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

Abstract

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,Bursaphelenchuscocophilus. 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 fromB. cocophilusnematode 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 ofB. cocophiluslysate. The primer set did not amplify the DNA from other aphelenchoidids, and sensitivity was achieved by ‘1:100 diluted’B. cocophilusDNA (roughly 1/1500 of total DNA from a single third-stage juvenile).