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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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 250-256 (March 2010) DOI: 10.2353/jmoldx.2010.090030 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2010 12, 250-256DOI: (10.2353/jmoldx.2010.090030) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Diagrammatic representation of the RD assay targets. The Journal of Molecular Diagnostics 2010 12, 250-256DOI: (10.2353/jmoldx.2010.090030) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2010 12, 250-256DOI: (10.2353/jmoldx.2010.090030) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions