Development of single-tube multiplex PCR for classification of Mycobacterium tuberculosis lineages based on large sequence polymorphisms.

Large sequence polymorphisms (LSPs) or regions of differences (RDs) are molecular epidemiological and evolutionary markers used to classify Mycobacterium tuberculosis (MTB) into East Asian (Beijing), Indo-Oceanic (IO), Euro-American (EuA) and East African Indian (EAI) lineages. The most used method is separate PCR and sequencing for each RD. We developed a single-tube multiplex PCR using four primer pairs specific to the four MTB lineages and a primer pair for species-specific RD9 with genomic DNA extracted from isolated colonies. The single-tube multiplex PCR produced lineage-specific amplicon patterns capable of differentiating the four MTB lineages. Sensitivity and specificity of the assay were 100% when differentiating MTB lineages from other species and strains of bacteria. The limit of detection of genomic MTB DNA was 12.5 ng. This single-tube multiplex PCR method offers a simple, rapid and reliable method for classification of MTB lineages based on LSPs.