Improved detection of barley yellow dwarf virus in single aphids using RT-PCR.
The detection of a British isolate of barley yellow dwarf virus (BYDV-G, PAV-like) from individual vector aphids, using a combined assay of reverse transcription and polymerase chain reaction (RT-PCR) is reported. The method makes use of a multiplex format, including internal control primers directed at conserved regions of insect actin. The actin primers serve as controls for each stage of the method and are suitable for use in a range of invertebrate species. Detection of BYDV in vector aphids for use in forecasting systems is at present carried out using an enzyme-amplified ELISA system. In direct comparisons with the amplified ELISA, RT-PCR shows an increase in sensitivity detecting 11 fg of purified virus. Detection of virus in ELISA-negative aphids by RT-PCR was also demonstrated, and its potential as a routine diagnostic tool for virus detection in aphids is discussed.
Canning
E S
ES
Department of Pure and Applied Biology, University of Leeds, UK.
Penrose
M J
MJ
Barker
I
I
Coates
D
D
D000818:Animals; D001042:Aphids / Q000821:virology*; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005240:Feasibility Studies; D001467:Hordeum / Q000821:virology; D017793:Luteovirus / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*
D004279:DNA, Viral; D012367:RNA, Viral