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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

Comparing RD 724 LSP-PCR and LRPS typing: Samples (N = 70) were analyzed by primers specific for RD 724 (lane M = DNA marker, 1 = MTB Uganda genotype, 2 = H37Rv, 3 = negative control (no DNA added), lane 4–72 = test samples) a band of 1.5 kb identifies MTB Uganda family, while that of 1.3 kb identifies other MTBC other than Uganda family (Panel a). For Panel b specific primers/probes (Rv004c primer/probe set) containing lineage specific SNP were used, the top panel shows the amplification (see also Additional file 5: Figure S2a) of the target region while the bottom panel shows the derived melting temperature. A peak of 62 °C shows the Uganda family while that of 68 °C shows the wild type (non-Uganda family)
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Fig1: Comparing RD 724 LSP-PCR and LRPS typing: Samples (N = 70) were analyzed by primers specific for RD 724 (lane M = DNA marker, 1 = MTB Uganda genotype, 2 = H37Rv, 3 = negative control (no DNA added), lane 4–72 = test samples) a band of 1.5 kb identifies MTB Uganda family, while that of 1.3 kb identifies other MTBC other than Uganda family (Panel a). For Panel b specific primers/probes (Rv004c primer/probe set) containing lineage specific SNP were used, the top panel shows the amplification (see also Additional file 5: Figure S2a) of the target region while the bottom panel shows the derived melting temperature. A peak of 62 °C shows the Uganda family while that of 68 °C shows the wild type (non-Uganda family)

Mentions: LSP-PCR and targeted sequencing reactions were run in parallel to validate the LRPS assay. A total of 70 MTBC isolates (confirmed by IS6110 PCR) were genotyped using LSP-PCR with primers specific for the RD 724 deletion in parallel with LRPS assay using Rv004ca primer/probes set (Table 1). While both assays were equally capable of identifying MTBC Uganda (Fig. 1 and Additional file 5 Fig S2(a)) only the LRPS assay was able to genotype all 70 samples the LSP-PCR assay failed to identify the MTBC lineages of several isolates (Fig. 1: lane 11 13, 14, 15, 21, 22, 26, 27, 33, 39, 43, 45, 56, 61, 62). With that result, both assays were re-evaluated using serially diluted H37Rv genomic DNA (10 ng–100 ng for LSP-PCR and 1 ng–10 ng for LRPS per PCR reaction) extracted by the Enzyme/CTAB method [29]. The detection limit of the LSP-PCR assay was 10-fold higher than that of the LRPS assay (30 ng approximately 7 × 106 copies/reaction and 3 ng approximately 7 × 105 copies/reaction) respectively (Additional file 4: Figure S1). Later, DNA from those samples that previously produced no LSP-PCR product (Fig. 1: lane 11,13,14,15, 21, 22, 26, 27, 33, 39, 43, 45, 56, 61, 62) were re-extracted and repeated the LSP-PCR genotyping reactions with at least 30 ng template DNA. This time, genotyping data were obtained for all isolates and were in agreement with those of the LRPS assay (data not shown).Fig. 1


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)

Comparing RD 724 LSP-PCR and LRPS typing: Samples (N = 70) were analyzed by primers specific for RD 724 (lane M = DNA marker, 1 = MTB Uganda genotype, 2 = H37Rv, 3 = negative control (no DNA added), lane 4–72 = test samples) a band of 1.5 kb identifies MTB Uganda family, while that of 1.3 kb identifies other MTBC other than Uganda family (Panel a). For Panel b specific primers/probes (Rv004c primer/probe set) containing lineage specific SNP were used, the top panel shows the amplification (see also Additional file 5: Figure S2a) of the target region while the bottom panel shows the derived melting temperature. A peak of 62 °C shows the Uganda family while that of 68 °C shows the wild type (non-Uganda family)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Comparing RD 724 LSP-PCR and LRPS typing: Samples (N = 70) were analyzed by primers specific for RD 724 (lane M = DNA marker, 1 = MTB Uganda genotype, 2 = H37Rv, 3 = negative control (no DNA added), lane 4–72 = test samples) a band of 1.5 kb identifies MTB Uganda family, while that of 1.3 kb identifies other MTBC other than Uganda family (Panel a). For Panel b specific primers/probes (Rv004c primer/probe set) containing lineage specific SNP were used, the top panel shows the amplification (see also Additional file 5: Figure S2a) of the target region while the bottom panel shows the derived melting temperature. A peak of 62 °C shows the Uganda family while that of 68 °C shows the wild type (non-Uganda family)
Mentions: LSP-PCR and targeted sequencing reactions were run in parallel to validate the LRPS assay. A total of 70 MTBC isolates (confirmed by IS6110 PCR) were genotyped using LSP-PCR with primers specific for the RD 724 deletion in parallel with LRPS assay using Rv004ca primer/probes set (Table 1). While both assays were equally capable of identifying MTBC Uganda (Fig. 1 and Additional file 5 Fig S2(a)) only the LRPS assay was able to genotype all 70 samples the LSP-PCR assay failed to identify the MTBC lineages of several isolates (Fig. 1: lane 11 13, 14, 15, 21, 22, 26, 27, 33, 39, 43, 45, 56, 61, 62). With that result, both assays were re-evaluated using serially diluted H37Rv genomic DNA (10 ng–100 ng for LSP-PCR and 1 ng–10 ng for LRPS per PCR reaction) extracted by the Enzyme/CTAB method [29]. The detection limit of the LSP-PCR assay was 10-fold higher than that of the LRPS assay (30 ng approximately 7 × 106 copies/reaction and 3 ng approximately 7 × 105 copies/reaction) respectively (Additional file 4: Figure S1). Later, DNA from those samples that previously produced no LSP-PCR product (Fig. 1: lane 11,13,14,15, 21, 22, 26, 27, 33, 39, 43, 45, 56, 61, 62) were re-extracted and repeated the LSP-PCR genotyping reactions with at least 30 ng template DNA. This time, genotyping data were obtained for all isolates and were in agreement with those of the LRPS assay (data not shown).Fig. 1

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