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LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine
Author(s) -
Kogovšek P.,
Hodgetts J.,
Hall J.,
Prezelj N.,
Nikolić P.,
Mehle N.,
Lenarčič R.,
Rotter A.,
Dickinson M.,
Boonham N.,
Dermastia M.,
Ravnikar M.
Publication year - 2015
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/ppa.12266
Subject(s) - loop mediated isothermal amplification , biology , phytoplasma , quarantine , dna extraction , homogenization (climate) , chromatography , botany , virology , polymerase chain reaction , dna , genetics , chemistry , gene , restriction fragment length polymorphism , ecology , biodiversity
In Europe the most devastating phytoplasma associated with grapevine yellows ( GY ) diseases is a quarantine pest, flavescence dorée ( FD p), from the 16SrV taxonomic group. The on‐site detection of FD p with an affordable device would contribute to faster and more efficient decisions on the control measures for FD p. Therefore, a real‐time isothermal LAMP assay for detection of FD p was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FD p in all leaf samples that were determined to be FD p infected using quantitative real‐time PCR . The whole procedure of sample preparation and testing was designed and optimized for on‐site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.