A rapid method for detection and identification of flaviviruses by polymerase chain reaction and nucleic acid hybridization.
A polymerase chain reaction (PCR) technique was developed and evaluated for the detection of flaviviruses. A set of sense and antisense oligomeric DNA primers were constructed from nucleotide sequences of the conserved region of the genome of several different flaviviruses. Virus specific complementary DNA (cDNA) was prepared by reverse transcription of total RNA extracted from infected cell cultures. Amplified cDNA was identified by nucleic acid hybridization with specific oligomeric internal probes. Various conditions, such as number of cycles and annealing temperature were examined to optimize the detection of viral RNAs from infected cell cultures. Slot blot hybridization with a radioactive probe was used to evaluate the sensitivity of PCR amplification. The PCR amplified RNA sequences of dengue 2 (DEN-2), West Nile (WN), St. Louis encephalitis (SLE) and Kunjin (KUN) virus and detected 0.1 to 1 pg of viral RNA. Japanese encephalitis (JE), Yellow Fever virus (YF), DEN-1, 3, and 4 viruses were not amplified. The more frequent occurrence of mismatches in the 3' primer binding site may explain the failure to amplify cDNA of these viruses.
Puri
B
B
Infectious Disease Threat Assessment Program, Naval Medical Research Institute, Bethesda, Maryland.
Henchal
E A
EA
Burans
J
J
Porter
K R
KR
Nelson
W
W
Watts
D M
DM
Hayes
C G
CG
D000330:Aedes; D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D004279:DNA, Viral; D003715:Dengue / Q000382:microbiology; D005069:Evaluation Studies as Topic; D018067:Flaviviridae / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral