Table 1.
List of viral RNA tested.
Table 2.
Sequence of primers and probes for the construction of molecular standards, real-time RT-PCR and RT-RPA assays.
Fig 1.
Differentiation between specific and non-specific signals of the RT-RPA assay.
A and C are real-time fluorescence intensity; B and D are the 1st derivative analysis. Specific DNA amplification represented by progressive fluorescence development in both views, A and B, while non-specific not. Black line shows specific amplification where blue line shows no amplification.
Fig 2.
The extraction area encompassing magnetic separator stand, vortex, rotator, 1.5–2 ml eppendorf tube rack, automatic 100–1000 μl micopipette, micropipette tips, digital timer, 1.5 ml disposable plastic Eppendorf tubes, and a waste container with autoclavable plastic bags. Both master mix and sample mix areas contain vortex, minicentrifuge, automatic 1–10 and 10–100 μl micopipettes, micropipette tips, scissor, and 0.2 ml tubes rack. The detection was done using the tubescanner (Twista device, TwistDx, Cambridge, UK). In addition to a waste container, gloves, disposable towels, and aluminum box with trolley (740x490x450 mm, ZARGES, Weilheim, Germany).
Fig 3.
Analytical sensitivity of DENV RT-RPA assays.
A, DENV1-3 and B, DENV4 RT-RPA assays. Fluorescence development via real-time detection in one RT-RPA run by using a dilution range of 107–101 RNA molecules/μl of the DENV1-3 and DENV4 RNA molecular standards (Graph generated by ESEquant tubescanner studio software). The sensitivity was 100 and 10 RNA copies for DENV1-3 and DENV4 RT-RPA, respectively. Data of 8 RT-RPA runs is compiled in Fig 4. The signal for ten RNA copies is very weak. The box in the lower right corner of Fig 3B magnifies the fluorescence signals for the ten RNA copies and the negative control. 107 represented by black line; 106, gray; 105, red; 104, blue; 103, green; 102, cyan; 101, dark khaki; negative control, orange.
Fig 4.
Reproducibility of DENV RT-RPA assays.
A, DENV1-3 and B, DENV4 RT-RPA assays. Semi-logarithmic regression of the data collected from eight DENV RT-RPA test runs on the RNA standard using PRISM. Both assays yielded results between 3–7 minutes. In DENV1-3 RT-RPA assay, 107–103 RNA molecules were detected 8 out of 8 runs, 102 in 1 out of 8 and 10 copies was not identified. In DENV4 RT-RPA assay, 107–102 RNA molecules were detected 8 out of 8 runs and 10 copies in 6/ out of 8. In Fig 4B, the value for 10 RNA copies was consistently 5.3 minutes in all 6 cases.
Fig 5.
Performance of DENV RT-RPA assays on spiked plasma samples.
A, sample spiked with DENV1; B, DENV2; C, DENV3; D, DENV4. DENV serotypes 1–4 were spiked into plasma samples. Serial dilutions of each of the spiked sample were tested simultaneously with real-time RT-PCR and RT-RPA assays (S2 Table). Limits of detection in RT-RPA assays were 237, 618, 363, and 383 RNA copies of DENV serotypes 1, 2, 3, and 4, respectively.
Fig 6.
Comparison between real-time RT-PCR (X-axis) and RT-RPA (Y-axis) for the detection of DENV in 31 clinical samples in Senegal.
Linear regression analysis of real-time RT-PCR cycle threshold values (Ct, X-axis) and RT-RPA threshold time in minutes (TT, Y-axis) were determined by PRISM (R2 = 0.39). The RT-RPA is much faster than the real-time RT-PCR even with samples with high Ct value.