Rapid Quantification of IS6110 Copy Number in M. tuberculosis Using Dual-Target qPCR Assay

PCR-based methods for detecting Mycobacterium tuberculosis ( M. tuberculosis ) have transformed tuberculosis (TB) diagnostics by allowing for the quick and sensitive identification of the pathogen’s DNA. The IS6110 insertion sequence is a popular molecular target due to its high specificity to the M. tuberculosis complex. However, the variability in IS6110 copy numbers among different strains raises questions about its reliability as a sole diagnostic marker. In this research, we devised a rapid qPCR-based technique to measure IS6110 copy numbers in clinical M. tuberculosis samples, comparing them with genome copy numbers estimated using a commercial MPB64-targeted kit. A custom standard curve was created using a linear DNA fragment representing the IS6110 sequence from the H37Rv strain. Analysis of 16 positive samples from 320 screened cases showed significant variability in IS6110 content. While most strains had 15–25 copies per genome, three samples had fewer than one copy per genome, likely due to mixed-strain populations dominated by IS6110-deficient bacilli. The method proved the feasibility of accurate IS6110 quantification and highlighted the importance of regional surveillance of IS6110 prevalence to prevent false negatives in PCR diagnostics. Incorporating multiple genetic targets and using dual fluorophores (e.g., FAM and VIC) in a single-tube multiplex qPCR can improve diagnostic accuracy and workflow efficiency. The findings advocate for a more nuanced, region-specific use of IS6110 in TB molecular diagnostics and emphasize the potential for automation through simple computational tools to enhance clinical decision-making.