Table 1.
Code, origin, host, and amount of DNA detected by the T. cruzi OligoC-TesT of 25 T. rangeli isolates.
Figure 1.
Presentation of the target DNA, primers and probes sequences.
Alignment of the internal control (IC) DNA sequence and the T. cruzi OligoC-TesT DNA target sequence (T.c.) within the satellite DNA (consensus sequence of GenBank accession numbers AY519985 to AY520098). The forward primer Tc-Sat-F (1), the biotinylated (Biot) reverse primers Tc-Sat-R (2), the T. cruzi detection probe (3) and the IC detection probe (4) are shown.
Figure 2.
Schematic overview of the T. cruzi OligoC-TesT.
Polymerase chain reaction (PCR) amplification of a sample containing T. cruzi DNA (1) is performed using a PCR mix containing single-stranded internal control (IC) template (2). The IC sequence is the same as the target sequence within the T. cruzi satellite DNA but with a specific internal sequence (dotted line). Both templates are amplified with the same primers of which the reverse primer is biotinylated (•). When the PCR and subsequent denaturation is completed, the PCR product solution contains single-stranded T. cruzi and IC DNA (3), and is mixed with an equal volume of migration buffer preheated at 55°C. The T. cruzi Oligo-Strip is dipped into the mixture and test results are read after 5 minutes migration at 55°C (4). During migration, the solution takes up the gold-labelled (○) detection probes. The T. cruzi detection probes (5) at the test side and the IC detection probes (6) at the control side hybridise with their respective amplicons. The biotinylated PCR products accumulate on the neutralite avidin lines at both sides of the dipstick and the T. cruzi and IC amplicons are visualized by their respective detection probes (7). In case of a negative sample, only the IC amplicon is present and visualized at the control side of the dipstick (8). The excess of detection probes migrate further and hybridise on the complementary probes coated at both sides of the dipstick as a control for migration (9).
Figure 3.
Analytical sensitivity of the T. cruzi OligoC-TesT.
The upper and lower line are the migration control and the T. cruzi test line respectively. A. Test results on a serial dilution of T. cruzi strain Y DNA in water containing 0.1 mg/ml acetylated bovine serum albumine (BSA). Dipsticks 1–10: 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 0.1 fg, 0.01 fg and 0 fg per assay. B. Test results on a serial dilution of T. cruzi epimastigotes in naïve human blood. Dipsticks 1–6: 10,000; 1,000; 100; 10; 1 and 0 parasites in 180 µl blood.
Table 2.
Diagnostic accuracy of the T. cruzi OligoC-TesT on biological samples from Chagas non-endemic controls and endemic controls and from T. cruzi infected persons, reservoir animals and vectors.