Abstract
RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PRC) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.
Original language | English (US) |
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Pages (from-to) | 260-267 |
Number of pages | 8 |
Journal | Journal of Medical Virology |
Volume | 33 |
Issue number | 4 |
State | Published - 1991 |
Externally published | Yes |
Keywords
- Dengue
- Japanese encephalitis
- Nesting primers
- Reverse transcriptase
- RNA
- St. Louis encephalitis
- Yellow fever
ASJC Scopus subject areas
- Virology