玻璃珠法提取堆肥微生物总DNA及其DGGE分析

Total DNA extraction of hot composting microorganism by glass beads for DGGE analysis

采用玻璃珠法从高温堆肥样品中进行了微生物总DNA的提取,以细菌16S rRNA基因V3区通用引物进行了PCR扩增,随后用变性梯度凝胶电泳(DGGE)技术对其微生物多样性进行评估.结果表明,玻璃珠法能够快速、有效地获得高质量的堆肥微生物总DNA,所得的DNA分子片段在15kb以上,使用细菌16S rRNA基因通用引物(27F和1492R)对总DNA进行PCR扩增,获得了近全长的16S rDNA序列(约1.5kb);DGGE分析结果表明,用该方法提取到的DNA种类较为丰富,多样性较好,能够进一步应用于群落结构分析.因此,玻璃珠法提取的DNA可以用于堆肥微生物的分子生态学研究及微生物群落结构分析.

Total DNA of the hot composting microorganism was directly extracted by glass beads method,and bacterial 16S rRNA genes were amplified with a pair of universal primers of the 16S rRNA gene V3 region by polymerase chain reaction（PCR）,then the diversity of the amplification products was evaluated by DGGE technology.The results indicaed that glass beads can extract microorganism total DNA from hot composting quickly,molecular size of DNA obtained using this protocol was about 23kb and a eubacterial 16S rRNA gene targeted primer pair（27F and 1492R） was used for PCR amplificaion,and got almost the full length 16S rDNA sequence（about 1.5 kb）.The results of DGGE analysis indicated that the DNA from the hot composting has high population diversity,and has further application in the community analyses.So the microbial DNA extracted by using glass beads could be used for molecular ecological study and community analyses.