A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.

Eddie M Wampande,Moses L Joloba,W Henry Boom,Mary Nsereko,Ezekiel Mupere,Stavroula K Hatzios,Beatrice Achan,Harriet K Mayanja,Sebastien Gagneux,Kathleen Eisenach

doi:10.1186/s12879-015-1121-7

Abstract

BackgroundAccurate 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.MethodGenotyping 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.ResultsThe 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.ConclusionThe LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-1121-7) contains supplementary material, which is available to authorized users.

Highlights

Mycobacterium tuberculosis (MTB) is an acid-fast bacillus that causes tuberculosis (TB) a chronically debilitating disease with a mortality rate approaching 2 million deaths per year [1,2,3]
Of the 300 sputum samples analyzed, 58 % contained Mycobacterium tuberculosis complex (MTBC) from the MTBC-Uganda family, 27 % from the MTBC lineage 4, 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage
A single nucleotide polymorphisms (SNPs) corresponding to a specific MTB lineage/sublineage was annotated by showing its position in the corresponding gene (ORF) and the associated nucleotide change (See Additional file 1: Table S1)

Summary

Introduction

Mycobacterium tuberculosis (MTB) is an acid-fast bacillus that causes tuberculosis (TB) a chronically debilitating disease with a mortality rate approaching 2 million deaths per year [1,2,3]. For example robust techniques such multi-locus sequence typing (MLST) [14] and whole genome sequencing (WGS) [15, 16], are difficult to adopt in resource-limited countries because they are prohibitively expensive [17] Other techniques, such as MIRU-VNTR, IS6110-RFLP, PGRS-RFLP, and CRISP [18, 19], can erroneously classify MTBC lineages [16, 20] due to homoplasy and are technically cumbersome. Some of these methods typically require prior culturing of MTB from sputum samples, a process that takes 1–2 months [21]. 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

Results

Discussion

Conclusion