一种适用于田间LAMP检测的橡胶树白粉菌DNA粗提取方法

Rough Extraction Method of Oidium heveae Steinm. DNA Applicable to LAMP Detection in the Field

为了探索建立一种快速、简便适用于田间LAMP (loop-mediated isothermal amplification)检测的橡胶树白粉菌DNA粗提取方法,在无液氮也不需要研磨的情况下,利用渗透压破壁的方法提取橡胶树白粉菌DNA。通过改变渗透压对橡胶树白粉菌DNA进行提取,使用特异性引物OH-666A (5'-ACTGACACCCCAT CACGACTAAAA-3'),OH-666S (5'-CATATTCTCACGAAGGAAAGCATTG-3')进行PCR扩增,并与试剂盒提取DNA的PCR产物进行比较,对提取的DNA进行LAMP检测。20μL甘油加入600μL ddH2O,离心10 min,去上清,加50μL水离心2 min即可提出橡胶树白粉菌的DNA,PCR扩增能获得清晰目的条带,对得到的扩增产物进行测序,同时对提取的DNA进行LAMP检测,并与实验室常规方法比对,所有结果均一致。破壁法可用于橡胶树白粉菌DNA的快速提取,由于不需要特殊仪器,且快速方便,在田间容易实现,所以更适用于田间橡胶树白粉菌DNA的田间粗提取,对开展橡胶树白粉病田间分子预测提供了技术支持。

In order to explore and establish a rapid and simple method to rough extract DNA from Oidium heveae, which is suitable for field LAMP （loop-mediated isothermal amplification） detection, this research extracted DNA from Oidium heveae without liquid nitrogen and grinding by osmotic pressure broken method. This research extracted Oidiura heveae DNA by changing the osmotic pressure. Specific primers, OH-666A （5＇-ACTGACACCC CATCACGACTAAAA-3）, OH-666S （5＇-CATATTCTCACGAAGGAAAGCATTG-3） were used to amplify PCR products for Oidium heveae, and compare with the PCR product of reagent DNA extraction kit. The LAMP detection was performed on DNA extracted. 20 ~L glycerol was added to 600 ~L ddI-I20. Centrifuged 10 min, and removed the supematant. Lastly added 50 ~L water and centrifuged 2 min to get Oidium heveae DNA. Clear target band could be obtained through PCR amplification, then the amplified products were sequenced. At the same time, we used Lamp reaction to detect the extracted DNA. Compared with the conventional laboratory methods, all the results were the same. The wall-breaking method could be used for rapid extraction of DNA from powdery mildewof rubber tree. Because it does not need special instruments and is easy to implement in the field, it might be more suitable for field crude extraction of Oidium heveae DNA in the field, and the establishment of this method might provide technical support for field molecular prediction of Oidium heveae.