Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection

Abstract

Background: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4.

Methodology/principal findings: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively.

Conclusions/significance: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

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 1^st 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. RPA mobile laboratory.

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.