A new diagnostic kit using real-time polymerase chain reaction for the detection of Mycobacterium tuberculosis.

UNLABELLED: The prevalence of infections by nontuberculous(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(MTB). A real-time PCR assay was developed using specific primers and probes targeting theregion. 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 intoTop10F. 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 theregion. The extracted plasmid concentration was 76.6 ng/µl. The assay utilized probes labeled with HEX for thegene and FAM for the internal control. The standard curve demonstrated a linear dynamic range with a correlation coefficient () ofand an efficiency of. The LOD was determined to be. 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-HEX and-FAM probes, effectively identifies. While the results show high specificity and potential for distinguishing MTB from NTM, further validation with clinical NTM isolates is recommended.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13568-026-02044-2.