Annealing sites for species-specific primers that targeted exons 3–5 of the calmodulin gene (<i>CAL</i>), and an internal positive control (IPC) that targeted the 5.8s region of ribosomal DNA.

<p>Predicted PCR products are shown for (A) <i>Sporothrix brasiliensis</i> primers, Sbra-F and Sbra-R; (B) <i>Sporothrix schenckii s</i>. <i>str</i>. primers, Ssch-F and Ssch-R; (C) <i>Sporothrix globosa</i> primers, Sglo-F and Sglo-R; (D) <i>Sporothrix mexicana</i> primers, Smex-F and Smex-R; (E) <i>Sporothrix pallida</i> primers, Spa-F and Spa-R; (F) <i>Ophiostoma stenoceras</i> primers, Oste-F and Oste-R. Above each depiction of the <i>CAL</i> gene, a scale indicates the sequence length, with ticks at intervals of 50 bp. (G) IPC primers IPC-F and IPC-R. ITS: internal transcribed spacer</p

<i>Sporothrix brasiliensis</i> distribution in mouse organs at 10 days post infection.

<p>Mice were challenged with intravenous injections of 1×10⁶ yeast cells (<i>S</i>. <i>brasiliensis</i>—CBS 132990). Gel electrophoresis (1.2% agarose) show PCR products (5-μL aliquots) amplified with primers, Sbra-F and Sbra-R (469 bp; yellow). Template DNAs were extracted and purified directly from fresh tissues (n = 5) dissected from (<i>left-to-right</i>, <i>top-to-bottom</i>) spleen, lungs, liver, kidneys, heart, brain, tail, and feces. Control reactions (347 bp; purple) used primers that recognized β-actin of the mouse genome. For PCR assays of DNA extracted from feces, the inhibition control was assessed by spiking the reaction solution with genomic DNA (100 ng) extracted from a <i>S</i>. <i>brasiliensis</i> culture (CBS 132990). Statistical analyses of the diagnostic accuracies are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004190#pntd.0004190.s003" target="_blank">S2 Table</a>.</p

Specificity in the presence of non-target templates.

<p>A pooled DNA sample (pool +) comprised equal volumes of the following DNA templates: <i>S</i>. <i>brasiliensis</i> (Sb: CBS 120339), <i>S</i>. <i>schenckii s</i>. <i>str</i>. (Ss: CBS 359.36), <i>S</i>. <i>globosa</i> (Sg: CBS 120340), <i>S</i>. <i>mexicana</i> (Sm: CBS 120341), <i>S</i>. <i>pallida</i> (Sp: CBS 302.73), and <i>O</i>. <i>stenoceras</i> (Os: Ss329). The specificity of amplification was confirmed by removing the target DNA from the pooled sample (pool -) for each set of primers. (<i>Left lane</i>) 50 bp DNA Ladder (Fermentas, USA) for sizing the amplicons.</p

Species-specific primer sequences that targeted the calmodulin gene (<i>CAL</i>) in members of the genus <i>Sporothrix/Ophiostoma</i>.

<p>IPC: internal positive control.</p><p>Species-specific primer sequences that targeted the calmodulin gene (<i>CAL</i>) in members of the genus <i>Sporothrix/Ophiostoma</i>.</p

Conidial PCR amplified with species-specific primers and internal positive control (IPC) primers for genotyping <i>Sporothrix</i> spp.

<p>A representative, 1.2% agarose gel shows PCR products (5-μL aliquots) amplified from DNA derived from conidia of <i>S</i>. <i>brasiliensis</i> (CBS 120339, CBS 132990, and CBS 133020), <i>S</i>. <i>schenckii</i> (CBS 359.36, CBS 132974, and CBS 132961), <i>S</i>. <i>globosa</i> (CBS 120340, CBS 132922, and CBS 132924), and <i>S</i>. <i>mexicana</i> (CBS 120341, CBS 132927, and CBS 132928). IPC bands are 101 bp; samples that contained IPC primers are indicated with an asterisk.</p

Phylogenetic analyses correlate with ecological behavior of <i>Sporothrix</i> species.

