T-RFLP技术在厌氧氨氧化菌群结构分析中的应用

Identification of anaerobic ammonium-oxidizing bacteria in environmental samples by T-RFLP

摘要:

为实现对样品中厌氧氨氧化菌群落组成的快速分析,提出一种基于末端限制性片段长度多态性技术(T-RFLP)分析环境样品中厌氧氨氧化菌群结构的方法．通过对SILVA (R108) SSU Ref数据库中已知厌氧氨氧化菌16S rDNA序列的模拟PCR和酶切分析,选取合适的PCR引物(Amx368f-Amx820r)和限制性内切酶(MspI和RsaI),使得不同厌氧氨氧化菌属对应不同末端片段长度(T-RFs),从而完成对厌氧氨氧化菌群落组成的快速分析．重复性和灵敏性检验结果表明,该方法能够有效、稳定地应用于环境样品中厌氧氨氧化菌群落组成的快速分析．

Abstract:

To rapidly identify anaerobic ammonium-oxidizing (anammox) bacteria in environmental samples, a cloning-independent method based on Terminal Restriction Fragment Length Polymorphism (T-RFLP) was proposed. Based on SILVA (R108) SSU Ref database containing 400 16S rRNA gene sequences of anammox bacteria, defined lengths of terminal restriction fragments (T-RFs) were predicted by virtual PCR and restriction enzyme digestion using suitable PCR primers (Amx368f-Amx820r) and restriction enzyme (MspI ＆ RsaI). Then the genus of anammox bacteria was identified by comparative analysis of T-RFs. The repeatability and sensibility analysis results show that the anammox bacteria specific T-RFLP analysis is a reliable tool to rapidly assess the complexity of anammox bacteria in environmental samples.