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A Rapid Method for Detection and Identification of Flaviviruses by Polymerase Chain Reaction and Nucleic Acid Hybridization
NAVAL MEDICAL RESEARCH INST BETHESDA MD
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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.
APPROVED FOR PUBLIC RELEASE