title: Development and application of single-tube multiplex real-time PCR for lineage classification of Mycobacterium tuberculosis based on large sequence polymorphism in Northeast Thailand.
abstract: An appreciation of the genetic diversity of Mycobacterium tuberculosis (Mtb) is needed for effective planning of strategies in tuberculosis (TB) control. Large sequence polymorphisms (LSPs) are the molecular epidemiological and evolutionary markers for classification of Mtb into East Asian (EA) or Beijing, Indo-Oceanic (IO), Euro-American (EuA) and East African-Indian (EAI) lineages. We aimed to develop a single-tube multiplex real-time PCR assay using melting curve analysis for lineage classification of Mtb based on LSPs. The technique was optimized and tested with well-characterized strains (n = 89). The developed technique was then applied to classify Mtb isolates from TB patients (n = 256) randomly recruited from 19 provinces covering Northeast Thailand in 2013-2014. The technique demonstrated 100% sensitivity and specificity based on well-characterized strains compared to conventional techniques. The detection limit of the technique is 0.05 ng of genomic DNA of Mtb. The 256 Mtb isolates represented IO (n = 178, 70%), Beijing (n = 60, 23%) and EuA (n = 18, 7%) lineages. Significant associations of the Beijing lineage with drug resistance (p < 0.001) and younger average age of TB patients (p < 0.001) compared to other lineages were shown. The single-tube multiplex real-time PCR technique provides a simple, rapid and high performance tool for characterizing Mtb based on LSPs.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address: kiatichai@kku.ac.th.
authors.forename: Kiatichai
authors.initials: K
authors.lastname: Faksri
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Rattanavinan
authors.initials: R
authors.lastname: Hanchaina
authors.affiliation: Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Arunnee
authors.initials: A
authors.lastname: Sangka
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Wises
authors.initials: W
authors.lastname: Namwat
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
mesh_terms: D017668:Age of Onset; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D017720:Molecular Epidemiology; D060885:Multiplex Polymerase Chain Reaction; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D010641:Phenotype; D011110:Polymorphism, Genetic; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013785:Thailand / Q000453:epidemiology; D014376:Tuberculosis / Q000175:diagnosis / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial
keywords: Large sequence polymorphism; Multiplex real-time PCR; Mycobacterium tuberculosis; Northeast Thailand