Detection of flaviviruses by reverse‐transcriptase polymerase chain reaction

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-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.

Applications of Polymerase Chain Reaction for Identification of Dengue Viruses Isolated from Patient Sera

“…Polymerase chain reaction (PCR), a new technique for an in vitro amplification of specific DNA segments (19,25), offers several obvious advantages for the diagnosis of viral infections. It has been used for the detection of many viruses such as human immunodeficiency virus (24), hepatitis B virus (14), influenza virus (26) and many others, including flaviviruses (4,5). We have developed a simple PCR method for rapid detection and identification of dengue viruses (18).…”

A rapid method for detection and identification of flaviviruses by polymerase chain reaction and nucleic acid hybridization

“…Previous investigators have described the PCR assay for St. Louis encephalitis (SLE), Japanese encephalitis (JE), yellow fever (YF), dengue 2 (DEN-2) and dengue-4 using different sets of primers for each virus [5]. Here, we evaluated RT/PCT amplification of RNA from four different viruses within the flavivirus group using a single set of flaviviral primers followed by nucleic acid hybridization with radiolabeled synthetic oligomeric probes.…”

“…Recently. Eldadah and others [5] adapted the RT-PCR method for the detection of flaviviruses. In the present study we describe the sensitivity and specificity of a unique set of primers which can be used for amplification of specific regions of the genomes of DEN-2, WN.…”

Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil

“…Traditionally, the diagnosis of Flavivirus infection has been done by virus isolation or serological testing. To overcome such problems, reverse transcription-polymerase chain reaction (RT-PCR) methods using universal primers have been described for certain flaviviruses [3][4][5][6][7] .…”