Geets Joke, Borremans Brigitte, Diels Ludo, Springael Dirk, Vangronsveld Jaco, van der Lelie Daniel, Vanbroekhoven Karolien
Limburg University Centrum, Department of Chemistry, Biology and Geology, Environmental Biology Group, Universitaire Campus, B-3590 Diepenbeek, Belgium.
J Microbiol Methods. 2006 Aug;66(2):194-205. doi: 10.1016/j.mimet.2005.11.002. Epub 2005 Dec 7.
A denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of dsrB (dissimilatory sulfite reductase beta-subunit)-genes in sulfate-reducing communities. For this purpose a PCR primer pair was optimized for the amplification of a approximately 350 bp dsrB gene fragment that after DGGE gel electrophoresis enabled us to discriminate between dsrB genes of different SRB-subgroups,-genera and -species. The dsrB-DGGE method revealed considerable genetic diversity when applied to DNA extracts obtained from aquifer samples that were derived from monitoring wells of an in situ metal precipitation (ISMP) pilot project conducted at the site of a non-ferrous industry or from environmental heavy metal contaminated samples. The sequences of the excised and sequenced DGGE bands represented dsrB genes of different SRB-subgroups,-genera and -species, thus confirming the broad applicability of the PCR primer pair. Linking the results of the physico-chemical follow-up of the field and lab experiments to the dsrB-DGGE data will provide a better understanding of the contribution of the SRB populations to the ongoing ISMP processes.
开发了一种变性梯度凝胶电泳（DGGE）方法来评估硫酸盐还原菌群中异化亚硫酸盐还原酶β亚基（dsrB）基因的多样性。为此，优化了一对PCR引物，用于扩增约350 bp的dsrB基因片段，经DGGE凝胶电泳后，我们能够区分不同SRB亚组、属和种的dsrB基因。当将dsrB-DGGE方法应用于从含水层样品中提取的DNA时，发现了相当大的遗传多样性，这些样品来自在有色金属工业场地进行的原位金属沉淀（ISMP）试点项目的监测井，或来自环境重金属污染样品。切除并测序的DGGE条带的序列代表了不同SRB亚组、属和种的dsrB基因，从而证实了该PCR引物对的广泛适用性。将现场和实验室实验的物理化学后续结果与dsrB-DGGE数据联系起来，将有助于更好地理解SRB群体对正在进行的ISMP过程的贡献。
