Real-Time PCRs that Discriminates Mycobacterium tuberculosis and Mycobacterium bovis Based on Single Nucleotide Polymorphism and Genome Analysis of Amplicons

Accurate identification of Mycobacterium strains is necessary from a diagnostic standpoint. Differences in gyrA 95 and gyrB (1410) genome due to specific single nucleotide polymorphism (SNP) were used to design Real-Time PCRs that can discriminate Mycobacterium tuberculosis (MT) from Mycobacterium bovis (MB) belonging to the Mycobacterium Tuberculosis Complex (MTBC). The Real-Time PCR employing the VIC labeled gyrA 95 probe specifically detected the ATCC reference strain of MT H37Rv and eight field strains as MT, but not MB or other members of MTBC and other micro-organism tested. The VIC labeled gyrA 95 assay could detect up to 450 fg of MT. The FAM labeled gyrB (1410) specifically detected MB, but not MT or other members of MTBC and other micro-organism tested. The assay could detect up to 600 fg of MB, but it could not detect MB from any field isolate. Both assays were completed in 78 minutes. The results of both Real-Time PCR assays did not differ significantly from culture by Student’s t-test. The analytical sensitivity of gyrA 95 assays determined by Receiver Operating Characteristic curve (ROC) was 100% at 95% Confidence Intervals (CI) |66.4 -100.0|, and analytical specificity 100% at 95% CI |75.3 -100.0|. The repeatability and reproducibility of both above assays tested by Bland-Altman Plot indicated that the mean Cq values were within acceptable statistical limits of + 1.96 SD. Comparison of partial genome sequences of gyrA 95 and gyrB (1410) of field strains of MT with MT H37Rv by Phylogenetic Tree and Disparity Index tests indicated that eight field strains were homologous to MT H37Rv, but one was homologous to Mycobacterium intracellulare. Real-Time PCR assays were found rapid, specific, sensitive, repeatable and reproducible. Further studies are necessary for conversion of these assays to quantitative formats and determine parameters of diagnostic estimates before their use under clinical settings.