An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses

G. J J Chang, D. W. Trent, A. V. Vorndam, E. Vergne, R. M. Kinney, C. J. Mitchell

Research output: Contribution to journalArticle

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Abstract

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Original languageEnglish (US)
Pages (from-to)477-483
Number of pages7
JournalJournal of Clinical Microbiology
Volume32
Issue number2
StatePublished - 1994

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Flavivirus
Viral RNA
Protein Sorting Signals
Reverse Transcriptase Polymerase Chain Reaction
Digoxigenin
St. Louis Encephalitis
Enzyme-Linked Immunosorbent Assay
Dengue
Biotin
Culicidae
St. Louis Encephalitis Viruses
Cell Culture Techniques
Immunosorbent Techniques
Viruses
Virology
Streptavidin
Aedes
Indirect Fluorescent Antibody Technique
Serum
Immune Sera

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses. / Chang, G. J J; Trent, D. W.; Vorndam, A. V.; Vergne, E.; Kinney, R. M.; Mitchell, C. J.

In: Journal of Clinical Microbiology, Vol. 32, No. 2, 1994, p. 477-483.

Research output: Contribution to journalArticle

Chang, G. J J ; Trent, D. W. ; Vorndam, A. V. ; Vergne, E. ; Kinney, R. M. ; Mitchell, C. J. / An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses. In: Journal of Clinical Microbiology. 1994 ; Vol. 32, No. 2. pp. 477-483.
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