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A single-nucleotide-polymorphism real-time PCR assay for genotyping of mycobacterium tuberculosis complex in peri-urban Kampala
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 3247023
Author(s) Wampande, Eddie M.; Hatzios, Stavroula K.; Achan, Beatrice; Mupere, Ezekiel; Nsereko, Mary; Mayanja, Harriet K.; Eisenach, Kathleen; Boom, W. Henry; Gagneux, Sebastien; Joloba, Moses L.; TB Res Unit
Author(s) at UniBasel Gagneux, Sebastien
Year 2015
Title A single-nucleotide-polymorphism real-time PCR assay for genotyping of mycobacterium tuberculosis complex in peri-urban Kampala
Journal BMC infectious diseases
Volume 15
Pages / Article-Number 396
Keywords Single nucleotide polymorphism, Lineages, Long sequence polymorphism, High-throughput
Abstract

Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients.; Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing.; The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage.; The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.

Publisher BioMed Central
ISSN/ISBN 1471-2334
edoc-URL http://edoc.unibas.ch/dok/A6438871
Full Text on edoc Available
Digital Object Identifier DOI 10.1186/s12879-015-1121-7
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/26423522
ISI-Number WOS:000362252200005
Document type (ISI) Article
 
   

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