ddPCR for the Detection and Absolute Quantification of Oropouche Virus

Abstract

Background: Oropouche virus (OROV) is a segmented RNA virus belonging to the genus Orthobunyavirus in the family Peribunyaviridae. Herein, an in-house droplet digital PCR (ddPCR) assay was used for the detection and quantification of OROV.

Methods: The ddPCR reaction was assessed as duplex assay using the human housekeeping gene RPP30. Limit of detection (LoD) analysis was performed in whole blood, serum, and urine. The assay was executed on a total of 28 clinical samples (whole blood n = 9, serum n = 11, and urine n = 8), of which 16 specimens were tested positive at the routine molecular diagnostics (endpoint and real-time PCRs).

Results: The LoD of the ddPCR performed using 10-fold serial dilution of OROV detected up to 1 cp/µL in all the biological matrices. Compared to the routine molecular diagnostics, the ddPCR assay showed 100% sensitivity for whole blood and serum and 75% for urine, highlighting higher positive rate of ddPCR.

Conclusion: We have established a quantitative RNA detection method of OROV with high sensitivity and specificity based on ddPCR. This test is capable of quantitatively monitoring the viral load of OROV and can contribute, in addition to laboratory diagnosis, to shed light on the pathogenesis, filling in the knowledge gaps of this neglected disease and to the vector control programs.

Keywords: Oropouche virus; RNA; ddPCR; quantification.

Figure 1

Evaluation of the ddPCR assay LoD performance. Standard curves were obtained from 10-fold serially diluted samples of the reference viral strain (10⁵ TCID₅₀/mL) in three biological matrices (whole blood (A), serum (B) and urine (C)). The curves include also the negative point (no viral strain). Pearson statistics was used.