Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on3 '-noncoding sequence

A fluorescent DNA probe (DV2.P1) specific to the conserved distal 3'-noncod ing region (nucleotides 10 653-10 678) of dengue 2 virus and a pair of flan king primers (DV2.L1 and DV2.U2) were designed to formulate a dengue 2-spec ific fluorogenic polymerase chain reaction (PCR). In addition, DV2.L1 was a lso used as a reverse transcription (RT) primer to generate superior cDNA f rom dengue viral RNA. Optimal assay conditions with zero background were es tablished to detect low levels of dengue 2 virus from clinical specimens. T he range of dengue virus detection in spiked human sera was determined to b e from 10 to 10(6) infectious virions per milliliter (plaque forming units determined using Vero cell line). Dengue 2 virus isolates from different ge ographic regions can be detected universally and identified by the fluoroge nic RT-PCR assay. Moreover, the assay is specific for dengue 2 virus and do es not recognize other related flaviviruses, including dengue serotypes 1, 3 and 4, Japanese encephalitis, St. Louis encephalitis, yellow fever, and K unjin viruses. The assay also detected efficiently immunocomplexed dengue v iruses. 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 proli feration and clearance, was also clearly illustrated by the assay. (C) 2000 Published by Elsevier Science B.V. All rights reserved.