Pine wilt disease (PWD), a serious threat to pine forests in East Asia and Europe, is caused by the pinewood nematode, Bursaphelenchus xylophilus. Because the nematodes are transmitted by insect vectors from dead to healthy trees, PWD spreads extremely rapid. Accurate and rapid diagnosis and then complete removal of the nematodeinfected trees from pine forests are thus very important for control of PWD. To diagnose PWD, B. xylophilus must be detected in infected trees. Generally, the nematode is extracted from wood tissues by using the Baermann funnel technique and is then identified under stereo and light microscopes. However, this method requires technical knowledge of nematode morphology and expensive equipment such as microscopes. In addition, nematode extraction takes a long time (usually 1 or 2 days), and identification of the nematode is possible only when adults (both male and female) are extracted. Several DNAbased protocols for the identification of B. xylophilus have recently been developed. Although these molecular techniques are sensitive and independent of the life stage of the nematode, they require both the use of a Baermann funnel to collect the nematodes from the wood tissues and expensive equipment such as a thermocycler or realtime PCR apparatus to amplify the DNA. We developed a simple and lowcost method of determining the presence of PWD by using loopmediated isothermal amplification (LAMP). This method consists of the following three processes: (1) DNA extraction. The DNA of B. xylophilus is directly extracted from small wood chips in DNA extraction buffer in a micro test tube for 30 min; (2) DNA amplification. Part of the extracted DNA solution is transferred to a reaction mixture in a 0.2 mL micro test tube, and then the B. xylophilus DNA is specifically amplified in the tube under a constant temperature for 60 min; (3) Judgment. Amplification of the target DNA is confirmed by the colour of reacted solution. The only piece of equipment that LAMP diagnosis requires is an incubator for maintaining a constant temperature, and the procedure is completed in only 90 min. Because this new method of diagnosing PWD is easier and cheaper than traditional method, its use is likely to become widespread.
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