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Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR.

Abstract：

:One of the major factors determining the incidence of Barley yellow dwarf virus (BYDV) on autumn-sown cereals is the viruliferous state of immigrant winged aphids. This variable is assessed routinely using the enzyme-linked immunosorbant assay (ELISA). However, the threshold for virus detection by ELISA can lead to false negative results for aphids carrying less than 10(6) particles. Although molecular detection techniques enabling the detection of lower virus quantities in samples are available, the relatively laborious sample preparation and data analysis have restricted their use in routine applications. A gel-free real-time one-step reverse transcription polymerase chain reaction (RT-PCR) protocol is described for specific detection and quantitation of BYDV-PAV, the most widespread BYDV species in Western Europe. This new assay, based on TaqMan technology, detects and quantifies from 10(2) to 10(8) BYDV-PAV RNA copies. This test is 10 and 10(3) times more sensitive than the standard RT-PCR and ELISA assays published previously for BYDV-PAV detection and significantly improves virus detection in single aphids. Extraction of nucleic acids from aphids using either phenol/chloroform or chelatin resin-based protocols allow the use of pooled samples or of a small part (up to 1/1600th) of a single aphid extract for efficient BYDV-PAV detection.

journal_name

J Virol Methods

journal_title

Journal of virological methods

authors

Fabre F,Kervarrec C,Mieuzet L,Riault G,Vialatte A,Jacquot E

doi

10.1016/s0166-0934(03)00097-1

subject

Has Abstract

pub_date

2003-06-09 00:00:00

pages

51-60

issue

1

eissn

0166-0934

issn

1879-0984

pii

S0166093403000971

journal_volume

110

pub_type

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