title: Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses.
abstract: A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.
authors.affiliation: Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias-IVIA, Carretera Moncada a Náquera km 5, 46113 Moncada, Valencia, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: López-Fabuel
authors.forename: Thierry
authors.initials: T
authors.lastname: Wetzel
authors.forename: Edson
authors.initials: E
authors.lastname: Bertolini
authors.forename: Alexandra
authors.initials: A
authors.lastname: Bassler
authors.forename: Eduardo
authors.initials: E
authors.lastname: Vidal
authors.forename: Luis B
authors.initials: LB
authors.lastname: Torres
authors.forename: Alberto
authors.initials: A
authors.lastname: Yuste
authors.forename: Antonio
authors.initials: A
authors.lastname: Olmos
mesh_terms: D029381:Closteroviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D017860:Nepovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013030:Spain; D046830:Tymoviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014773:Virology / Q000379:methods; D027843:Vitis / Q000821:virology*