Selection of IS6110 conserved regions for the detection of Mycobacterium tuberculosis using qPCR and LAMP

IS6110 insertion sequence is a frequently used target for Mycobacterium tuberculosis detection. However, its sequence variability is studied insufficiently. We aimed to identify the most conservative and variable regions in IS6110 sequences and develop qPCR and LAMP oligonucleotide sets for the conservative regions. Using in-house Python scripts, 3609 M. tuberculosis genome sequences from the NCBI database were aligned; conservative regions were identified to design oligonucleotide sets. IS6110 fragments located within the 31-231 bp region were the most conservative and represented in genomes and were used to design qPCR and LAMP oligonucleotides. The in silico sensitivity of the qPCR oligonucleotides on the whole genome set was 99.1% and 98.4%. For the LAMP primers developed, the sensitivity was 96.9%. For qPCR, the limit of detection with 95% confidence (LoD_95%) was four IS6110 copies per reaction, with LoD_90% being 200 BCG cells per ml of artificial sputum. For LAMP, LoD_95% was 16 copies per reaction, with LoD_90% being 400 Mycobacterium bovis Bacille Calmette-Guerin (BCG) cells per ml of artificial sputum. We have demonstrated the IS6110 sequence variability and designed highly sensitive qPCR and LAMP oligonucleotides to detect M. tuberculosis.