Stephanie L. Angione, Aartik A

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A Novel Subtyping Assay for Detection of Clostridium difficile Virulence Genes Stephanie L. Angione, Aartik A. Sarma, Aleksey Novikov, Leah Seward, Jennifer H. Fieber, Leonard A. Mermel, Anubhav Tripathi The Journal of Molecular Diagnostics Volume 16, Issue 2, Pages 244-252 (March 2014) DOI: 10.1016/j.jmoldx.2013.11.006 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Components of the droplet sandwich platform and workflow. A: Exploded view of the platform components: ITO-coated glass (1) with a compound droplet (2) surrounded by a spacer (3) and covered with a coverslip (4). B: The fully assembled platform effectively sandwiches the compound droplet in a reaction chamber. The slide dimensions are 40 mm × 40 mm, and the compound droplet is approximately 2.8 mm in diameter. C: Heating profile generated by COMSOL Multiphysics 4.3b simulation software. The ITO-coated glass resistive surface heats radially up to 95°C when 15 V is applied to the resistive surface. D: Plot of DNA amplification with fluorescent intercalating dye shows positive samples (blue), negative samples with no change over time (red), and the calculated threshold (black). Fluorescence is collected in real time during the extension phase of PCR, using a photomultiplier tube. AU, arbitrary units; Norm., normalized. The Journal of Molecular Diagnostics 2014 16, 244-252DOI: (10.1016/j.jmoldx.2013.11.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Electrophoresis gel plot of single-gene and multiplex PCR using C. difficile reference strain 630 and the NAP1/027/BI cultured isolate VA17. The template is indicated at the top of each lane. Lanes 1 and 4, tcdB primer set; lanes 2 and 5, tcdC primer set; lanes 3 and 6, cdtB primer set; lanes 7 and 8, multiplex results for the reference strain 630 and the hypervirulent VA17 isolate, respectively. The Journal of Molecular Diagnostics 2014 16, 244-252DOI: (10.1016/j.jmoldx.2013.11.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Electrophoresis gel plot of multiplex PCR. A different clinical stool DNA isolate was used as template for each amplification, from eight positive samples and two negative controls. A sample identifier for the template is indicated at the top of each lane. Each positive sample (lanes 1 to 8; samples A–C, E–G, L, and M) shows the 328-bp amplicon for tcdB, followed by either a 300-bp amplicon for tcdC (lanes 1, 3, 7, and 8; samples A, C, L, and M) or a 282-bp amplicon for tcdC with the NAP1/027/BI deletion (lanes 2, 5, and 6; samples B, F, and G). Sample E (lane 4) shows a greater deletion in tcdC at 261 bp. Samples with any deletion in tcdC were also positive for binary toxin, indicated by the amplicon at 190 bp for cdtB (B, E, F, and G). Lanes 9 and 10 (samples N and O) show representative samples of clinical negative controls. B: Electropherogram for samples E, F, and L. The electropherogram image shows the peaks of the three amplicons cdtB, tcdC, and tcdB for samples E, F, and L, respectively. The base-pair shifts in the tcdC amplicon are easily visible between 70.0 seconds and 77.5 seconds; sample L shows the longest amplicon at 75.25 seconds, followed by sample F (18-bp deletion) at 73.55 seconds and then sample E (39-bp deletion) at 71.25 seconds. The peak for tcdB appears in all three samples at 79.0 seconds and cdtB in samples E and F at 64.4 seconds. The Journal of Molecular Diagnostics 2014 16, 244-252DOI: (10.1016/j.jmoldx.2013.11.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Singleplex qPCR results for tcdB, tcdC, and cdtB amplification in C. difficile 630. The tcdB and tcdC amplicons have CT values of 12.2 and 12.7, respectively, for a starting concentration of 1.0 ng/μL. The cdtB gene is not present in the C. difficile 630 genome, and amplification did not occur. B: The corresponding electrophoresis gel plot for PCR performed on the droplet sandwich platform shows appropriate-size amplicons for tcdB at 328 bp and tcdC at 300 bp. Amplification of cdtB shows no product, and the assay is negative for that gene. The Journal of Molecular Diagnostics 2014 16, 244-252DOI: (10.1016/j.jmoldx.2013.11.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 A: Singleplex qPCR results for tcdB, tcdC, and cdtB amplification in the VA17 NAP1/027/BI hypervirulent strain of C. difficile. The tcdB, tcdC, and cdtB amplicons have CT values of 11.8, 15.2, and 11.3, respectively, for a starting concentration of 1.0 ng/μL. B: The corresponding electrophoresis gel plot for PCR performed on the droplet sandwich platform shows, the amplicons are of the appropriate size: 328 bp for tcdB, 282 bp for tcdC, and 190 bp for cdtB. The amplicon size of tcdC indicates the presence of the 18-bp deletion associated with NAP1/027/BI. The Journal of Molecular Diagnostics 2014 16, 244-252DOI: (10.1016/j.jmoldx.2013.11.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions