Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay.
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.
Johnson
Barbara W
BW
Diagnostic and Reference Laboratory, Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, 3150 Rampart Rd., Fort, Collins, CO 80521. bfj9@cdc.gov
Russell
Brandy J
BJ
Lanciotti
Robert S
RS
D017931:DNA Primers; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D006801:Humans; D012367:RNA, Viral / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012703:Serotyping; D013045:Species Specificity
D017931:DNA Primers; D012367:RNA, Viral