Dataset Information

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

ABSTRACT: Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/?L. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/?L were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ?10 copies/?L (8/14 vs 0/13, respectively, P?=?0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.

SUBMITTER: Rojas A

PROVIDER: S-EPMC6906250 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Real-time RT-PCR for the detection and quantitation of Oropouche virus.

Rojas Alejandra A Stittleburg Victoria V Cardozo Fátima F Bopp Nathen N Cantero César C López Sanny S Bernal Cynthia C Mendoza Laura L Aguilar Patricia P Pinsky Benjamin A BA Guillén Yvalena Y Páez Malvina M Waggoner Jesse J JJ

Diagnostic microbiology and infectious disease 20190909 1

Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/μL. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/μL were de ...[more]

PMID: 31727377

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Project description:Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log?? cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Dataset Information

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

Publications

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

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