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

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

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

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

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

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