Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis
Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis
复制标题
DOI：
10.1128/jcm.35.12.3232-3239.1997
复制
发表时间：
1997-12
影响因子：
9.4
通讯作者：
Christian Carriere;Paul F. Riska;O. Zimhony;J. Kriakov;S. Bardarov;Judah Burns;John Chan;William R. Jacobs
中科院分区：
文献类型：
--
作者：
Christian Carriere;Paul F. Riska;O. Zimhony;J. Kriakov;S. Bardarov;Judah Burns;John Chan;William R. Jacobs
TM4是一种感染具有临床意义的分枝杆菌的溶解性噬菌体。利用TM4构建了荧光素酶报告噬菌体phAE40，该噬菌体可用于结核分枝杆菌的快速检测和药敏试验。然而，噬菌体的溶解性质导致可检测光输出的损失，并限制了检测的灵敏度。我们描述了几种旨在提高TM4荧光素酶噬菌体产生的荧光素酶活性的策略，包括(i)改变荧光素酶基因在噬菌体基因组中的位置，（ii）分离噬菌体的宿主突变体，以及（iii）在噬菌体中引入温度敏感突变，使其在感染温度下不会复制。与第一代报告噬菌体phAE40相比，通过这些方法产生的几种新噬菌体显示出更高的荧光素酶产生强度，其中一种噬菌体phAE88也显示出更长的荧光素酶活性持续时间。这种方法可以在短短1天内检测到120个卡介苗细胞并确定结核分枝杆菌的药物敏感性。
TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day.