Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay

被引:344
作者
Johnson, BW [1 ]
Russell, BJ [1 ]
Lanciotti, RS [1 ]
机构
[1] Ctr Dis Control & Prevent, Diagnost & Reference Lab, Arbovitrus Dis Branch, Div Vector Borne Infect Dis, Ft Collins, CO 80521 USA
关键词
D O I
10.1128/JCM.43.10.4977-4983.2005
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 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.
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页码:4977 / 4983
页数:7
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