Novel targeting of the lepB gene using PCR with confronting two-pair primers for simultaneous detection of Mycobacterium tuberculosis complex and Mycobacterium bovis.

Abstract

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTC), is the leading cause of infectious disease-related mortality worldwide. The standard method for TB diagnosis usually requires long periods of mycobacteria cultivation, leading to delayed diagnosis, inefficient treatment and widespread occurrence of the disease. Therefore, a rapid method for the detection and differentiation of MTC from other mycobacteria is essential for disease diagnosis. Here, we describe the potential of using the type I signal peptidase (lepB) gene as a novel target for TB diagnosis, based on confronting two-pair primers PCR (PCRCTPP) that can detect MTC and simultaneously differentiate M. bovis. The limit of detection of the developed technique was equivalent to 12–120 bacilli. PCR-CTPP was highly specific to only MTC and M. bovis, and no cross-reaction was detected in 27 DNA of the non-tuberculous mycobacterial and bacterial strains tested. Thirty-nine blinded clinical isolates and 72 sputum samples were used to validate the PCR-CTPP in comparison with the standard mycobacterial culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP were equal to 95, 100, 100 and 95 %, respectively, when tested with clinical isolates. Furthermore, upon testing with the sputum samples, the sensitivity, specificity, PPV and NPV were observed to be 84, 76, 90 and 67 %, respectively. Hence, this highly sensitive novel technique, which is rapid, easy to conduct and cost-effective, is a potential method for TB diagnosis and epidemiological studies, especially in resource-limited countries with a high TB burden.