Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements.
We developed robust, ultrasensitive, and accurate quantitative assays for maedi-visna virus (MVV) RNA and DNA genomic sequences and mRNA's expressed at various stages of lentiviral replication. Assay design was based on PCR with real-time fluorescence resonance energy transfer measurements. Specific assays were developed for gag-pol (genomic), tat, rev, env, and vif transcripts. Assay linearity ranged from 60 to 6 x 10(7) copies of target DNA. All assays were able to detect and measure corresponding mRNA's in MVV-infected FOS cells, whereas no signal was detected in mock-treated cells. In addition, RT-PCR based on amplification of gag sequences could be used to quantify RNA genomic sequences in supernatants from infected cells. These quantitative assays can be used to study the role of genetic elements in MVV infection and pathogenesis. They also allow rapid testing of lentiviral vectors and packaging systems based on MVV.
Gudmundsson
Bjarki
B
Department of Biochemistry and Molecular Biology, University of Iceland Faculty of Medicine, Reykjavik, Iceland.
Bjarnadottir
Helga
H
Kristjansdottir
Steinunn
S
Jonsson
Jon J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D008264:Macrophages / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014790:Visna-maedi virus / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger