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

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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).

Affiliations: 1: 1Forestry and Forest Products Research Institute (FFPRI), 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan ; 2: 2Tropical Fruits Program, International Center for Tropical Agriculture (CIAT), Km 17, Recta Cali-Palmira Apartado Aéreo 6713, Cali, Colombia ; 3: 3Fort Lauderdale Research and Education Center, University of Florida/IFAS, 3205 College Avenue, Davie, FL 33314-7799, USA

*Present address: National Museum of Nature and Science, 4-1-1, Amakubo, Tsukoba, Ibaraki 305-0005, Japan
**Corresponding author, e-mail: nkanzaki@affrc.go.jp
∗∗∗Present address: Kansai Research Center, FFPRI, 68 Nagaikyutaroh, Momoyama, Fushimi, Kyoto, 612-0855, Japan
∗∗∗∗Present address: Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA
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2017-06-06
2018-09-22

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