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Journal of Clinical Microbiology, October 2005, p. 4977-4983, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.4977-4983.2005

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

Diagnostic and Reference Laboratory, Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado

Received 15 June 2005/ Returned for modification 11 July 2005/ Accepted 26 July 2005

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 cocirculating 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 10⁶ 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.

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