Polyvalent Degenerate Oligonucleotides Reverse Transcription-Polymerase Chain Reaction: A Polyvalent Detection and Characterization Tool for Trichoviruses, Capilloviruses, and Foveaviruses

Abstract

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations

“…However, most studies using degenerate primers to detect plant viruses are purely qualitative and degeneracies may not cover all possible variations within primer binding sites. Inosine bases can be used to add polyvalent degeneracies in primers targeting potentially variable positions within conserved sites (Foissac et al, 2005;Teycheney et al, 2007), although this approach appears to have had limited use in the field of plant virology. An additional disadvantage to using both degenerate primers and polyvalent inosine-containing primers is the reduction in sensitivity of the PCR (Nam et al, 2016;Teycheney et al, 2007).…”

Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR

“…Detecting more than one virus in an assay can accelerate testing procedures and decrease the cost of diagnosis and the quantity of plant material required (Al Rwahnih et al, 2004;Foissac et al, 2005;Hassan et al, 2006).…”

Further Characterization of Tulip Severe Mosaic Virus Supports Classification as a Member of Genus Ampellovirus in the Family Closteroviridae

“…Citrus tatter leaf virus (CTLV), foveaviruses, i.e. Apple stem pitting virus (ASPV) and closteroviruses (Tian et al, 1996;Saldarelli et al, 1998;Foissac et al, 2005 Based on these molecular and biological data, the taxonomic structure of the family of Closteroviridae was revised (Martelli et al, 2002). Within the family Closteroviridae, viruses were reclassified in 3 genera: (i) Closterovirus, (ii) Ampellovirus, and (iii) Crinivirus.…”