苹果炭疽菌DNA改良提取法及PCR体系的优化

Comparison of Several Modified DNA Extraction Methods and Optimization of PCR System for Colletotrichum gloeosporioides in Apple

以苹果炭疽病胶孢炭疽菌(Colletotrichum gloeosporioides)为试材,采用改良的SDS法、改良的CTAB法和改良的尿素法等对基因组DNA进行了提取和比较,并对ITS1-5.8S-ITS2区基因PCR扩增体系中模板的添加量以及退火温度进行了调整和优化。结果表明:采用这3种方法均能提取得到目的 DNA,但采用改良的SDS法提取的基因组DNA产率最高、质量最好;当在25μL PCR体系中添加0.75μL粗提DNA 10倍稀释液以及退火温度设定为51℃时PCR产物的质量最高。

The genomic DNA of Colletotrichum gloeosporioides in apple was extracted and compared by modified SDS method,modified CTAB method and modified urea method,and the additive amount of template and the annealing temperature in the PCR system for the amplification of ITS1-5.8S-ITS2 gene were adjusted and optimized. The results showed that using the above three methods all could get the target DNA,but the yield of genomic DNA extracted by modified SDS method was the highest and its quality was the best. When 0.75 μL 10-fold dilution of the extracted crude DNA was added into 25 μL PCR system and the annealing temperature was set as 51 ℃,the quality of the PCR product was the highest.