title: Development of single-tube multiplex PCR for classification of Mycobacterium tuberculosis lineages based on large sequence polymorphisms.
abstract: 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.
authors: Rattanavinan
authors: R
authors: Hanchaina
authors: Viraphong
authors: V
authors: Lulitanond
authors: Wises
authors: W
authors: Namwat
authors: Kiatichai
authors: K
authors: Faksri
mesh_terms: D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D013785:Thailand
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial