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污染控制技术及原理

Comparison of methods for total microbial DNA extraction from solid-state fermentation

基金项目：国家高技术研究发展计划(863)项目(No.2004AA649370);国家重点基础研究发展规划(973)项(No.2005CB724203)

摘要：为了分析固态发酵过程中微生物群落的多样性及演替情况,对比研究了从固态发酵中提取细菌和真菌DNA的3种方法——溶壁酶法、超声波法、液氮研磨+CTAB法.使用紫外分光光度计测定了由不同提取方法得到的DNA的产量与纯度;使用细菌16SrDNA基因通用引物(341F和907R)和真菌18SrDNA基因通用引物(NU-SSU-0817和NU-SSU-119)对DNA进行了PCR扩增;采用DGGE(变性梯度凝胶电泳)法对固态发酵中细菌和真菌的多样性进行了分析.结果显示,3种方法得到的粗提和纯化DNA长度均约为23kb;细菌和真菌PCR产物长度分别约为586bp和422bp.细菌和真菌PCR产物的DGGE分析表明,3种方法提取的DNA所反映的微生物多样性比较一致;但紫外分光光度计测定结果表明溶壁酶法提取固态发酵中微生物总DNA产量最高,超声波法次之,液氮研磨+CTAB法最低.

Abstract：To analyze the diversity and succession of microbes during solid-state fermentation,different genomic DNA extraction methods for bacteria and fungi were employed,including lyticase lysis,ultrasonic lysis and grinding lysis in liquid nitrogen with CTAB.The quantity and purity of the DNA were measured by ultraviolet spectrophotometer.PCR amplification was carried out with a eubacterial 16SrDNA targeted primer pair(341F and 907R)and a fungi 18SrDNA targeted primer pair(NU-SSU-0817 and NU-SSU-1196).The bacterial and fungal diversity during solid-state fermentation was analyzed by denaturing gradient gel electrophoresis(DGGE).The results show that the crude and purified DNA fragments extracted during solid-state fermentation have a length of about 23 kb.The length of the DNA fragments after bacterial and fungal PCR amplification was about 586 bp and 422 bp,respectively.DNA band profiles by DGGE analysis for either bacterial or fungal PCR products testify to the similar microbial diversity observed for all three DNA extraction methods.Lyticase lysis yielded the highest quantity of DNA,followed by ultrasonic lysis,followed by grinding lysis in liquid nitrogen with CTAB.