一种快速制备丝状真菌PCR反应模板的方法

A method for rapid preparation of PCR templates of different filamentous fungi

为建立一种快速、简便制备丝状真菌PCR反应模板的方法,提高丝状真菌菌株鉴定与转化子筛选效率,本试验以10种不同种丝状真菌为材料,比较了3种不同试剂微波法制备丝状真菌PCR反应模板的效果,并优化了微波处理的时间。试验结果表明,取少量菌丝,加入50 mmol/L NaOH,无菌吸头将菌丝打散,微波炉高火处理30 s,离心取上清,即可获得用作PCR反应的模板。该方法制备的10种不同种丝状真菌PCR反应模板用ITS引物进行扩增,都可得到与传统CTAB法提取的DNA模板同样清晰明亮的扩增条带。该法制备的PCR模板可用于不同引物和不同公司的PCR反应产品扩增。将该方法制备的PCR模板用于囊状匍柄霉Stemphylium vesicarium转化子的筛选,其结果与传统CTAB法提取的DNA结果一致。此法为丝状真菌提供了更加快速简便的PCR模板制备方法,极大减少了DNA提取的人力和物力成本。

A rapid and simple method for preparing PCR reaction template of filamentous fungi and improving the efficiency of filamentous fungal strain identification and transformant screening was established.Ten different species of filamentous fungi were used as materials to compare the effects of microwave preparation of filamentous fungi PCR templates using three different reagents,and the duration of microwave treatment were optimized.The templates for PCR could be obtained by adding 50 mmol/L NaOH to a small of mycelium,breaking up the mycelium with sterile sucker,treating it with high heat in microwave oven for 30 s,and centrifuging the solution to get the supernatant.The PCR templates of 10 different species of filamentous fungi obtained by this method can be amplified by ITS primers,and the same clear and bright target bands as the DNA templates extracted by the traditional CTAB method can be obtained.The templates prepared by this method are suitable for different primers and different PCR amplification products.The PCR template prepared by this method was used to screen the transformants of Stemphylium vesicarium,and the results were consistent with the DNA extracted by the traditional CTAB method.This rapid and simple PCR template preparation method for screening large number of transformants and molecular identification of filamentous fungi greatly reduces the labor and material resources cost in the process of DNA extraction.