸定序與同位素雜交技術，但其在確認所增殖放大的核酸片準確性上可扮演決定性的角色。結果：在選擇各逶限制酵素具有獨特不對稱切點與使用簡單的特性條件下，SacII適用於IS6110和mtp40的增放大核酸片段分析；Eco O109I 則適用於65kDa的增植放大核酸片段分析。結論：當結合使用時，此核酸聚合酵素連鎖反應暨限制酵素分解片段分析分法已成功的利用在核性腦膜炎患者的腦脊髓液檢體中結核分枝桿菌的共測與鑑定。(慈濟醫學 1999; 11:31-320)

關鍵字

分子診斷；結核枝桿菌；核酸聚合酵素連鎖反應；限酵素分解片段分析

並列摘要

Objective: We combined the PCR assays developed previously with restriction fragment analysis (PCR-RFA) to detect and identify Mycobacterium tuberculosis. Patients and Methods: The PCR assay itself targets three DNA: the 65 kDa antigen protein gene for detecting a wide spectrum of mycobacterial strains; the IS6110 insertion sequence for detecting the Mycobacterium tuberculosis complex, mainly M. tuberculosis and M. bovis BCG; and the mtp40 genomic fragment for distinguishing M. tuberculosis from M. bovis BCG. RFA, on the other hand, requiring no DNA sequencing or radioactive hybridization, plays a confirmation role to ensure the correctness of the amplified DNA products. Results: Several restriction enzymes, with properties of unique cutting site at the asymmetrical location of the expected amplicon and simple digestion conditions, have been selected and tested in this method. Among these, Sac II is suitable for both of the amplicons from the IS6110 and the mtp40 targets, whereas Eco O109I is suitable for the amplicon from the 65 kDa target. Conclusions: Taken together, this PCR-RFA method had been used successfully in the detection and identification of M. tuberculosis in a CSF sample from a patients with tuberculous meningitis. (Tzu Chi Med J 1999; 11:311-320)

並列關鍵字

molecular diagnosis ； Mycobacteriuym tuberculosis ； PCR ； restriction fragment analysis

國際替代計量

Use of Polymerase Chain Reaction-Restriction Fragment Analysis for Detection and Identification of Mycobacterium tuberculosis

全文下載

主題瀏覽