The prevalence of infections by nontuberculous mycobacteria (NTM), which present symptoms similar to tuberculosis (TB), is rising worldwide, highlighting the critical need for precise differential diagnosis. To minimize false-positive TB diagnoses, this study focused on a novel target unique to Mycobacterium tuberculosis (MTB). A real-time PCR assay was developed using specific primers and probes targeting the IS6110 region. Specific primers and probes were designed using Primer3plus and evaluated in silico using the NCBI database. An internal control was incorporated by synthesizing and cloning a fragment of the 16 S rRNA gene into the pcDNA3.1(+) vector, which was then transformed into Escherichia coli Top10F. Standards were prepared to determine the limit of detection (LOD) and assay efficiency. The kit’s accuracy and reproducibility were evaluated using real-time PCR on 15 MTB-positive DNA samples. The designed primers and probes specifically amplified a 365 bp fragment of the IS6110 region. The extracted plasmid concentration was 76.6 ng/µl. The assay utilized probes labeled with HEX for the IS6110 gene and FAM for the internal control. The standard curve demonstrated a linear dynamic range with a correlation coefficient (R²) of > 0.98 and an efficiency of 100%. The LOD was determined to be 10 copies/µL. Evaluation of accuracy and repeatability indicated high consistency, with minimal variation in Ct values across repetitions. Furthermore, the kit demonstrated stability across different manufacturing LOTs. In conclusion, this study demonstrates that the developed quantitative Real-Time PCR assay, utilizing IS6110-HEX and 16 S rRNA-FAM probes, effectively identifies M. tuberculosis. While the results show high specificity and potential for distinguishing MTB from NTM, further validation with clinical NTM isolates is recommended.
Mousavi-Kohnesari et al. (Thu,) studied this question.