环境微生物群落分析的T-RFLP技术及其优化措施

Application and Optimization of T-RFLP Method for Microbial Community Analysis

末端限制性酶切片段长度多态性分析(terminal restriction fragment length polymorphism, T-RFLP)是近年来发展起来的、不依赖于培养的微生物群落分析方法之一.由于其在微生物群落结构分析方面的特点,包括分辨率高、易于实现自动化及互联网海量数据共享等优势,自1997年最先被报道以来得到了广泛的应用,成为环境微生物群落分析的最强有力的工具之一.本文详细介绍了T-RFLP技术的原理,并从环境样品群落DNA的提取、引物设计和PCR扩增、限制性酶切、电泳分离检测和T-RFLP图谱解析等5个方面讨论了用该技术解析环境微生物群落的方法和技巧,简述了近8 a来国外T-RFLP技术在群落分析中的研究进展.类似于其他的分子微生物生态学技术, T-RFLP也有自身的缺陷,因此重点分析了该技术的局限性及相应的解决办法.

Terminal restriction fragment length polymorphism （T-RFLP） analysis is a culturing-independent approach for analyzing microbial community in environments. It has been proved to be powerful and widely applied since developed in 1997. The principle and general procedure of the T-RFLP technique for the analysis of microbial community are discussed in detail, including DNA extraction of environmental samples, amplification of genes encoding the 16S rRNA, 18S rRNA or enzymes with fluorescently labeled primers, the restriction enzyme digestion of PCR products, capillary electrophoresis and theanalysis of T- RFLP profile. A brief review is also made on the research of microbial community with the T-RFLP technique in recent 8 years. As with other PCR-based molecular techniques, the T-RFLP is not free of pitfalls. The limits of the T-RFLP approach are also summarized as well as their possible solutions in this paper. In addition, some valuable web resources are introduced for a better understanding and handling of T-RFLP method. With the advantages of high sensitivity, simplicity for automatization and availability to share the substantive data on Internet, the T-RFLP is nowadays one of the most powerful tools for analyzing microbial community. Fig 2, Tab 1, Ref 62