Automated type specific ELISA probe detection of amplified NS3 gene products of dengue viruses.
OBJECTIVE
To apply an automated system of nucleic acid hybridisation coupled with the enzyme linked immunosorbent assay (ELISA) for the type specific detection of amplification products of dengue viruses.


METHODS
Non-structural 3 (NS3) gene targets of reference strains of all four dengue and other flaviviruses, as well as dengue patient viraemic sera, were subjected to reverse transcription and polymerase chain reaction using consensus and dengue type specific primers and digoxigenin-11-dUTP label incorporation. The amplification products were detected by biotinylated type specific primers which served as ELISA capture probes bound to streptavidin coated tubes.


RESULTS
Significantly high spectrophotometric absorbance readings were obtained by hybridisation of the consensus and seminested amplification products of all four dengue viruses with their respective capture probes. In contrast, extremely low absorbances were observed for consensus products of Japanese encephalitis, yellow fever, and Kunjin viruses, which served as negative controls. These ELISA data correlated well with agarose gel electrophoresis of dengue type specific amplified products of diagnostic sizes.


CONCLUSIONS
The combination of in vitro amplification and antibody based detection offers rapid, type specific, high throughput, and gel-free detection of amplified products of dengue viruses.
Chow
V T
VT
Department of Microbiology, Faculty of Medicine, National University of Singapore, Republic of Singapore.
Yong
R Y
RY
Ngoh
B L
BL
Chan
Y C
YC
D003715:Dengue / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005416:Flavivirus / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D020365:RNA Helicases; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases