Global tuberculosis (TB) control and eradication is hampered by the unavailability of simple, rapid and affordable diagnostic tests deployable at low infrastructure microscopy centers. We have developed and evaluated the performance of a nucleic acid amplification test for detection of Mycobacterium tuberculosis (MTB), the NWU-TB test, in clinical sputum specimens from 306 patients with suspected pulmonary tuberculosis. The test involves sputum sample processing using a Lyser device within 7 min, followed by rapid multiplex-PCR on a fast thermal cycler within 25 min, and amplicon resolution on agarose gel electrophoresis. Samples were also examined for presence of MTB using smear microscopy, GeneXpert and MGIT culture. Results were assessed in comparison to a MGIT culture as gold standard. Of the 306 patients, 174 had a previous TB history or already on treatment, and 132 were TB naïve cases. The NWU-TB system was found to have an overall sensitivity and specificity of 80.8% (95% CI: 75-85.7) and 75.6% (95% CI: 64.9-84.4) respectively, in comparison to 85.3% (95% CI: 79.9-89.6) and 73.2% (95% CI: 62.2-82.4) respectively for GeneXpert; and 62.1% (95% CI: 55.3-68.4) and 56.1% (95% CI: 44.7-67) respectively for smear microscopy. The study has shown that the NWU-TB system allows detection of TB in less than two hours and can be utilized at low infrastructure sites to provide quick and accurate diagnosis at a very low cost.