土壤镰刀菌种群检测的巢式PCR-DGGE方法的建立

本试验比较了4种提取小麦-玉米轮作免耕田土壤DNA的方法,并对巢式PCR反应体系和扩增条件进行了优化,建立了适合检测土壤中镰刀菌多样性的PCR-DGGE体系。结果表明:土壤DNA的质量是影响PCR扩增结果的关键因子,试剂盒Fast DNA SPINfor Soil所提土壤DNA片段接近23 bp,A260/A280比值为1.84,DNA产量为18.1μg/g,更适合进行PCR反应。DNA模板的浓度决定PCR扩增结果,第一轮PCR扩增的DNA模板适宜量为2～5 ng,以第一轮PCR产物的原液为模板进行第二轮PCR扩增,在退火温度为67℃时扩增结果最好。PCR产物在变性范围为45%～60%的丙烯酰胺梯度凝胶中电泳7～8 h,其条带能够完全分离,可以用作土壤中镰刀菌多样性的检测。

Nested PCR-DGGE method for detecting population of Fusarium spp. in untiliged soil

Four methods of extracting DNA from soil were compared and the conditions of nested PCR amplification for soil DNA were optimized.The PCR-DGGE method for detecting the diversity of Fusarium spp.in soil was developed.The results showed that the quality of DNA extracted from soil was the key factor influencing the result of PCR amplification.The quality of PCR production was due to the concentration of template DNA.In the first round of PCR,the suitable concentration of template DNA was 2-5 ng.In the second amplification,two factors were important for the concentration of the template DNA and annealing temperature.In the DGGE detecting,the product was fully separated with the denaturant acrylamide concentration from 45% to 60%,which was suitable to detect the population of Fusarium spp.in soil.