重新评估用于通过变性梯度凝胶电泳（DGGE）对反硝化细菌进行群落调查的靶向nirS、nirK和nosZ基因的聚合酶链式反应（PCR）引物。

Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE.

作者信息

Throbäck Ingela Noredal, Enwall Karin, Jarvis Asa, Hallin Sara

机构信息

Department of Microbiology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden.

出版信息

FEMS Microbiol Ecol. 2004 Sep 1;49(3):401-17. doi: 10.1016/j.femsec.2004.04.011.

DOI:10.1016/j.femsec.2004.04.011

Abstract

We re-evaluated PCR primers targeting nirS, nirK and nosZ genes for denaturing gradient gel electrophoresis as a tool to survey denitrifying community composition in environmental samples. New primers for both nirS and nosZ were combined with existing primers, while for nirK the previously published F1aCu:R3Cu set was chosen for denaturing electrophoresis. All three sets yielded amplicons smaller than 500 bp and amplified the correct fragment in all environmental samples. The denaturing gradient gel electrophoresis worked satisfactorily for nirK and nosZ, but not for nirS. This was probably due to the multiple melting domains in this particular nirS fragment. From the excised and sequenced bands, only sequences related to the target genes were detected and tree analysis showed that the selected primers acted as broad range primers for each of the three genes. By use of the new nirS primers it was demonstrated that agricultural soil harbours a substantial diversity of nirS denitrifiers.

摘要

我们重新评估了用于变性梯度凝胶电泳的靶向nirS、nirK和nosZ基因的PCR引物，以此作为一种调查环境样品中反硝化群落组成的工具。针对nirS和nosZ的新引物与现有引物相结合，而对于nirK，选择先前发表的F1aCu:R3Cu引物组用于变性电泳。所有这三组引物产生的扩增子均小于500 bp，并且在所有环境样品中均扩增出正确的片段。变性梯度凝胶电泳对nirK和nosZ的效果令人满意，但对nirS却不行。这可能是由于该特定nirS片段中存在多个解链结构域。从切下并测序的条带中，仅检测到与目标基因相关的序列，并且系统发育分析表明，所选引物对这三个基因中的每一个都起着广泛引物的作用。通过使用新的nirS引物证明，农业土壤中含有大量不同的nirS反硝化细菌。