Background. Tuberculosis, a global health problem and highly prevalent in India, has always been a serious problem with respect to definitive diagnosis. Polymerase chain reaction (PCR) techniques are now widely used for early detection and species differentiation of mycobacteria, but mostly with their own limitations. We aim to detect and differentiate Mycobacterium tuberculosis (Mtb) infections by choosing appropriate target sequences, ideally present in all mycobacterial species (MTB complex) and absent in others. Methods. Amplification of three target sequences from unrelated genes, namely, hsp 65 (165？bp), dnaJ (365？bp), and insertion element IS 6110 (541？bp) by PCR was carried out in clinical samples from suspected cases of tuberculosis/ mycobacterioses and healthy controls. Results. The sensitivity of this method ranged from 73.33% to 84.61%, and the specificity was 80%. The PCR method was significantly better ( and ) than both smear and culture methods. Conclusion. Our trimarker-based PCR method could specifically detect M. tuberculosis and MTB complex infection from that of major pathogenic NTM and nonpathogenic mycobacteria. This method, by well distinguishing between MTB complex and NTM, presented a fast and accurate method to detect and diagnose mycobacterial infections more efficiently and could thereby help in better patient management particularly considering the increase in mycobacterial infections due to emergence of NTM over the past decades. 1. Introduction Tuberculosis is a major public health problem with a total of 9.2 million new cases and 1.7 million deaths from tuberculosis (TB) in 2006. India accounts for one-fifth of the global TB burden (WHO 2008), which has been on the rise due to multidrug-resistant and highly virulent strains of Mycobacterium tuberculosis (Mtb) [1] and combined effect of HIV. An accurate diagnosis of tuberculosis is desirable before the start of anti-tuberculosis therapy [2]. The laboratory diagnosis of Mtb depending on acid-fast bacillus (AFB) smear can yield a result within 24？h. However, smear is not very specific for Mtb and also requires 103 to 104 organisms per mL of sputum. Bacterial culture is superior to AFB smear, both in terms of sensitivity and specificity. But, since mycobacteria have very strict growth requirements, culture-based diagnostic methods are slow. Further, diagnoses involving radiological examinations and Tuberculin test help to detect the disease to some extent, but are not very reliable in case of extrapulmonary tuberculosis. The BACTEC system however, gives a very quick result
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