Optimization of PCR Conditions for Soil Microbial Diversity Study

研究土壤微生物多样性的PCR条件优化(英文)

[Objective] The research aimed to optimize the test condition of PCR-DGGE method to analyze genetic diversity of soil microorganism. [Method] The amplified results of PCR were compared by improving high salt method for extracting soil DNA, improving primer design, changing the annealing temperature and amplification system of PCR reaction process. [Result] The result of extracting soil micro-bial DNA was better by high salt method which was improved. A single band was obtained and operation was easy when choosing 20 μl system for PCR amplification. There was no nonspecific amplification when choosing annealing temperature at 55 ℃, and cycle number of 35 was easy for following DGGE analysis. [Conclusion] The optimized PCR reaction system has high specificity and reliability.

[目的]对PCR-DGGE法的试验条件进行优化,以更好地分析土壤微生物遗传多样性。[方法]改良高盐法提取土壤DNA,改进引物设计,改变PCR反应过程中退火温度、扩增体系,比较PCR扩增结果。[结果]经过改良的高盐法提取的土壤微生物DNA效果更好。PCR扩增选择20μl体系条带单一,易于操作。退火温度选择在55℃时无非特异性扩增,35个循环次数易于后续DGGE分析。[结论]已经优化的PCR基因扩增体系特异性高且稳定可靠。