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 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.
Wu
S J
SJ
Viral Diseases Department, Naval Medical Research Center, Silver Spring, Maryland 20910-7500, USA. wus@nmrc.navy.mil
Lee
E M
EM
Putvatana
R
R
Shurtliff
R N
RN
Porter
K R
KR
Suharyono
W
W
Watts
D M
DM
King
C C
CC
Murphy
G S
GS
Hayes
C G
CG
Romano
J W
JW
D000818:Animals; D002522:Chlorocebus aethiops; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D014709:Vero Cells; D010948:Viral Plaque Assay
D012367:RNA, Viral