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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (3): 170-173.

• 农学 农业基础科学 • 上一篇    下一篇

冬虫夏草RAPD反应体系的建立及优化

张德利 钱敏 曾纬 陈仕江   

  • 收稿日期:2010-07-20 修回日期:2010-08-23 出版日期:2011-02-05 发布日期:2011-02-05
  • 基金资助:

    重庆市“十一五”重大科技攻关项目;重庆市自然科学基金

Establishment and optimization of RAPD reaction system of Cordyceps Sinensis

  • Received:2010-07-20 Revised:2010-08-23 Online:2011-02-05 Published:2011-02-05

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摘要：

为建立并优化冬虫夏草RAPD反应体系，通过改变RAPD反应体系中Mg2+、dNTP、引物、模板等主要成分的浓度，结合RAPD扩增效果，建立冬虫夏草RAPD反应体系，然后通过改变主要热循环参数，优化冬虫夏草RAPD反应体系。结果表明：适合冬虫夏草的RAPD反应体系为25 μL体系中内含1×PCR缓冲液、1.5 μmol/μL Mg2+、320 μmol/L dNTP、24 ng引物、20 ng模板、1 U Taq酶；扩增程序的优化结果为：95℃预变性5 min，然后35个循环（94℃变性45 s，36℃复性1 min，72℃延伸2 min），循环结束后72℃延伸7 min。综上，RAPD技术可用于冬虫夏草的鉴定、评价分析。

关键词: 精异丙甲草胺, 精异丙甲草胺, 菜豆, 气相色谱法, 残留

Abstract:

To establish RAPD reaction system of Cordyceps sinensis. Through changing the density of Mg2+, dNTP, primer, template DNA in RAPD reaction system, with the result of RAPD amplify, established the RAPD reaction system of Cordyceps sinensis. Then through changing the main thermal cycle parameter, optimize RAPD reaction system of Cordyceps sinensis. The results showed that an RAPD reaction system suitable for Cordyceps sinensis was established, that was: 20 μL amplification containing 1×PCR buffer, 1.5 μmol/μL Mg2+, 320 μmol/L dNTP, 24 ng premer、20 ng template DNA、1 U Taq DNA polymerase. The optimization result of amplifying the procedure was that: pre-Modified 5 min at 95℃, and then circulated 35 times (denatured 45 s at 94℃, renatured 1 min at 36℃, elongated 2 min at 72℃), and then elongated 2 min at 72℃ after the cycle. The RAPD reaction system can be used to RAPD analysis in Cordyceps sinensis.

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