Single-Reaction, Multiplex, Real-Time RT-PCR for the Detection, Quantitation, and Serotyping of Dengue Viruses

Abstract

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 log₁₀ 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. Dengue, or break-bone fever, is the most common mosquito-borne viral disease of humans with over half the world's population at risk for infection. Dengue has a wide spectrum of clinical manifestations, from self-limited febrile illness to fatal hypovolemic shock, and because of this, dengue is difficult to distinguish from many other infections based on clinical characteristics alone. Diagnostic testing is therefore critical to accurately identify dengue virus (DENV)-infected patients and also rule out dengue in patients with undifferentiated fever. Unfortunately, current diagnostics for early DENV detection consist of point-of-care or laboratory-based antigen tests that lack sensitivity or molecular assays that are laborious to perform or lack the test characteristics necessary for routine use. To address these limitations, we developed a single-reaction, multiplex, real-time RT-PCR for the detection, quantitation, and serotyping of dengue viruses from patient serum or plasma. We demonstrate that this diagnostic test is more analytically sensitive than a commonly used reference molecular assay, and is able to detect viral RNA and provide serotype information in specimens collected more than 5 days after fever onset and from patients who had already developed anti-DENV IgM antibodies. This unique combination of sensitivity and serotyping capability in a simple, single-reaction format represents a step forward in dengue diagnostics.