title: Development of one-tube real-time qRT-PCR and evaluation of RNA extraction methods for the detection of Eggplant mottled dwarf virus in different species.
abstract: A one-tube real-time qRT-PCR assay was developed, for the detection and quantification of Eggplant mottled dwarf virus (EMDV), a pathogen affecting cultivated and ornamental plants. The amplification efficiency of the assay was 98% and the linear range of quantification was from 20 to 2×10(8) RNA transcripts. Total RNA extraction methods (three developed methods and one commercially available RNA extraction kit) were evaluated using tissues from seven different plant species and synthetic EMDV RNA transcripts of known concentration. The recovery rates of RNA and the effect of co-extracted inhibitors revealed that methods involving PVPP and phenol-chloroform extraction were the most efficient. These modifications were necessary for processing samples containing high phenolic and polysaccharide compounds such as woody plants. The developed EMDV detection protocol was successfully applied in forty naturally infected woody and herbaceous plants belonging to six different species. The protocol comprises a useful method for low-cost detection of ssRNA viruses in diverse plant tissues.
authors.affiliation: Laboratory of Plant Pathology, School of Agriculture, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece. Electronic address: pappi@agro.auth.gr.
authors.forename: Polyxeni G
authors.initials: PG
authors.lastname: Pappi
authors.affiliation: Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra str., 54627, Thessaloniki, Greece. Electronic address: schainto@vet.auth.gr.
authors.forename: Serafeim C
authors.initials: SC
authors.lastname: Chaintoutis
authors.affiliation: Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra str., 54627, Thessaloniki, Greece. Electronic address: dovas@vet.auth.gr.
authors.forename: Chrysostomos I
authors.initials: CI
authors.lastname: Dovas
authors.affiliation: Laboratory of Plant Pathology, School of Agriculture, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece. Electronic address: eftimiou@agro.auth.gr.
authors.forename: Konstantinos E
authors.initials: KE
authors.lastname: Efthimiou
authors.affiliation: Laboratory of Plant Pathology, School of Agriculture, Faculty of Agriculture, Forestry and Natural Environment, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece. Electronic address: katis@agro.auth.gr.
authors.forename: Nikolaos I
authors.initials: NI
authors.lastname: Katis
mesh_terms: D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012209:Rhabdoviridae / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D032323:Solanum melongena / Q000821:virology*; D014773:Virology / Q000379:methods
chemical_list: D012367:RNA, Viral
keywords: Eggplant mottled dwarf virus; One-tube real-time qRT-PCR; Quantification; Total RNA extraction