Toxin–Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64 M. tuberculosis and 26 M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

毒素-抗毒素（TA）系统是细菌中的一些小型遗传模块，在抗生素耐药性和耐受性发展中发挥着重要作用。全基因组测序（WGS）是分析致病性分枝杆菌TA系统的有效方法。然而，本研究旨在使用简单且廉价的 PCR 测序方法来研究 II 型 TA 系统。利用来自结核分枝杆菌( M. tuberculosis ) 菌株 H37Rv 和牛分枝杆菌( M. bovis ) 菌株 BCG 的WGS 数据，通过 Primer3 软件设计了relJK、mazEF3和vapBC3基因家族特异性的引物。随后，使用新开发的 PCR 检测方法对总共 90 个分离株进行了检查，其中包含 64 M 。结核病和 26 M 。牛分离株，包括 45 种利福平敏感菌株和 45 种利福平耐药菌株。最后，将28个分离株（包括14个利福平耐药分离株）送去测序，并将其序列与上述参考序列进行比对和比较。mazEF3、relJK和vapBC3基因的扩增子大小分别为 825、875 和 934 bp。此外，所有测试的分离株都显示出这些 TA 家族的特定扩增子。为了评估引物的特异性，对金黄色葡萄球菌和大肠杆菌分离株进行了 PCR。所检查的样品均不具有所需的扩增子。因此，引物具有可接受的特异性。结果表明，所开发的 PCR 测序方法可用于有效研究某些类型的 TA 系统。考虑到全基因组测序的成本较高且难以解释其结果，这种简单且廉价的方法有利于分枝杆菌TA系统的评估。