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

Citation
Sjl. Wu et al., Detection of dengue viral RNA using a nucleic acid sequence-based amplification assay, J CLIN MICR, 39(8), 2001, pp. 2794-2798
Citations number
18
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
39
Issue
8
Year of publication
2001
Pages
2794 - 2798
Database
ISI
SICI code
0095-1137(200108)39:8<2794:DODVRU>2.0.ZU;2-Q
Abstract
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.