目的应用T-RFLP分析胆固醇结石中微生物群落结构。方法应用末端标记限制性片段长度多态性（Terminal Restriction Fragment Length Polymorphism，T-RFLP）和克隆文库分析，以微生物群落16S rRNA基因（16S rDNA）为目标，对8例常规细菌培养阴性的纯胆固醇患者胆囊结石和胆汁中的微生物群落结构进行解析和比较。结果发现8例纯胆固醇结石患者胆汁中未找到细菌存在的证据，结石中细菌16S rDNA阳性率为37.5%（3/8），细菌16S rDNA片段测序表明细菌群落与肠杆菌科和微球菌科的微生物有较高的相似性，同时细菌群落中检出了大量的未培养微生物（Uncultured bacterium clone）。结论运用T-RFLP方法分析16S rDNA克隆片段能够有效评估纯胆固醇结石中的细菌群落结构的多样性。

Objective To analysis of microbial community structure in cholesterol calculus by T-RFLP (Terminal Restriction Fragment Length Polymorphism). Methods Small subunit rRNA gene (16S rDNA) was retrieved from 8 patients'' gallstone and bile with cholesterol calculus. The microbial community structure of these cholesterol calculus patients'' gallstone and bile were investigated by T-RFLP and clone libraries approaches. Results There was no bacterium found in bile from 8 patients with cholesterol calculus. The positive rate of bacterial 16S rDNA in stone was 37.5%(3/8). Bacterial 16S rDNA sequencing fragments showed that microbial community and Enterobacteriaceae and Micrococcaceae microorganisms had high similarity. Meanwhile, there were detected a number of uncultured bacterium from microbial community. Conclusion Analysis of 16S rDNA cloned fragment could effectively evaluate the diversity of bacterial community structure by T-RFLP method in cholesterol stone.

马铃薯是宁夏回族自治区南部山区重要的粮菜兼用作物,随栽培面积持续扩大,轮作倒茬困难,由此带来的连作障碍问题日渐突出。为探明马铃薯连作障碍机制,分别采集马铃薯正茬、连作1年、6年和10年的根际土壤样本,利用末端标记限制性片段长度多态性(terminal restriction fragment length polymorphism, T-RFLP)技术研究连作栽培对根际土壤微生物多样性的影响,以期揭示马铃薯根际土壤主要菌群动态变化规律及连作障碍的可能原因。结果表明：连作栽培后马铃薯土壤细菌和真菌DNA仍具有较高的T-RFLP多态性,但不同连作年限根际土壤中优势T-RFs片段发生变化,多年连作使某些T-RFs消失；随连作年限增加,根际土细菌Shannon-Wienen指数、Simpson指数和Sorenson指数下降,而真菌Shannon-Wienen指数和Simpson指数上升。菌群分析发现,土壤细菌中以厚壁菌门(Firmicutes)芽孢杆菌纲(Bacilli)和梭菌纲(Clostridia)所占比例最大。连作使细菌和真菌菌群发生变化,芽孢杆菌纲、鞘氨醇纲(Sphingobacteria)等比例下降甚至消失,β-变形菌纲(Beta proteobacteria)和异常球菌纲(Deinococci)比例上升；土壤真菌中座囊菌纲(Dothideomycetes)煤炱目(Capnodiales)随连作年限增加比例下降,粪壳菌纲(Sordariomycetes)肉座菌目(Hypocreales)随连作年限增加比例上升。马铃薯连作使根际土壤中芽孢杆菌属(Bacillus)等有益菌属的细菌减少,罗尔斯通菌属(Ralstonia)等致病菌属的细菌增加。连作导致马铃薯根际土壤细菌多样性水平降低,真菌多样性水平升高,根际土壤微生物多样性存在明显差异,连作破坏了根际土壤微生物群落的平衡,使其根际土壤微生态环境恶化。连作障碍可能是多因素综合互作用的结果,持续的研究有利于做出较合理的解释。

Because of its high nutritive value and extensive use, potato has become a critical crop in the southern mountain areas of Ningxia, China. However, the expansion of cultivated land areas under potato has made not only crop rotation difficult, but as has also worsened the obstacles of potato continuous cropping in recent years. In order to alleviate and eventually resolve the obstacles of potato continuous cropping, rhizosphere soils were sampled under the following potato treatments after 2 years, 7 years and 11 years of continuous cropping in the southern mountain areas of Ningxia, China. Terminal restriction fragment length polymorphism (T-RFLP) was used to study the genetic diversity of soil microorganisms, to explore the dynamics of main soil microbial groups in rhizosphere soils and to find the possible reasons for the obstacles of potato continuous cropping in the region. The results showed that some specific T-RFs fragments changed or disappeared in rhizosphere soil sa