title: Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control.
abstract: Apple scar skin viroid (ASSVd) is an important quarantine pathogen for international movement of pome germplasm as it can cause significant damage to pip fruit. A one-step real-time RT-PCR assay was developed for the rapid and sensitive detection of ASSVd. The assay was able to detect a wide range of ASSVd isolates and was highly specific compared to a published conventional RT-PCR. The detection limit of the new assay was estimated to be about 100 copies of the ASSVd target. The assay can be run as a duplex with the nad5 internal control primers and probe to simultaneously check the PCR competency of the samples therefore reducing the risk of false negatives. It is expected that this real-time RT-PCR assay will facilitate efficient testing for ASSVd by regulatory services, and will also have a wider use for the general detection of ASSVd in a range of pip fruit.
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: dnature diagnostics & research Ltd., 24 Island Road, Gisborne 4010, New Zealand.
authors.forename: John
authors.initials: J
authors.lastname: Mackay
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand.
authors.forename: Lia
authors.initials: L
authors.lastname: Liefting
authors.affiliation: Plant Health and Environment Laboratory, Ministry for Primary Industries, P.O. Box 2095, Auckland 1140, New Zealand. Electronic address: lisa.ward@mpi.govt.nz.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ward
mesh_terms: D027845:Malus / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014772:Viroids / Q000235:genetics / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: ASSVd; Apple scar skin viroid; Quarantine; Real-time RT-PCR, RT-qPCR