Additional file 1: Figure S1. of Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity

Expression and purification of the recombinant proteins rLbSCD6 (a) and rLbRBP42 (b). Analysis by Coomassie blue staining of their expression in E. coli-M15 cells (Lanes 1–3) and purified fractions (Lane 4). Lane 1: total cell extract before induction; Lane 2: total cell extract after induction with IPTG; Lane 3: insoluble fraction of the cell extract; Lane 4: eluted protein after affinity chromatography and in-column refolding procedure. Abbreviations: MW, molecular weight marker. (TIFF 673 kb

Additional file 1: Figure S1. of Leishmania braziliensis SCD6 and RBP42 proteins, two factors with RNA binding capacity

Expression and purification of the recombinant proteins rLbSCD6 (a) and rLbRBP42 (b). Analysis by Coomassie blue staining of their expression in E. coli-M15 cells (Lanes 1–3) and purified fractions (Lane 4). Lane 1: total cell extract before induction; Lane 2: total cell extract after induction with IPTG; Lane 3: insoluble fraction of the cell extract; Lane 4: eluted protein after affinity chromatography and in-column refolding procedure. Abbreviations: MW, molecular weight marker. (TIFF 673 kb

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p

Inclusivity of LAMP assay tested in purified DNA samples from <i>T</i>. <i>cruzi</i> strains representative of the different discrete type units.

<p>Left panels: Visualization by the naked eye of <i>T</i>. <i>cruzi</i> DNA stocks representative of different DTUs. Tc I (A: 1.0 x10^-2 fg/test; B: 1.0 x10^-3 fg/test); Tc II (C: 2.5 fg/test; D: 2.5x10^-1 fg/test); Tc III (E: 7.5 x 10⁻² fg/test; F: 7.5 x 10⁻³ fg/test); Tc IV (G: 5.0 x 10⁻¹ fg/test, H: 5.0 x 10⁻² fg/test); Tc V (I: 1.5 x 10⁻¹ fg/test; J: 1.5 x 10⁻² fg/test); Tc VI (K: 1.0 x 10⁻¹ fg/test; L. 1.0 x 10⁻² fg/test) Right panels: Amplification plots obtained in the LAMP reaction after analyzing the samples in a Rotor Gene 3000 thermocycler. Y axis represents fluorescence and x axis represents Cts (Threshold cycles). Only the highest dilution giving amplification and the next dilution giving non detectable results are shown.</p

INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).

<p>INCLUSIVITY in <i>Trypanosoma cruzi</i> strains representative of different DTUs (fg/test).</p

Clinical specimens results from LAMP test and qPCR as a comparator.

<p>Clinical specimens results from LAMP test and qPCR as a comparator.</p

Analytical sensitivity of LAMP assay in spiked EDTA blood samples.

<p>EDTA blood samples spiked with different quantities of purified <i>T</i>. <i>cruzi</i> DNA were processed using RAS columns or the Boil & Spin method. (A) EDTA blood processed using RAS columns. (B): EDTA blood processed by Boil & Spin method. Upper panels: LAMP reaction products analyzed by electrophoresis in 1.2% agarose gels and stained with ethidium bromide. Bottom panels: pictures of LAMP reaction products visualized by the naked eye.</p

Analytical sensitivity of LAMP assay.

<p>Top panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from <i>CL</i> Brener stock (DTU VI). Bottom panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from Sylvio X10 stock.The aliquots were expressed in fg/μL. A: 0; B: 1.0 x 10⁻³; C: 1.0 x 10⁻²; D: 1.0 x 10⁻¹, E: 1. NC: Non template control.</p

Exclusivity of LAMP assay tested in purified DNA samples from <i>T</i>. <i>rangeli</i>, <i>Leishmania major</i> stocks and non-infected human DNA.

<p>Top Panel. (A): <i>Leishmania major</i> DNA was tested at 1.0 x 10¹fg/ μL; (B) 1.0 x .10² fg/ μL; (C) 1.0 x 10³ fg/ μL; (D) 1.0 x. 10⁴ fg/ μL. <i>T</i>. <i>rangeli</i> DNA was tested (E) at 1.0 x 10⁴ fg/ μL and (F) 1.0 x .10³ fg/μL. Bottom Panel. (A): Mixture containing equal volumes of <i>T</i>. <i>cruzi</i> and <i>Leishmania major</i> DNA at 1.0 x 10⁴ fg/μL. (B): Mixture containing equal volumes of <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i> DNA at 1.0 x 10⁴ fg/μL. (C): <i>T</i>. <i>cruzi</i> DNA tested at 10 fg/μL. (D) and (E): <i>T</i>. <i>rangeli</i> DNA tested at 10 and 100 fg/μL, respectively. NIHB: Non-infected human DNA. PC: Positive Control. NC: Negative Control.</p