Rapid, single-step RT-PCR typing of dengue viruses using five NS3 gene primers.
In order to detect and type dengue viruses in serum specimens, four type-specific downstream primers were designed for use with a consensus upstream primer in a reverse transcription and polymerase chain reaction (RT-PCR) assay. RT-PCR using these five primers amplified NS3 gene fragments of diagnostic sizes of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively, but not for Japanese encephalitis, Kunjin and yellow fever viruses. The conventional two-step RT-PCR procedure was simplified by combining RT and PCR in a single-step format with a "hot start". This RT-PCR protocol was applied successfully to dengue virus-spiked serum and dengue patient serum samples, and could detect as few as one PFU of dengue virus. This assay offers a rapid, specific and sensitive molecular technique for the simultaneous detection and typing of dengue viruses.
Seah
C L
CL
Department of Microbiology, Faculty of Medicine, National University of Singapore.
Chow
V T
VT
Tan
H C
HC
Can
Y C
YC
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D003715:Dengue / Q000175:diagnosis; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020365:RNA Helicases; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases; D013045:Species Specificity; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000097:blood / Q000235:genetics; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017931:DNA Primers; D007106:Immune Sera; D020365:RNA Helicases; D012367:RNA, Viral; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases