Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity 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 assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque 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 specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.

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Nucleic acid amplification technologies allow for the development of highly sensitive and specific diagnostic assays. The capacity to amplify and detect analyte targets, which may be present in a clinical sample as a single copy; is characteristic of many of these amplification technologies. NASBA is an isothermal method of nucleic acid amplification with such capability, and is particularly well suited for the amplification of RNA analytes. NASBA utilizes the coordinated activities of three enzymes (AMV-RT, RNase H, T7 RNA polymerase), and two oligonucleotide primers which are specific for the analyte target. The amplification process is part of a total system which includes a versatile nucleic acid isolation procedure, and powerful detection methodology. In this report, the development of NASBA technology for the detection of human Retrovirus RNA will be discussed. Specifically, a qualitative NASBA assay for the RNA of HTLV I, and a quantitative NASBA assay for HIV-1 will be described.