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Detection of Fomitiporia torreyae and Fulviformes umbrinellus by multiplex loop‐mediated isothermal amplification ( mLAMP ) for diagnosis of Japanese pear dwarf
Author(s) -
Fukuta S.,
Nagai H.,
Suzuki R.,
Matsumoto Y.,
Kato S.,
Saka N.,
Horikawa H.,
Kato S.,
Miyake N.
Publication year - 2017
Publication title -
annals of applied biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.677
H-Index - 80
eISSN - 1744-7348
pISSN - 0003-4746
DOI - 10.1111/aab.12325
Subject(s) - loop mediated isothermal amplification , biology , primer (cosmetics) , pear , amplicon , multiplex , microbiology and biotechnology , multiplex polymerase chain reaction , detection limit , polymerase chain reaction , dna , genetics , botany , chromatography , gene , chemistry , organic chemistry
Abstract Japanese pear dwarf, caused by the fungi Fomitiporia torreyae or Fulviformes umbrinellus, is one of the most important diseases affecting Japanese pear ( Pyrus pyrifolia var. culta). To diagnose this disease, a multiplex loop‐mediated isothermal amplification ( mLAMP ) reaction using primer sets designed from the rDNA internal transcribed spacer ( ITS ) sequences of F. torreyae and F. umbrinellus was developed. The optimal conditions for simultaneous detection of the two pathogens were investigated. The best results were obtained at a reaction temperature of 65°C and a primer ratio of 1:1.5 ( F. torreyae : F. umbrinellus ). Fluorescently labelled mLAMP amplicons were precipitated using polyethyleneimine. As a result, multiplex detection was enabled by the fluorescent colour of precipitate under ultraviolet light. The detection limit of mLAMP was 100 fg of genomic DNA , which was 10 times more sensitive than the polymerase chain reaction ( PCR ) method. The mLAMP assay was applied to Japanese pear trunk samples from Aichi Prefecture, Japan, and the results were compared with those obtained using PCR . As a result, mLAMP was observed to be effective for the specific detection of F. torreyae or F. umbrinellus .