Development of pyrazinamide susceptilbility testing method for Mycobacterium tuberculosis by using gene analysis technique

吡津酰胺(PZA)是烟酰胺的类似物，是一种治疗结核病的前药，需要通过细菌吡津酰胺酶的活性转化为杀菌化合物吡嗪酸。导致吡嗪酰胺酶活性丧失的突变导致结核分枝杆菌对PZA的耐药性。因此，吡嗪酰胺酶活性的检测为耐PZA结核病的鉴别提供了可能。然而，检测结核分枝杆菌分离株的吡嗪酰胺酶活性需要大量的细菌，因此耗时较长。在这项研究中，我们建立了一种新的方法来检测吡嗪酰胺酶活性的基础上，基于PCR的系统。采用5‘端含有噬菌体T7启动子序列的正向引物，对30株耐药临床分离株的吡嗪酰胺酶基因(pncA基因)进行了聚合酶链式反应。然后将扩增产物直接用于体外转录-翻译耦合系统。与敏感的结核分枝杆菌类型菌株H37Rv相比，所有对PZA耐药的菌株都显示出吡津酰胺酶活性降低。相比之下，所有15株敏感的临床分离株都表现出与H37Rv相似的吡嗪酰胺酶活性。这一事实表明，该系统对于快速检测耐PZA的结核分枝杆菌是可行的。

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidaseactivity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this study, we developed a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at the 5' ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.