Location: Vegetable Crops Research Unit
Title: Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription Authors
|German, Thomas - UW MADISON|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: February 10, 2008
Publication Date: July 26, 2008
Citation: Willis, D.K., German, T.L. 2008. Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription [abstract]. Phytopathology. 98:S171. Technical Abstract: Efforts to analyze the replicative RNA produced by Maize fine streak virus (MFSV) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not distinguish between virion (vRNA) and virion-complementary (vcRNA) or viral mRNA. Detection and quantitation of the products of viral replication requires strand-specific cDNA synthesis. However, auto-priming (generation of cDNA without primers) of the vRNA or vcRNA and false priming of the incorrect strand complicate detection and quantitation of viral replicative RNAs. Our work describes the identification of efficient primers specific to each of the seven MFSV ORFs as well as vRNA and vcRNA. Strand-specificity was improved by increasing cDNA reaction temperature from 42°C to 60°C with tagged primers which reduced auto-priming 23-fold and non-specific priming by 21 to 315-fold. Using this methodology, we established that MFSV vRNA is 30 to 60-fold more abundant than the replicative vcRNA in maize leaf tissue exhibiting fine streak symptoms. In contrast to mRNA ratios established in the well studied rhabdovirus Vesticular stomatitis virus, the N, P and L gene messages of MFSV were statistically equivalent in symptomatic maize leaf tissue.