Sequences and concentrations of primers and probes used in the multiplex real-time PCR assays.

<p>^a[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref044" target="_blank">44</a>]; ^b[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref045" target="_blank">45</a>]; SL-IR, spliced leader intergenic region; 18S, 18S-ribosomal ADN; COII, cytochrome oxidase II; 24Sα, 24Sα-ribosomal ADN; MTq, multiplex Real-Time PCR; BHQ, Black Hole Quencher. The + in front of the nucleotide indicates an LNA (Locked Nucleic Acid) monomer substitution.</p><p>Sequences and concentrations of primers and probes used in the multiplex real-time PCR assays.</p

Inclusivity and specificity assays for the multiplex real-time PCR genotyping algorithm.

<p>Cycle threshold (Ct) values obtained for each TaqMan probe in the analysis of <i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> sp. stocks and human DNA. 0.1–10 ng of each <i>T</i>. <i>cruzi</i> strain and 2–10 ng of <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. stocks were used in the reaction tube.</p><p>DTU, Discrete Typing Unit; neg, negative; nd, not done.</p><p>Inclusivity and specificity assays for the multiplex real-time PCR genotyping algorithm.</p

<i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. isolates used to evaluate the analytical performance of the multiplex real-time PCR genotyping assays.

<p>References: ^a[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref006" target="_blank">6</a>]; ^b[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref026" target="_blank">26</a>]; ^c[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref027" target="_blank">27</a>]; ^d[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref028" target="_blank">28</a>]; ^e[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref029" target="_blank">29</a>]; ^f[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref030" target="_blank">30</a>]; ^g[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref031" target="_blank">31</a>]; ^h[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref032" target="_blank">32</a>]; ⁱ[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref033" target="_blank">33</a>] ^j[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref034" target="_blank">34</a>]; ^k[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref022" target="_blank">22</a>]; ^l[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref023" target="_blank">23</a>]; ^m[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref035" target="_blank">35</a>]; ⁿ[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref036" target="_blank">36</a>]; ^o[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref037" target="_blank">37</a>]; ^p[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref038" target="_blank">38</a>]; ^q[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref039" target="_blank">39</a>]; ^r[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref040" target="_blank">40</a>]; ^s[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref041" target="_blank">41</a>]. DTU, Discrete Typing Unit; nd, no data.</p><p><i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. isolates used to evaluate the analytical performance of the multiplex real-time PCR genotyping assays.</p

Multiplex real-time PCR flowchart for identification of <i>Trypanosoma cruzi</i> DTUs in biological samples.

<p>SL-IR, spliced leader intergenic region; 18S, 18S-ribosomal ADN; COII, cytochrome oxidase II; 24Sα, 24Sα-ribosomal DNA; MTq, multiplex TaqMan Real-Time PCR.</p

Linear range and analytical sensitivity of the second round multiplex real-time PCR tests.

<p><b>A.</b> 18S-COII MTq PCR assay for reference stocks representing <i>T</i>. <i>cruzi</i> DTUs TcII, TcV and TcVI. Detection of TcII stock is shown for both TaqMan probes 18S-FAM and COII-Cy5. <b>B.</b> 24Sα MTq PCR for reference stocks representing <i>T</i>. <i>cruzi</i> DTUs TcIII, TcIV-SA and TcIV-NA. X-axis represents serial dilutions of whole genomic DNA from each stock and Y-axis represents the obtained Ct value. Linear regression analysis, equation and R² are shown for each graph. TcII, strain Tu18; TcV, strain PAH265; TcVI, strain CL Brener; TcIII, strain M5631; TcIV-SA (TcIV from South America), strain CanIII; TcIV-NA (TcIV from North America), strain Griffin.</p

Linear range and analytical sensitivity of the first round SL-IR MTq PCR for <i>T</i>. <i>cruzi</i> DTUs and TcI SL-IR genotypes.

<p>X-axis represents serial dilutions of whole genomic DNA from each stock and Y-axis represents the obtained Ct value. Linear regression analysis, equation and R² are shown for each graph. Inserts inside plots represent the Ct values obtained for the complete DNA concentration range tested (1 fg—10 ng/ reaction tube). TcIa, strain K98; TcIb, strain Cas16; TcId, strain G; TcIe, strain PALV1 cl1; TcII, strain Tu18; TcIII, strain M5631; TcIV, strain CanIII; TcV, strain PAH265; and TcVI, strain CL Brener.</p

Multiplex real-time PCR genotyping algorithm validation with biological samples.

<p>Positive results obtained with the Real-Time and conventional PCR assays for the DTU characterization of biological samples were compared. The number of samples belonging to each DTU group corresponds to the Real-Time PCR algorithm results.</p><p>^aEleven peripheral blood samples and one skin biopsy sample</p><p>^bSixty three urine/feces samples on filter paper and 25 abdomen/tissue samples</p><p>^cForty peripheral blood and 4 heart explant samples; pos, positive results. Mixed infections were characterized as ^dTcV plus TcVI,</p><p>^e6 TcI plus TcIV, 1 TcI plus TcIII/IV and 2 TcIII plus TcIV,</p><p>^fTcI plus TcII. AI, acute <i>T</i>. <i>cruzi</i> infection; ACD, asymptomatic chronic Chagas disease; SCD, symptomatic chronic Chagas disease; CI, congenitally infected children; and RCD, patients with reactivation in the context of immunosuppression.</p><p>Multiplex real-time PCR genotyping algorithm validation with biological samples.</p