Visual Format for Detection of Mycobacterium tuberculosis and M

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Visual Format for Detection of Mycobacterium tuberculosis and M Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons  Parameet Kumar, Kapili Nath, Bimba Rath, Manas K. Sen, Potharuju Vishalakshi, Devender S. Chauhan, Vishwa M. Katoch, Sarman Singh, Sanjay Tyagi, Vishnubhatla Sreenivas, Hanumanthappa K. Prasad  The Journal of Molecular Diagnostics  Volume 11, Issue 5, Pages 430-438 (September 2009) DOI: 10.2353/jmoldx.2009.080135 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Position of primers and beacons in the mce3 operon11 used for the detection and identification of M. tuberculosis and M. bovis. 12.7-kb insert prevents Taq polymerase-mediated amplification in M. tuberculosis, hence amplification seen only in M. bovis. The Journal of Molecular Diagnostics 2009 11, 430-438DOI: (10.2353/jmoldx.2009.080135) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Signal-to-noise ratio of molecular beacons. The signal-to-noise ratio of M. tuberculosis and M. bovis beacons was determined by the visual format assay. Tube 1, buffer (negative control); tube 2, buffer + M. tuberculosis beacon (negative control); tube 3, buffer + M. tuberculosis beacon + target oligonucleotide of M. tuberculosis (positive control); tube 4, buffer + M. tuberculosis beacon + target oligonucleotide of M. bovis; tube 5, buffer (negative control); tube 6, buffer + M. bovis beacon (negative control); tube 7, buffer + M. bovis beacons + target oligonucleotide M. bovis (positive control); tube 8, buffer + M. bovis beacon + target oligonucleotide M. tuberculosis. B: Thermo-denaturation profile for molecular beacons. Dotted line denotes M. bovis beacon with its target oligonucleotide melt curve. Continuous line denotes M. tuberculosis beacon with its target oligonucleotide melt curve. All other lines denotes various controls. The Journal of Molecular Diagnostics 2009 11, 430-438DOI: (10.2353/jmoldx.2009.080135) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Amplification plots show the detection of the serially diluted DNA of M. tuberculosis (A) and M. bovis (B) by real-time PCR using molecular beacons. Tenfold serial dilution of the mycobacterial DNA, ranging from 5 × 106 to 5 bacilli/reaction (from left to right) and the negative control were amplified in individual tubes. PCR cycles have been plotted against fluorescence intensity. The Journal of Molecular Diagnostics 2009 11, 430-438DOI: (10.2353/jmoldx.2009.080135) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Limit of detection of M. tuberculosis and M. bovis DNA by PCR assay using molecular beacons by the visual format (A) and gel-based assays (B). M. tuberculosis and M. bovis DNA were added as follows: tube 1, 50 ng (5 × 106 bacilli); tube 2, 5 ng (5 × 105 bacilli); tube 3, 500 pg (5 × 104 bacilli); tube 4, 50 pg (5 × 103 bacilli); tube 5, 5 pg (5 × 102 bacilli); tube 6, 500 fg (5 × 101 bacilli); tube 7, 50 fg (5 bacilli); tube 8, negative control in visual format; tube 8′, 5 fg (∼1 bacilli) in gel-based; tube 9, negative control. The Journal of Molecular Diagnostics 2009 11, 430-438DOI: (10.2353/jmoldx.2009.080135) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 A: CM-PCR for detecting and differentiating M. tuberculosis and M. bovis in the CSF sample, CS-112. The ethidium bromide stained amplification products of M. tuberculosis and M. bovis generated by using FP, RPT, and RPC primers were electrophoresed on 2.2% agarose gel. The 162- and 127-bp products obtained in M. tuberculosis and M. bovis respectively, are indicated. Lane 1, PF-7; lane 2, CSF; lane 3, M. tuberculosis (positive control, H37Rv); lane 4, M. bovis (positive control AN-5); lane M, 50-bp molecular weight marker; lanes 6 and 7, negative control. B: Visual format using molecular beacons for detecting and differentiating M. tuberculosis and M. bovis in the CSF sample, CS-112. Tubes 1–4 showing fluorescence obtained using primer pair FP and RPT with M. tuberculosis beacon and tubes 5–8 showing fluorescence obtained using primer pair FP and RPC with M. bovis beacon. Tubes 1 and 5, PF-7; tubes 2 and 6, CSF; tube 3, M. tuberculosis positive control; tube 7, M. bovis positive control; tubes 4 and 8, negative control. The Journal of Molecular Diagnostics 2009 11, 430-438DOI: (10.2353/jmoldx.2009.080135) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions