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

Wampande EM, Hatzios SK, Achan B, Mupere E, Nsereko M, Mayanja HK, Eisenach K, Boom WH, Gagneux S, Joloba ML, Tuberculosis Research Un - BMC Infect. Dis. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. wamps@covab.mak.ac.ug.

ABSTRACT

Background: 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.

Method: 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.

Results: 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.

Conclusion: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.

No MeSH data available.


Related in: MedlinePlus

Pair wise alignment of H37Rv (wild type) and MTB lineage (mutant) sequences: BioEdit Version 7.2.5 (Ibis Biosciences, USA)was used to align the wild type (Rv004c, Rv2962 and Rv0129c) and the corresponding mutant (MTB L3, MTB L4-NU or MTB L4-U) sequences. The shaded and bold nucleotide shows the point mutation
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Fig3: Pair wise alignment of H37Rv (wild type) and MTB lineage (mutant) sequences: BioEdit Version 7.2.5 (Ibis Biosciences, USA)was used to align the wild type (Rv004c, Rv2962 and Rv0129c) and the corresponding mutant (MTB L3, MTB L4-NU or MTB L4-U) sequences. The shaded and bold nucleotide shows the point mutation

Mentions: To ascertain the accuracy of LRPS in genotyping MTBC lineages PCR products of 9 MTBC isolates (3 isolates for each lineage) were sequenced. The resulting sequences of the gene containing the lineage-specific SNP were compared with the corresponding H37Rv sequences using Bio Edit software (Ibis Biosciences, USA) the data confirmed that the sequenced PCR products contained lineage-specific SNPs (See Fig. 3).Fig. 3


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

Wampande EM, Hatzios SK, Achan B, Mupere E, Nsereko M, Mayanja HK, Eisenach K, Boom WH, Gagneux S, Joloba ML, Tuberculosis Research Un - BMC Infect. Dis. (2015)

Pair wise alignment of H37Rv (wild type) and MTB lineage (mutant) sequences: BioEdit Version 7.2.5 (Ibis Biosciences, USA)was used to align the wild type (Rv004c, Rv2962 and Rv0129c) and the corresponding mutant (MTB L3, MTB L4-NU or MTB L4-U) sequences. The shaded and bold nucleotide shows the point mutation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590274&req=5

Fig3: Pair wise alignment of H37Rv (wild type) and MTB lineage (mutant) sequences: BioEdit Version 7.2.5 (Ibis Biosciences, USA)was used to align the wild type (Rv004c, Rv2962 and Rv0129c) and the corresponding mutant (MTB L3, MTB L4-NU or MTB L4-U) sequences. The shaded and bold nucleotide shows the point mutation
Mentions: To ascertain the accuracy of LRPS in genotyping MTBC lineages PCR products of 9 MTBC isolates (3 isolates for each lineage) were sequenced. The resulting sequences of the gene containing the lineage-specific SNP were compared with the corresponding H37Rv sequences using Bio Edit software (Ibis Biosciences, USA) the data confirmed that the sequenced PCR products contained lineage-specific SNPs (See Fig. 3).Fig. 3

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. wamps@covab.mak.ac.ug.

ABSTRACT

Background: 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.

Method: 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.

Results: 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.

Conclusion: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.

No MeSH data available.


Related in: MedlinePlus