Detection of Fomitiporia torreyae and Fulviformes umbrinellus by multiplex loop ‐mediated isothermal amplification (mLAMP) for diagnosis of Japanese pear dwarf

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.

Source: Annals of Applied Biology - December 14, 2016 Category: Biology Authors: S. Fukuta, H. Nagai, R. Suzuki, Y. Matsumoto, S. Kato, N. Saka, H. Horikawa, S. Kato, N. Miyake Tags: RESEARCH ARTICLE Source Type: research