Combined Molecular Gram Typing and High-Resolution Melting Analysis for Rapid Identification of a Syndromic Panel of Bacteria Responsible for Sepsis-Associated.

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Combined Molecular Gram Typing and High-Resolution Melting Analysis for Rapid Identification of a Syndromic Panel of Bacteria Responsible for Sepsis-Associated Bloodstream Infection Hani Ozbak, Paul Dark, Satyanarayana Maddi, Paul Chadwick, Geoffrey Warhurst The Journal of Molecular Diagnostics Volume 14, Issue 2, Pages 176-184 (March 2012) DOI: 10.1016/j.jmoldx.2011.12.004 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Grouping of bloodstream infection pathogens by Tm. Melting curves for 21 bacterial species in the bloodstream infection panel show separation into three distinct groups (Gp1, Gp2, and Gp3). Tm data for each species are given in Table 2. Gp, group. The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 HRMA of Tm group 1 organisms. Differentiation of S. aureus from coagulase-negative staphylococci (CoNS) species, by differences in curve shape against K. pneumoniae as the reference organism, is shown. The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Decision-tree identification of Tm group 2 organisms by HRMA. Differentiation of six pathogen species, based on sequential analysis of difference curve shape using multiple reference organisms [S. aureus (A, C, and E), E. faecalis (B), and C. freundii (D)] and primer PP3 (A and B) or PP4 (C, D, and E), is shown. E. aerogenes, Enterobacter aerogenes; H. influenzae, Haemophilus influenzae; K. oxytoca, Klebsiella oxytoca; S. marcescens, Serratia marcescens. The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Decision-tree identification of Tm group 3 organisms by HRMA. Differentiation of 11 pathogen species based on Gram classification, followed by sequential analysis of difference curve shape using multiple reference organisms [K. pneumoniae (A and B), S. maltophilia (C), and primer PP3 (A and B) or PP4 (C)], is shown. The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Concentration dependence of real-time PCR-HRMA: amplification curves for different quantities of K. pneumoniae DNA (250 fg to 250 ng) using the PP3 primer pair (A) and difference plots (against S. aureus as the reference) after HRMA of the samples shown in A (B). The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Reproducibility of difference curve shape for S. aureus. Intraindividual variability in difference curve for S. aureus against the K. pneumoniae reference. A: Results of 20 replicate real-time PCR-HRMAs of a preparation of S. aureus DNA (25 ng per assay). B: Interindividual variability. Data show a difference plot of six different isolates of S. aureus against the K. pneumoniae reference. The Journal of Molecular Diagnostics 2012 14, 176-184DOI: (10.1016/j.jmoldx.2011.12.004) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions