末端限制性片段长度多态性(T-RFLP)技术的系统误差分析

System Error Analysis for Terminal Restriction Fragment Length Polymorphism (T-RFLP)

摘要:

T-RFLP技术是一种新近发展起来的分析微生物多样性的分子生物学方法, 与其它多样性分析技术相比, 具有一些不可比拟的优势, 但T-RFLP技术操作流程将对结果产生系统误差的程度鲜有报道。实验以紫茎泽兰入侵过程中4种土样中的nifH基因多样性分析为例, 进行了只改变T-RFLP操作流程中一个步骤的3次重复分析, 结果表明: 限制性内切酶种类对T-RFLP分析结果的可重复性影响最大, PCR次之, 而毛细管电泳对结果的可重复性几乎没有影响。

Abstract:

T-RFLP is a newly developed molecular technique for microbial diversity analysis. Compared with other techniques, T-RFLP has several unparalleled advantages. However, there is no report on measuring the system errors generated in T-RFLP operational process. In this study, diversity analysis of nifH gene from 4 soil samples which represented different invaded stages after Ageratina adenophora (Sprengel) invasion was chosen as a case. In order to determine possible system errors in our study, multiple analysis in which only one procedure of the whole operational processes was changed each time were conducted. Results showed that repeatability for the results of T-RFLP analysis was influenced mostly by the types of Restriction Endonucleases, followed by the PCR procedure, while capillary electrophoresis has almost no effect.