<p>Comparing the clades over the entire tree reveals a consistent decrease of bark beetle and soil association outside <i>Ophiostoma</i>, with a concomitant increase of vertebrate infectivity in <i>S</i>. <i>brasiliensis</i>, <i>S</i>. <i>schenckii</i>, and <i>S</i>. <i>globosa</i>. Phylogenetic tree generated by neighbor-joining analysis using partial nucleotide sequences of the rDNA operon (ITS1+5.8s+ITS2). Bootstrap value (1,000 replicates) was added to respective branches. Each species is indicated at its respective position in the phylogenetic tree. Bar = total nucleotide differences between taxa.</p

The sensitivity of primer sets for amplification tested with 10-fold serial dilutions of <i>Sporothrix</i> spp. DNA.

<p>Agarose gel electrophoresis shows different amounts of DNA templates (100 ng to 0.01 fg) amplified with the corresponding primer sets. (<i>Top row</i>) Sbra-F and Sbra-R with <i>S</i>. <i>brasiliensis</i> DNA (CBS 132990); (<i>row 2</i>) Ssch-F and Ssch-R with <i>S</i>. <i>schenckii s</i>. <i>str</i>. DNA (CBS 132974); (<i>row 3</i>) Sglo-F and Sglo-R with <i>S</i>. <i>globosa</i> DNA (CBS 132922); (<i>row 4</i>) Smex-F and Smex-R with <i>S</i>. <i>mexicana</i> DNA (CBS 132927); (<i>row 5</i>) Spa-F and Spa-R with <i>S</i>. <i>pallida</i> DNA (CBS 302.73); (<i>row 6</i>) Oste-F and Oste-R with <i>O</i>. <i>stenoceras</i> DNA (Ss329).</p

<i>Sporothrix/Ophiostoma</i> strains used in this study for standardizing the PCR-based identification test.

<p>IPEC, Instituto de Pesquisa Clínica Evandro Chagas, Fiocruz, Brazil; FMR, Facultat de Medicina i Ciències de la Salut, Reus, Spain; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; ^T, type strain. All “Ss” strains belong to the culture collection of Federal University of São Paulo (UNIFESP).</p><p><i>Sporothrix/Ophiostoma</i> strains used in this study for standardizing the PCR-based identification test.</p

<i>Sporothrix schenckii</i> distribution in mouse organs at 10 days post infection.

<p>Mice were challenged with intravenous injections of 5×10⁶ yeast cells (<i>S</i>. <i>schenckii</i>—CBS 132974). Gel electrophoresis (1.2% agarose) show PCR products (5-μL aliquots) amplified with primers, Ssch-F and Ssch-R (331 bp; red). Template DNAs were extracted and purified directly from fresh tissues (n = 5) dissected from (<i>left-to-right</i>, <i>top-to-bottom</i>) spleen, lungs, liver, kidneys, heart, brain, tail, and feces. Control reactions (347 bp; purple) used primers that recognized β-actin of the mouse genome. For PCR assays of DNA extracted from feces, the inhibition control was assessed by spiking the reaction solution with genomic DNA (100 ng) extracted from a <i>S</i>. <i>schenckii</i> culture (CBS 132974). Statistical analyses of the diagnostic accuracies are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004190#pntd.0004190.s003" target="_blank">S2 Table</a>.</p

Agarose gel electrophoresis shows successful amplification with specific primer sets and templates.

<p>(A) (<i>Lane 1</i>) 50 bp DNA ladder (Fermentas, USA) for size determinations; (<i>Lane 2</i>) Sbra-F and Sbra-R with <i>S</i>. <i>brasiliensis</i> DNA (CBS 132990); (<i>Lane 3</i>) Ssch-F and Ssch-R with <i>S</i>. <i>schenckii s</i>. <i>str</i>. DNA (CBS 132974); (<i>Lane 4</i>) Sglo-F and Sglo-R with <i>S</i>. <i>globosa</i> DNA (CBS 132922); (<i>Lane 5</i>) Smex-F and Smex-R with <i>S</i>. <i>mexicana</i> DNA (CBS 132927); (<i>Lane 6</i>) Spa-F and Spa-R with <i>S</i>. <i>pallida</i> DNA (CBS 302.73); (<i>Lane 7</i>) Oste-F and Oste-R with <i>O</i>. <i>stenoceras</i> DNA (Ss329). (B) A second pair of primers was added to each reaction as an internal positive control (IPC), which targeted the 5.8s region of ribosomal DNA. IPC primers generated a 101 bp amplicon; samples that contained IPC primers are indicated with an asterisk. The absence of an IPC product indicated that PCR was inhibited. The presence of IPC product and absence of the target indicated interference from a polymorphism in the DNA target of the species-specific primers.</p