SNP-Based Identification of Mycobacterium tuberculosis Strains using TaqMan PCR

Within the WHO framework of genomic epidemiological surveillance of tuberculosis, we have developed a panel of primers and TaqMan probes for PCR-based identification of SNPs in major epidemiological genotypes of Mycobacterium tuberculosis responsible for the spread of MDR/XDR strains in North Eurasia. Validation was performed on a dataset of 590 randomly selected strains circulating in the Krasnoyarsk Krai and Irkutsk region in 2021-2025. We identified 308 and 164 Beijing (L2) strains in the Krasnoyarsk and Irkutsk regions, respectively. The identified Beijing (L2) genotypes included B0/W148 (19 and 76); Central Asian (85 and 47) and its Central Asian Outbreak subtype (12 and 25); and orphan L2 genotypes (14 and 16). We also revealed 88 and 30 Euro-American (L4) strains in the Krasnoyarsk Krai and Irkutsk region, respectively. The identified L4 genotypes included Haarlem (9 and 6); LAM (35 and 7); Ural (19 and 5); S (2 and 0); and orphan L4 genotypes (15 and 12). We developed an approach employing two PCR to discriminate the principal M. tuberculosis genotype set into L2 and L4 lineages covering over 99% of all tuberculosis strains circulating in Russian Federation and other North Eurasian countries. Within the proposed framework, the required and sufficient number of PCR for genotyping and in-depth subtyping does not exceed eight. The feasibility of developing tests for further subtyping of epidemiological M. tuberculosis genotypes is discussed.