Detection of flaviviruses by reverse-transcriptase polymerase chain reaction.
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-PCR) 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.
Eldadah
Z A
ZA
Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
Asher
D M
DM
Godec
M S
MS
Pomeroy
K L
KL
Goldfarb
L G
LG
Feinstone
S M
SM
Levitan
H
H
Gibbs
C J
CJ
Gajdusek
D C
DC
D000330:Aedes; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000382:microbiology; D001923:Brain Chemistry; D002478:Cells, Cultured; D004279:DNA, Viral / Q000737:chemistry; D005416:Flavivirus / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014036:Togaviridae Infections / Q000175:diagnosis
D004279:DNA, Viral; D009841:Oligonucleotides; D012194:RNA-Directed DNA Polymerase