Serotype-Specific Detection of Dengue Viruses in a Fourplex Real-Time Reverse Transcriptase PCR Assay

TLDR

Dengue viruses are positive‑strand RNA flaviviruses comprising four serologically distinct serotypes (DEN‑1 to DEN‑4) that co‑circulate worldwide and exhibit genotypic variation within each serotype. The study aimed to develop a real‑time quantitative nucleic acid amplification assay capable of detecting the RNA of a single dengue serotype. Serotype‑specific primer‑probe sets were designed to detect all genotypes, and the assay was evaluated in single‑plex and four‑plex reactions, achieving limits of detection as low as 0.002 PFU for DEN‑2, ‑3, and ‑4 and 0.5 PFU for DEN‑1 across 40 viremic serum specimens. The assay detected viral RNA in all 40 viremic serum samples, demonstrating its suitability for differential diagnosis of dengue serotypes and rapid identification of isolates.

Abstract

The dengue (DEN) viruses are positive-strand RNA viruses in the genus Flavivirus. Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by infection with one of four closely related but serologically distinct DEN viruses, designated DEN-1, DEN-2, DEN-3, and DEN-4 viruses. All four DEN serotypes are currently co-circulating throughout the subtropics and tropics, and genotypic variation occurs among isolates within a serotype. A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction (0.4 to 1.2 x 10(6) PFU/ml). Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.