Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay

Faster techniques are needed for the early diagnosis of dengue fever and de ngue hemorrhagic fever during the acute viremic phase of infection. An isot hermal nucleic acid sequence-based amplification (NASBA) assay was optimize d to amplify viral RNA of all four dengue virus serotypes by a set of unive rsal primers and to type the amplified products by serotype-specific captur e probes. The NASBA assay involved the use of silica to extract viral nucle ic acid, which was amplified without thermocycling. The amplified product w as detected by a probe-hybridization method that utilized electrochemilumin escence. Using normal human plasma spiked with dengue viruses, the NASBA as say had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specif icity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The "gold standard" used for comparison and evaluation was the mosquito C6/36 cell culture ass ay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The N ASBA assay was able to detect dengue viral RNA in the clinical samples at p laque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specifi city of 100% based on the negative test results for the 21 normal human ser um or plasma samples. These results indicate that the NASBA assay is a prom ising assay for the early diagnosis of dengue infections.