利用环介导恒温扩增法简易诊断松萎蔫病  

Simple diagnosis of Pine Wilt Disease using loop-mediated isothermal amplification

作　　者：Takuya Aikawa Natsumi Kanzaki Taisei Kikuchi 

机构地区：[1]Tohoku Research Center,Forestry and Forest Products Research Institute,Morioka, Iwate 020-0123, Japan [2]Forestry and Forest Products Research Institute,Tsukuba, Ibaraki 305-8657, Japan

出　　处：《南京林业大学学报（自然科学版）》2011年第4期148-149,共2页Journal of Nanjing Forestry University：Natural Sciences Edition

摘　　要：诊断松萎蔫病的传统方法是从木质组织中分离松材线虫,然后用显微镜进行形态学鉴定。基于DNA分子检测的方法尽管很灵敏,不需要固定发育阶段的松材线虫,但是仍然需要用Baer-mann漏斗法分离线虫,并且需要昂贵的仪器。笔者研发了利用环介导恒温扩增法检测松材线虫的存在,该方法包括:(1)从木块中提取DNA;(2)DNA扩增;(3)通过反应溶液的颜色做出诊断。该方法仅使用培养箱且整个过程只需要90 min,比以往方法更加简便和快速。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 nematode-infected 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 DNA-based 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 real-time PCR apparatus to amplify the DNA.We developed a simple and low-cost method of determining the presence of PWD by using loop-mediated 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 maint

关 键 词：松萎蔫病 松材线虫 DNA 环介导恒温扩增法 

分 类 号：S764[农业科学—森林保护学]