基于RPA等温扩增的松材线虫快速检测技术体系建立  

Development of a point-of-care testing technology for pine wood nematode based on recombinase polymerase amplification

作　　者：张伟[1,2] 车吉明[3] 理永霞 马健[4] 李东振 张星耀 ZHANG Wei;CHE Jiming;LI Yongxia;MA Jian;LI Dongzhen;ZHANG Xingyao(Key Laboratory of Forest Protection of National Forestry and Grassland Administration,Ecology and Nature Conservation Institute,Chinese Academy of Forestry,Beijing 100091,China;Co-Innovation Center for Sustainable Forestry in Southern China,Nanjing Forestry University,Nanjing 210037,China;Kunyushan Forest Farm of Yantai,Yantai 264100,China;Jiaozhou Bureau of Natural Resources and Planning,Jiaozhou 266300,China)

机构地区：[1]中国林业科学研究院森林生态环境与自然保护研究所,国家林业和草原局森林保护学重点实验室,北京100091 [2]南京林业大学,南方现代林业协同创新中心,江苏南京210037 [3]山东省烟台市昆嵛山林场,山东烟台264100 [4]胶州市自然资源和规划局,山东胶州266300

出　　处：《中国森林病虫》2024年第3期1-8,共8页Forest Pest and Disease

基　　金：“十四五”国家重点研发计划项目“松材线虫病灾变机制与可持续防控技术研究”(2021YFD1400900);国家自然科学基金项目“松墨天牛协助松材线虫扩散型四龄幼虫发育成熟的作用机理”(32271896)。

摘　　要：利用松材线虫Bursaphelenchus xylophilus基因组分析和等温扩增技术,设计生产松材线虫野外快速检测技术产品并测试其效果。结果表明:比较分析松材线虫和拟松材线虫Bursaphelenchus mucronatus基因组发现松材线虫特异性基因Bx-gene12,以此为基础设计特异性扩增引物和探针,对采集自不同地区的松材线虫、拟松材线虫和松树内其他线虫株系采用研磨颗粒以1500 r/min转速震荡5 min的破碎方法有效释放线虫DNA,40℃等温扩增15 min反应条件下可有效区分松材线虫和其他线虫,检测灵敏度达到100 fg或1条线虫;该快速检测方法在25 min内有效检测出染病黑松Pinus thunbergii和赤松Pinus densiflora松木内的松材线虫。利用RPA等温扩增技术实现25 min内对野外环境下松材线虫的快速检测,可为松材线虫病及时有效防控提供支持。Through the analysis of the genome of Bursaphelenchus xylophilus and recombinase polymerase amplification(RPA)technology,a point-of-care testing product on pine wood nematode for field test was designed and produced.The results indicated that a specific gene,Bx-gene12,was identified through genome analysis of B.xylophilus.Based on this,specific primers and probes were designed.Grinding reagents were used to break nematode samples collected from pine trees in different regions.The deoxyribonucleic acids(DNA)of nematodes were effectively released by shaking at 1500 revolutions per minute for 5 minutes.Under isothermal amplification at 40℃for 15 minutes,the detection sensitivity reached 100 fg or 1 nematode,effectively distinguishing B.xylophilus from other nematodes.Samples were taken from the wilted Pinus thunbergii and Pinus densiflora,the point-of-care testing method could effectively identify B.xylophilus in 25 minutes.This study utilized RPA technology to achieve point-of-care testing of B.xylophilus in the wild within 25 minutes,which can provide support for the timely and effective prevention and control of pine wilt disease.

关 键 词：松材线虫 快速检测 等温扩增 技术体系 

分 类 号：S763.16[农业科学—森林保护学]