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林业科学 ›› 2005, Vol. 41 ›› Issue (2): 91-95.doi: 10.11707/j.1001-7488.20050215

• 论文及研究报告 • 上一篇    下一篇

疫木中松材线虫的免疫学检测方法的研究

白钢1 王玉嬿2 马洪周1 李海燕2 杨文博1   

  1. 1.南开大学生命科学学院,天津300071;2.国家林业局森林病虫害防治总站,沈阳110034
  • 收稿日期:2003-03-03 修回日期:1900-01-01 出版日期:2005-03-25 发布日期:2005-03-25

Development and Application of an Immunoassay Method for Identification of Bursaphelenchus xylophilus in Pinewood

Bai Gang1,Wang Yuyan2,Ma Hongzhou1,Li Haiyan2,Yang Wenbo1   

  1. 1.College of Life Science, Nankai University Tianjin 300071; 2.General Station of Forest Pest and Disease Control, The State Administration of Forestry Shenyang 11003
  • Received:2003-03-03 Revised:1900-01-01 Online:2005-03-25 Published:2005-03-25

摘要:

以松材线虫的粉碎虫体免疫家兔制备抗血清,通过免疫印迹考察抗血清的特异性。利用固相松材线虫抗原和游离线虫抗原同线虫抗体相竞争,建立竞争型ELISA法用于疫木中松材线虫的分析。该方法可以直接检测出10mg的木屑中0.1μg微量的线虫蛋白,约7条线虫的存在。利用该方法对20个不同地区、树种及类型的松材样品进行检测,疫木中松材线虫的检出率为100% ,但拟松材线虫也呈现了一定的交叉反应。研究结果表明,以松材线虫抗原蛋白为指标的免疫学检测方法简便快速、灵敏度高,为松材线虫的快速检疫提供一种可能的途径

关键词: 松材线虫, 线虫鉴定, 免疫印迹, ELISA

Abstract:

Taking the specimens of Bursaphelenchus xylophilus (BX) and B. mucronatus (BM) fragments as research objects, an immunoassay method for nematode antigen analysis was developed in this paper. The rabbit antiserum against BX was prepared, and the antigen proteins were assayed by SDS-PAGE and western blot. Using BX fragments as solid-phase, the assay principle was based on competition between an analyte and nematode solid-phase antigen for anti-BX antibody. Then using HRP labeled goat anti-rabbit IgG as a tracer antibody, an enzyme-linked immunosorbent assay (ELISA) was developed. The method was specific to BX antigen harvested from different areas. Although the ELISA showed a little cross reactivity with BM, it could identify 0.1μg nematode protein in 10 mg pinewood specimen. The result suggested that the ELISA method using nematode antigen as a detection objective showed a new potential for the quarantine inspection and forest pest control of BX.

Key words: Bursaphelenchus xylophilus, nematode analysis, western blot, ELISA