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A Two-Tube Combined TaqMan/SYBR Green Assay to Identify Mycobacteria and Detect Single Global Lineage-Defining Polymorphisms in Mycobacterium tuberculosis 

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Presentation on theme: "A Two-Tube Combined TaqMan/SYBR Green Assay to Identify Mycobacteria and Detect Single Global Lineage-Defining Polymorphisms in Mycobacterium tuberculosis "— Presentation transcript:

1 A Two-Tube Combined TaqMan/SYBR Green Assay to Identify Mycobacteria and Detect Single Global Lineage-Defining Polymorphisms in Mycobacterium tuberculosis  Eddy S.G. Cheah, Joanne Malkin, Robert C. Free, Su-Min Lee, Nelun Perera, Gerrit Woltmann, Hemu Patel, Patrick T. Kimmitt, Rebecca J. Smith, Kumar Rajakumar, Michael R. Barer  The Journal of Molecular Diagnostics  Volume 12, Issue 2, Pages (March 2010) DOI: /jmoldx Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Schematic illustration of the combined 16S-RD assay. A: For the 16S assay (tube 1), TaqMan (T) signals show the presence or absence of MTBC-specific 16S rDNA sequence, whereas the SYBR Green (S) signals indicate the presence or absence of Mycobacterium genus-specific 16S rDNA sequence. B: For the RD assay (tube 2), T signals show the presence or absence of RD750, whereas the S signals act as further confirmation of MTBC-specific sequence and a positive assay control when the T signal is negative. T+ S− signals would be anomalous in both assays, indicating amplicons that do not interact with SYBR Green; no results were obtained in this category. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Diagrammatic representation of the RD assay targets.
The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Effect of combining TaqMan and SYBR Green chemistries in single real-time PCRs. M. tuberculosis CDC 1551 target at 107 gene copies and two 100-fold serial dilutions were tested. Dotted lines represent reactions containing both TaqMan and SYBR Green, whereas solid lines represent reactions containing either SYBR Green (A) or TaqMan (B) alone. A shows 16S SYBR Green signals, and B shows RD TaqMan Cy5 signals. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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