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

A fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) syst em was developed for use as a rapid diagnostic lest for determining dengue viremia. The dengue virus 3 ' -noncoding sequence was utilized to formulate serotype-specific RT-PCR assays for quantitative identification of the fou r different dengue virus serotypes. A generic RT primer set containing two dengue specific anti-sense primers (DV-LI and DV-L2) could be used to trans cribe extracted viral RNA of all four dengue virus types to complimentary D NA (cDNA). The resultant dengue viral cDNA could be quantitatively identifi ed at the serotype level by the 5 ' -3 ' exonuclease assay using four serot ype-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 d engue reference serotypes and 134 clinical samples were used to validate de tection sensitivity and specificity of the dengue serotype RT-PCR assay, us ing 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. (C) 2001 Elsevier Science B.V. All rights reserved.