Results of the EQA for molecular detection of CCHFV – Part 1.

<p>+: positive (17): Midilli et al., 2007.</p><p>−: negative (30): Wölfel et al., 2007.</p><p><b>bold</b>: quantified result (32): Drosten et al., 2002.</p>*<p>: correct strain (35): Rodriguez et al., 1997.</p><p>FN: false negative result (36): Schwarz et al., 1996.</p><p>qRT: real-time RT-PCR (c): commercial assay.</p><p>nRT: nested RT-PCR (h): in house assay.</p><p>cRT: conventional RT-PCR.</p

Odd ratios obtained for each method by using real-time PCR as the reference.

<p>OR: odd ratio; CI: confidence interval.</p

Results of the EQA for molecular detection of CCHFV – Part 2.

<p>+: positive (21): Duh et al., 2006.</p><p>−: negative (30): Wölfel et al., 2007.</p><p>n.d.: not determined (32): Drosten et al., 2002.</p><p><b>bold</b>: quantified result (33a): Midilli et al., 2009 (Europe 1).</p>*<p>: correct strain (33b): Midilli et al., 2009 (Europe 2).</p><p>FN: false negative result (34): Papa. et al., 2007.</p><p>PN: false positive result (35): Rodriguez et al., 1997.</p><p>qRT: real-time RT-PCR (36): Schwarz et al., 1996.</p><p>nRT: nested RT-PCR (37): Deyde et al., 2006.</p><p>cRT: conventional RT-PCR (38): Burt et al.,1998.</p><p>(c): commercial assay (39): Burt et al., 2005.</p><p>(h): in house assay.</p

Phylogenetic tree of the <i>bont/A</i> sequence retrieved in contaminated food and several toxinotype A strains.

<p>A neighbor-joining tree was constructed based on the <i>bont/A</i> sequence retrieved by the RMA for the enrichment culture of the <i>C. botulinum</i> strain in a naturally contaminated salad specimen, and the corresponding sequences of 6 strains representative of 5 known <i>C. botulinum</i> toxinotype A subtypes: strain Hall (A1), NCTC 2916 (A[B]), Kyoto (A2), Loch Maree (A3), 657 (Ba4) and A661222 (A5). Bootstrap values and genetic distance (bar) are shown. The Genbank accession number of each strain is indicated in parentheses. The <i>bont</i> sequence obtained from the contaminated salad specimen is marked in bold.</p

Description of the sequences tiled on the PathogenID v2.0 microarray for the detection and characterization of BoNT-producing clostridia.

<p>Description of the sequences tiled on the PathogenID v2.0 microarray for the detection and characterization of BoNT-producing clostridia.</p

Neurotoxin gene cluster organization.

<p>In the gene cluster encoding the botulinum neurotoxin complex, the “ha cluster” appears to be associated with type A1, A5, B, C, D and G <i>bont</i> genes, and the “orf-X cluster” with type A1, A2, A3, A4, F and E <i>bont</i> genes. ¹Although the type A1 <i>bont</i> gene is mostly found in a ha cluster, it has been found associated with an orf-X cluster in some single toxin strains and in all bivalent strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067510#pone.0067510-Carter1" target="_blank">[18]</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067510#pone.0067510-Raphael1" target="_blank">[17]</a>.²The botR gene is in front of the ha genes in all toxinotype C and D strains.³The Ha33 gene is absent in all toxinotype G strains.⁴The botR gene is absent in all toxinotype E strains</p

Identification and characterization of <i>C. botulinum</i> strains based on the sequences retrieved by the PathogenID v2.0 microarray and the BLAST analysis results.

<p>Identification and characterization of <i>C. botulinum</i> strains based on the sequences retrieved by the PathogenID v2.0 microarray and the BLAST analysis results.</p

Detection spectrum of the PathogenID_v2.0 according to the nucleotide diversity of the tested BoNT-producing strains.

<p>The spectrum of detection of BoNT-producing clostridia by the PathogenID v2.0 was assessed using 9 well-characterized strains representative of all toxinotypes and several toxin A subtypes. Each sequence obtained by the RMA for which the percentage of nucleotide divergence compared to the corresponding tiled sequence is known (<i>n</i> = 118), is indicated by a blue diamond, and presented according to the percentage of nucleotide bases determined by the RMA (call rate). The linear association between these two parameters is shown, and demonstrates a good correlation (correlation coefficient R² of 0.79).</p

Call-rates observed for the different <i>C. botulinum</i> sequences retrieved by the PathogenID V2.0 microarray for all tested bacterial strains.

<p>Sequences retained after filtering using the defined threshold are designated in bold.</p

Phylogenetic tree derived from nucleotide sequence data of the entire S segment.

The phylogenetic analyses were carried out after multiple alignments of the obtained sequences along with the GenBank reference sequences (including all published sequences). Maximum likelihood (ML) methods were used to construct trees based on full sequences of the S segment (1690 nt). The GenBank accession numbers for the S gene are OR528950, OR528951, OR528952, OR528953 for samples 7, 108, 120 and 166, respectively.</p