Comparative evaluation of pncA gene, IS6110 and 12.7-Kb fragment based PCR assays for simultaneous detection of Mycobacterium tuberculosis complex (M. tuberculosis and M. bovis) in cultured strains and clinical specimens

Abstract

PCR based molecular techniques help in discrimination of two closely related Mycobacterium tuberculosis and M.bovis. Here, we analyzed 24 M. bovis, 39 M. tuberculosis, 21 fresh acid-fast positive sputum samples and standardmycobacterial strains with pncA, 12.7 Kb and IS6100 based PCR assays. DNA from cultures and sputum yielded a positiveamplification of 185 bp with M. tuberculosis specific reverse primer pncAMT-2 but not with M. bovis specific reverseprimer pncAMB-2 and all M. bovis strains showed a positive amplification of 185 bp with M. bovis specific reverse primerpncAMB-2 but not with M. tuberculosis specific reverse primer pncAMT-2. The 12.7 Kb fragment based PCR performed onDNA extracted from cultures of M. tuberculosis and sputum yielded product of 168 bp while M. bovis showed 262 bpproducts. M. tuberculosis complex specific IS6110 PCR assay performed on DNA extracted from M. tuberculosis, M. boviscultures and sputum samples yielded M. tuberculosis complex specific 123-bp amplified products. The sequence analysis ofrepresentative PCR products of IS6110 and 12.7 Kb fragment showed 99-100% and 100% identity in amplicon products,respectively. To test reliability of primers, M. tuberculosis and M. bovis cultures were mixed and subjected to IS6110, pncAand 12.7 Kb PCR assay. pncA primers could not successfully and reliably discriminate the mixed culture, however, 12.7 Kbfragment primers successfully discriminated the mixed culture of M. tuberculosis and M. bovis.