Introduction: Delay in diagnosis of paucibacillary extra pulmonary tuberculosis (TB) and of smear negative TB has hampered the efforts taken by Control Programs to curb its spread. Better efforts to control spread of TB require more accurate and rapid diagnostic test. Aims: To facilitate early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of a single tube in-house nested PCR in comparison against culture and composite reference standard (CRS). Methods: Single tube nested PCR was performed using primers targeting Insertion Sequence (IS) 6110 of Mycobacterium tuberculosis complex. Microbiological techniques includes AFB smear microscopy and cultivation on solid egg-based medium (Lowenstein-Jensen [LJ]) and on liquid culture medium using BACTEC MGIT 960 system, BD Microbiology Systems. Results: The sensitivity and specificity of PCR against culture was observed to be 89.7% [95% CI: 84.1-93.5] and 73.1% [95% CI: 67.4-78.1] respectively and that against CRS criteria was 80.2% [95% CI: 75.1-84.5] and 97.1% [95% CI: 92.9-98.9] respectively. PCR showed 100% [111/111, 95% CI: 97-100] sensitivity for smear positive specimens. For smear negative specimens sensitivity and specificity of PCR against culture was observed to be 78.4% [69/88, 95% CI: 68.4-86.5] and 77.3% [204/264, 95% CI: 71.7-82.2] respectively and that against CRS was 68.1% [124/182, 95% CI: 60.8-74.8] and 97.1% [165/170, 95% CI: 93.3-99] respectively. Conclusion: CRS criteria were observed to be better than culture for assessing the diagnostic accuracy of PCR test. (copyright) 2013.