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- Title
A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.
- Authors
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.; Tuberculosis Research Unit
- Abstract
<bold>Background: </bold>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.<bold>Method: </bold>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.<bold>Results: </bold>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.<bold>Conclusion: </bold>The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.
- Subjects
UGANDA; SPUTUM microbiology; TUBERCULOSIS microbiology; DNA analysis; DOCUMENTATION; GENETIC polymorphisms; GENETIC techniques; MYCOBACTERIUM tuberculosis; NUCLEOTIDES; POLYMERASE chain reaction; RESEARCH funding; TUBERCULOSIS; SEQUENCE analysis; GENOTYPES
- Publication
BMC Infectious Diseases, 2015, Vol 15, Issue 1, p1
- ISSN
1471-2334
- Publication type
journal article
- DOI
10.1186/s12879-015-1121-7