Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method

We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing.