香蕉穿孔线虫双重PCR快速检测技术研究  

Development of duplex PCR assay for detection of burrowing nematode,Radopholus similis

作　　者：葛建军[1] 曹爱新[1] 周国梁[2] 赵国柱[3] 谢丙炎[4] 

机构地区：[1]中国检验检疫科学研究院动植物检疫研究所,北京100029 [2]上海出入境检验检疫局,上海200135 [3]北京林业大学生命科学与技术学院,北京100083 [4]中国农业科学研究院蔬菜研究所,北京100081

出　　处：《植物病理学报》2007年第5期472-478,共7页Acta Phytopathologica Sinica

基　　金：上海市科委重大攻关项目(o3dz19315);科研院所公益研究专项(2004DIB3J096;2003DIB3J108);"863"项目(2006AA10Z434)

摘　　要：香蕉穿孔线虫(Radopholus similis)是国际上公认的植物检疫性有害生物之一,是热带地区果树上最重要的线虫。通过设计的属水平上的特异性引物R1、R2(R1/2)和种水平上的特异性引物RS1、RS2(RS1/2)进行双重PCR扩增香蕉穿孔线虫,可扩增出长度为362和291 bp的2个特异性片段谱带。同时分别对检测体系中反应参数进行优化,最佳延伸温度为51.4℃,引物R1/2与RS1/2最佳浓度配比为0.2μm o.lL-1/1.2μmol.L-1,并测试检测引物的灵敏度,能进行有效检测的最低起始核酸量为100 pg;同时,也能对单条线虫进行检测以及样品中线虫的检测。专化性测试证明,2对引物能有效地进行香蕉穿孔线虫的快速分子诊断。The burrowing nematode, Radopholus similis（Cobb） Thorne, is one of considerable plant quarantine pests, it may be the most important nematode pest on fruit crops （ especially citrus and banana） in the tropics. Two pairs of primers R1, R2 （R1/2） and RS1, RS2 （RS1/2） were developed to identify the burrowing nematode. The primer R1/2 and RS1/2 could produce 362 bp band for all nematodes of genus Radopholus, but only R. similis produced 291 bp band. At the same time, the parameters of PCR were optimized and sensitivity were tested. The optimized conditions were an extension temperature of 51.4℃ and primers concentrations of R1/2 and RS1/2 at 0.2 μmoL/L and 1.2 μmol/L, individually. The minimum amount of DNA of R. similis could be detected was as low as 100 pg, and it also could detect DNA from individual nematode and nematodes from samples of plant. Thus, it demonstrates that duplex PCR approach will provided an accurate and effective method for quick detection of burrowing nematode.

关 键 词：香蕉穿孔线虫 分子检测 双重PCR 

分 类 号：S436.6[农业科学—农业昆虫与害虫防治]