Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3′-noncoding sequence

Abstract

A fluorescent DNA probe (DV2.P1) specific to the conserved distal 3′-noncoding region (nucleotides 10 653–10 678) 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 to10 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.