Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3'-noncoding sequence.
A fluorescent DNA probe (DV2.P1) specific to the conserved distal 3'-noncoding region (nucleotides 10653-10678) of dengue 2 virus and a pair of flanking primers (DV2.L1 and DV2.U2) were designed to formulate a dengue 2-specific fluorogenic polymerase chain reaction (PCR). In addition, DV2.L1 was also used as a reverse transcription (RT) primer to generate superior cDNA from dengue viral RNA. Optimal assay conditions with zero background were established to detect low levels of dengue 2 virus from clinical specimens. The range of dengue virus detection in spiked human sera was determined to be from 10 to 10(6) infectious virions per milliliter (plaque forming units determined using Vero cell line). Dengue 2 virus isolates from different geographic regions can be detected universally and identified by the fluorogenic RT-PCR assay. Moreover, the assay is specific for dengue 2 virus and does not recognize other related flaviviruses, including dengue serotypes 1, 3 and 4, Japanese encephalitis, St. Louis encephalitis, yellow fever, and Kunjin viruses. The assay also detected efficiently immunocomplexed dengue viruses. In practice, the fluorogenic RT-PCR assay detected readily viremia in sera collected from individuals ill with dengue fever. The rise and fall of dengue 2 virus concentrations in rhesus monkeys, reflecting viral proliferation and clearance, was also clearly illustrated by the assay.
Houng
H H
HH
Department of Enteric Infections and Virus Diseases, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20307, USA.
Hritz
D
D
Kanesa-thasan
N
N
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D000936:Antigen-Antibody Complex; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006801:Humans; D008253:Macaca mulatta; D009500:Neutralization Tests; D012367:RNA, Viral / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D020413:3' Untranslated Regions; D000936:Antigen-Antibody Complex; D005456:Fluorescent Dyes; D012367:RNA, Viral