Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1–4 using conserved and serotype-specific 3′ noncoding sequences

Abstract

A fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) system was developed for use as a rapid diagnostic test for determining dengue viremia. The dengue virus 3′-noncoding sequence was utilized to formulate serotype-specific RT-PCR assays for quantitative identification of the four different dengue virus serotypes. A generic RT primer set containing two dengue specific anti-sense primers (DV-L1 and DV-L2) could be used to transcribe extracted viral RNA of all four dengue virus types to complimentary DNA (cDNA). The resultant dengue viral cDNA could be quantitatively identified at the serotype level by the 5′–3′ exonuclease assay using four serotype-specific sense primers. The fluorogenic dengue type-specific RT-PCR can detect each of the four dengue types at similar low detection limits, i.e. 20–50 plaque forming units per milliliter of serum. Two panels with four dengue reference serotypes and 134 clinical samples were used to validate detection sensitivity and specificity of the dengue serotype RT-PCR assay, using virus isolation in cell culture as the criterion standard. By analyzing sera samples from Puerto Rico that were collected from 1999 through 2000, the assay demonstrated high level detection sensitivity and specificity of 92.8 and 92.4%, respectively, for all four dengue virus serotypes.