冬虫夏草RAPD反应体系的建立及优化

Establishment and Optimization of RAPD Reaction System of Cordyceps sinensis

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25μL体系中内含1×PCR缓冲液、1.5μmol/μL Mg2+、320μmol/L dNTP、24ng引物、20ng模板、1U Taq酶;扩增程序的优化结果为:95℃预变性5min,然后35个循环(94℃变性45s,36℃复性1min,72℃延伸2min),循环结束后72℃延伸7min。综上,RAPD技术可用于冬虫夏草的鉴定、评价分析。

To establish RAPD reaction system ofCordyceps 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 ofCordyceps sinensis.The results showed that an RAPD reaction system suitable forCordyceps 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 inCordyceps sinensis.