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Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium.

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Presentation on theme: "Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium."— Presentation transcript:

1 Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile  Wing Huen A. Chow, Cindy McCloskey, Yanhong Tong, Lin Hu, Qimin You, Ciarán P. Kelly, Huimin Kong, Yi-Wei Tang, Wen Tang  The Journal of Molecular Diagnostics  Volume 10, Issue 5, Pages (September 2008) DOI: /jmoldx Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Schematic diagram of the mechanisms for IsoAmp CDAD test. A: Using a biotin-labeled excess primer, a 3′-FITC-labeled tcdA gene probe and a 3′-DIG-labeled IC probe can bind to the biotin-labeled single-stranded DNA generated from the asymmetric HDA reaction in the amplification of C. difficile toxin gene tcdA and an IC. These probe-target hybrids then bind to streptavidin-conjugated color particles and are captured by an anti-FITC and an anti-DIG antibody coated on the DNA detection strip. B: The procedure of using a BESt Cassette for amplicon detection. The amplification reaction vessel is placed in an amplicon cartridge (step 1); the cartridge is closed to immobilize the reaction vessel (step 2); the amplicon cartridge is inserted into the detection chamber (step 3); the handle of the detection chamber is closed to seal the vessel into the chamber and to cut open the running buffer reservoir and the reaction vessel (step 4); and after 10 to 15 minutes, the detection window of the chamber is read by eye to score the assay result (step 5). The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Determining the analytical sensitivity of IsoAmp CDAD test. Different amounts of genomic DNA of an A+/B+ C. difficile strain (ATCC 9689) mixed with 5 × 103 copies of IC plasmid and 2 μl of pooled stool DNA from CDAD-negative fecal samples were used as the template in the IsoAmp CDAD test. The amounts of C. difficile genomic DNA per reaction used in the test are as follows: 1, 200 copies; 2, 20 copies; 3, 10 copies; 4, 5 copies; 5, 2 copies; and 6, 0 copies. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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