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 transcript:

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 452-458 (September 2008) DOI: 10.2353/jmoldx.2008.080008 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 452-458DOI: (10.2353/jmoldx.2008.080008) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 452-458DOI: (10.2353/jmoldx.2008.080008) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions