Table 1.
Characteristics of trypanosomatid parasites used in this study.
Table 2.
Primers and probes employed in this study.
Fig 1.
Schematic representation of the TevRPA-LF.
A: RPA-based generation of a T. evansi specific RoTat1.2 VSG amplicon for detection by a lateral flow (LF) assay. Step 1: two oligonucleotide primers (TevRPA-Fw and TevRPA-Rv-biotin) form a complex with the recombinase. Step 2: the primer-recombinase complexes invade the homologous sequences on the target DNA. Step 3: A DNA polymerase with a strand displacement activity performs amplification of the target sequence under isothermal conditions, resulting in the generation of a biotinylated amplicon. Step 4: the generated amplicons are again invaded by primer-recombinase complexes in a self-perpetuating cycle fueled in ATP by creatine kinase. Step 5: an oligonucleotide (FAM-probe) carrying a 5’ FAM tag, a spacer sequence and a 3’ blocking group forms a complex with the recombinase and invades the biotinylated amplicon generated in the previous steps. Step 6: only when the FAM-probe has successfully invaded the biotinylated amplicon and bound its complementary sequence, can the Nfo endonuclease bind and cleave the spacer region and 3’ blocking group. Step 7: after removal of the 3’ region of the FAM probe, the Nfo endonuclease dissociates. This allows the DNA polymerase to employ the cleaved FAM-probe as a forward primer. Together with the biotinylated reverse primer (TevRPA-Rv-biotin) this leads to the formation of an amplicon bearing both the FAM and biotin tags. B: Read-out of the RPA via LF. The FAM- and biotin-tagged RPA product is mixed with the LF buffer, loaded onto the sample pad and is transported to the adsorbent pad through capillary flow. The RPA product is first bound by gold-labeled rabbit anti-FAM antibodies and later captured by a streptavidin-coated test line (TL). The control line (CL) is coated with anti-rabbit antibodies. While a valid negative test only contains a reddish band at the CL, a valid positive test will display bands at both the TL and CL.
Fig 2.
A: Initial RPA incubated at 37°C for 30 minutes on various samples. Lane 1, T. evansi purified genomic DNA; Lane 2, naïve mouse purified genomic DNA; Lane 3, sample without any template; Lane 4, RPA kit positive control; Lane 5, RPA kit negative control. B: RPA reaction on T. evansi purified genomic DNA incubated at different temperatures for a constant time of 30 minutes. C: RPA reaction on T. evansi purified genomic DNA incubated at a constant temperature of 39°C for various times. In all panels Lane M indicates the molecular mass marker, whereas Lane N in panels B and C represents a negative control sample (no template DNA).
Fig 3.
Read-out of the TevRPA via a lateral flow assay (TevRPA-LF) and agarose gel electrophoresis.
A: Selection of a suitable probe for the development of the TevRPA-LF. P1 and P2 refer to FAM probes 1 and 2, respectively. Lane 1, T. evansi purified genomic DNA; Lane 2, naïve mouse purified genomic DNA. B: Assessment of the specificity of the TevRPA-LF. Lanes 1-7, various T. evansi strains as listed in Table 1; Lane 8, T. congolense; Lane 9, T. vivax; Lane 10, T. brucei; Lane 11, L. donovani. C: Comparison of the sensitivities of the TevRPA by a lateral flow assay and agarose gel electrophoresis. Lanes 1-8, 10-fold dilution series of T. evansi purified genomic DNA starting at 10 ng μl−1 (1 μl was loaded onto the gel). Lane 1, 10 ng; Lane 2, 1 ng; Lane 3, 100 pg; Lane 4, 10 pg; Lane 5, 1 pg; Lane 6, 100 fg; Lane 7, 10 fg; Lane 8,1 fg. All panels display the read-out of the TevRPA by a lateral flow assay (left) and agarose gel electrophoresis (right). In all panels Lane M indicates the molecular mass marker, whereas Lane N represents a negative control sample (no template DNA). CL and TL refer to the control and test lines, respectively.
Fig 4.
Evaluation of the TevRPA-LF as a test-of-cure tool in T. evansi infections in mice.
A: C57BL/6 mice were infected with T. evansi RoTat1.2 (n = 5) and the presence of parasites was monitored over the course of the infection by microscopy (top panel), the TevPCR (middle panel, performed on parasite genomic DNA purified from the collected blood samples), and TevRPA-LF (bottom panel, executed on crude parasite genomic DNA extracted from the collected blood). The results are displayed as the percentages of mice that scored positive or negative as determined by the above-mentioned techniques. B: C57BL/6 mice infected with T. evansi RoTat1.2 (n = 5) were treated with Berenil at 5 days post-infection. The presence of parasites was followed by microscopy, the TevPCR and the TevRPA-LF throughout the experiment. The panels and color codes are the same as for panel A. The TevPCR and TevRPA-LF read-outs are shown in Fig 5.
Fig 5.
TevPCR and TevRPA-LF read-outs.
The TevPCR (bottom panels) and TevRPA-LF (upper panels) read-outs displayed in Fig 4. A: TevPCR and TevRPA-LF results for the mouse infection trial of Group 1 mice (corresponds to the data set shown in Fig 4A). B: TevPCR and TevRPA-LF results for the mouse infection trial of Group 2 mice (corresponds to the data set shown in Fig 4B). In all panels Lane M indicates the molecular mass marker, Lanes 1-6 indicate the individual mice (mouse 6 was used as a negative control within each data set and was not infected), Lane N is a negative control sample (no template DNA) and Lane P is the positive control (T. evansi purified genomic DNA). CL and TL refer to the control and test lines, respectively.