Enzyme-Mediated Amplification (EMA) for Detection of the Pinewood Nematode Bursaphelenchus xylophilus

Abstract

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a notorious parasitic nematode of pine trees that causes pine wilt disease (PWD), leading to extensive mortality of different pine species around the world and considerable economic losses, thus posing a threat to healthy pines worldwide. Fast and accurate detection technology is necessary for the management of PWD spread. This study describes the development of a new DNA extraction method and detection technology, enzyme-mediated amplification (EMA), using primers and a newly designed probe according to the rRNA internal transcribed spacer gene ITS2. The detection process can be completed within 40 min, including DNA extraction for 10 min and detection for 30 min, by exploiting the synergistic action of multiple enzymes. This method can detect PWNs from different geographic areas quickly and accurately at all life stages, singly or in a mixture, and can distinguish PWNs from other species of the Bursaphelenchus group, showing that it is not only reliable but also rapid, greatly improving the efficiency and speed of PWN detection. Therefore, the technology is expected to be highly beneficial in PWN quarantine testing.