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

吡嗪酰胺（PZA）是烟酰胺的类似物，是结核病的前药，需要通过细菌吡嗪酰胺酶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使得鉴别PZA耐药结核病成为可能。而对M.结核病分离物需要大量的杆菌，因此是耗时的。在这项研究中，我们建立了一个新的方法检测吡嗪酰胺酶的活性与PCR为基础的系统。用5'端含T7噬菌体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PCR扩增。然后将PCR产物直接进行体外转录-翻译偶联系统。与敏感的M相比，所有测试的PZA抗性分离株均显示吡嗪酰胺酶活性降低。结核型菌株H37 Rv。与此相反，所有15个敏感的临床分离株表现出类似的吡嗪酰胺酶活性的H37 Rv。这一事实表明，该系统用于快速检测抗PZA的M。结核

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.