Loop-mediated isothermal amplification (LAMP) for detection of the red ring nematode, Bursaphelenchus cocophilus

in Nematology
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As a first step in developing a quick, accurate and simple method for the diagnosis of red ring disease, the loop-mediated isothermal amplification (LAMP)-based identification procedure was applied to the causative agent, Bursaphelenchus cocophilus. Two LAMP primer sets were designed using two loci of ribosomal RNA genes, i.e., D2-D3 expansion segments of the large subunit (D2-D3 LSU), and internal transcribed spacers (ITS). Within those two sets of primers, the D2-D3 LSU primer set successfully yielded amplicons from B. cocophilus nematode lysate prepared from 3-year-old DESS-fixed specimens. The specificity of the primers was examined using 18 species of confamilial Aphelenchoididae nematodes and primer sensitivity was tested using a diluted series of B. cocophilus lysate. The primer set did not amplify the DNA from other aphelenchoidids, and sensitivity was achieved by ‘1:100 diluted’ B. cocophilus DNA (roughly 1/1500 of total DNA from a single third-stage juvenile).

Loop-mediated isothermal amplification (LAMP) for detection of the red ring nematode, Bursaphelenchus cocophilus

in Nematology



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    Visual inspection of LAMP reaction tube by fluorescence after 60 min of incubation at 63°C. Upper lane from left to right: positive reactions to Bursaphelenchus cocophilus (three individuals of ST01; two replicates for one individual of NT25; two replicates for one individual of NT26; and two individuals of NT32); negative reaction to B. platzeri; B. penai; B. tadamiensis; B. xylophilus; B. mucronatus; B. conicaudatus. Lower lane from left to right: negative reaction to B. firmae; B. luxuriosae; B. doui; B. sinensis; B. osumiana; B. parvispicularis; B. gerberae; B. niphades; B. sakishimanus; undescribed Bursaphelenchus sp. ‘Sugadaira’ (B. eggersi group); undescribed Bursaphelenchus sp. ‘Esashi’ (B. eremus group); Aphelenchoides xylocopae.

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    Agarose gel electrophoresis showing the sensitivity of LAMP for the detection of Bursaphelenchus cocophilus using diluted series of the DNA lysates. Left to right: Markers, A single individual-derived lysate, 1:5, 1:10, 1:25, 1:50, 1:100, 1:250 and 1:500 distilled lysates, and negative control (distilled water).

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