title: Sensitive detection of Tomato ringspot virus by real-time TaqMan RT-PCR targeting the highly conserved 3'-UTR region.
abstract: A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand. Electronic address: joe.tang@mpi.govt.nz.
authors.forename: Joe
authors.initials: J
authors.lastname: Tang
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand.
authors.forename: Subuhi
authors.initials: S
authors.lastname: Khan
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand.
authors.forename: Catia
authors.initials: C
authors.lastname: Delmiglio
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand.
authors.forename: Lisa I
authors.initials: LI
authors.lastname: Ward
mesh_terms: D020413:3' Untranslated Regions; D017931:DNA Primers / Q000235:genetics; D017860:Nepovirus / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D020413:3' Untranslated Regions; D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral
keywords: Detection; Real-time; TaqMan; Virus