Rapid detection and discrimination of fabaviruses by flow‐through hybridisation with genus‐ and species‐specific riboprobes

Abstract

Viruses cause significant damage in agricultural crops worldwide. Disease management requires sensitive and specific tools for virus detection and identification. Also, detection techniques need to be rapid to keep pace with the continuous emergence of new viral diseases. The genus Fabavirus is composed of five viruses infecting many economically important crops worldwide. This research describes the development of a procedure based on flow-through hybridisation (FTH), which is faster than and as sensitive as conventional hybridisation for virus detection in tissue-prints from infected plants. Six digoxigenin-labelled RNA probes were synthesised with two levels of specificity: (a) five specific for each viral species within this genus, and (b) a genus-specific probe that hybridises with a nucleotide sequence signature only found in the 5′-untranslated region of the genus Fabavirus, which is the first of this type reported for plant viruses. The new procedure developed is useful for rapid detection and discrimination of the five fabaviruses identified so far and opens the possibility of discovering new species of this genu

Detection of Southern tomato virus by molecular hybridisation

“…RT‐PCR can be a sensitive technique of detection but is prone to false positives by contamination or false negatives by reaction inhibition of polymerases and is not appropriate for massive sample analyses. Molecular hybridisation has enough sensitivity to detect many plant viruses and it is suitable to analyse a high number of samples simultaneously by using tissue‐prints or sap extracts as targets which avoid nucleic acid extraction from plant samples (Galipienso et al ., ; Ferriol et al ., ). In this work, we developed a method for STV detection by molecular hybridisation with a digoxigenin‐labelled RNA probe.…”

Molecular and biological characterization of highly infectious transcripts from full-length cDNA clones of broad bean wilt virus 1

“…2). Infection was confirmed by flow-through hybridization of tissue prints of the upper non-inoculated leaves (Ferriol et al, 2015). All C. quinoa plants were infected for both transcripts and the wt virus (Table 1).…”

“…2). BBWV-1 infection was also detected by flow-through hybridization of tissue-prints (Ferriol et al, 2015).…”

“…Finally, the genome integrity of the viral progeny in C. quinoa and V. faba plants derived from the full-length cDNA clones pBenR1 and pBenR2 was evaluated by Northern blot hybridization. Total RNAs from transcripts and wt virus progenies were separated by 1.2% formaldehyde agarose gel electrophoresis in MOPS (3-Morpholinopropane-1-sulfonic acid) buffer, transferred by capillarity onto a positively charged nylon membrane (Roche Diagnostics, Basel, Switzerland) and hybridized with probes Fab1 and Fab2 for RNA1 and RNA2, respectively (Ferriol et al, 2015). Probe Fab1 and Fab2 were obtained using primers Fab5 R1F (Ferriol et al, 2015) and T7-5 R1R for the RNA1 and Fab5 R1F/T7-5 R2R for the RNA2 (Supplementary Table S1).…”

“…Total RNAs from transcripts and wt virus progenies were separated by 1.2% formaldehyde agarose gel electrophoresis in MOPS (3-Morpholinopropane-1-sulfonic acid) buffer, transferred by capillarity onto a positively charged nylon membrane (Roche Diagnostics, Basel, Switzerland) and hybridized with probes Fab1 and Fab2 for RNA1 and RNA2, respectively (Ferriol et al, 2015). Probe Fab1 and Fab2 were obtained using primers Fab5 R1F (Ferriol et al, 2015) and T7-5 R1R for the RNA1 and Fab5 R1F/T7-5 R2R for the RNA2 (Supplementary Table S1). BBWV-1 transcript progeny showed the same size for the two genomic RNAs as was seen for the wt virus progeny (Fig.…”