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采用化学裂解和酶解相结合的方法,以加入PVPP的高盐缓冲液作为细胞裂解反应体系,用PEG- 8000进行DNA沉淀.从双孢蘑菇堆肥后发酵的4个代表时期培养料样品中提取微生物总DNA,再以总DNA为模板,以专用引物F27和R1498进行PCR扩增,获得16S rDNA片段,经纯化后构建4个时期样品的细菌16S rDNA文库。试验结果表明,本试验获得的总DNA质量较好。采用PCR扩增可获得多个细菌、放线菌和真菌特异片段;细菌16S rDNA文库的目的片段插入效率在90%以上。
The method of chemical lysis and enzymolysis combined with high salt buffer of PVPP was used as cell lysis reaction system and DNA was precipitated by PEG-8000. The samples were extracted from the culture medium samples of Agaricus bisporus fermentation The total DNA was extracted from the total DNA. The 16S rDNA fragments were obtained by PCR with specific primers F27 and R1498. After purification, the 16S rDNA libraries of bacteria were constructed. The test results show that the quality of the total DNA obtained in this experiment is better. Multiple bacterial, actinomycetes and fungal-specific fragments were obtained by PCR amplification. The insertion efficiency of bacterial 16S rDNA library was over 90%.