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Development and use of molecular tool for rapid enumeration of sulphate-reducing bacteria and for assessment of their activity

开发和使用分子工具快速计数硫酸盐还原菌并评估其活性

基本信息

批准号：
15580170
负责人：
KONDO Ryuji
金额：
$ 2.37万
依托单位：
Fukui Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We developed a new PCR primer set and a new quantitative method using PCR are useful tool for the detection and the enumeration of sulpahte-reducing bacteria (SRB) in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment and water samples from anaerobic aquatic environments. High densities of SRB were detected by the competitive PCR assuming that all SRB have a single copy of dsr. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.We also developed a competitive RT-PCR method for the quantification of dsr mRNA in Desulfovibrio desulfuricans. The amount of dsr mRNA was determined at different growth conditions to find the relationship between the dsr mRNA content per cell and the rate of metabolism (cell specific sulphate reduction rate, csSRR). The maximum dsr mRNA content in SRB cell correlated with csSRR, indicating that dsr mRNA may be of value as molecular proxy assessing cellular sulphate reduction rates in nature.

我们开发了一套新的PCR引物和一种新的PCR定量方法，为自然环境中硫酸盐还原菌（SRB）的检测和计数提供了有用的工具。设计了一对特异性针对硫酸盐还原菌（SRB）异化亚硫酸盐还原酶基因（dsr）的PCR引物。使用单组dsr特异性引物进行PCR扩增，从27个供试SRB菌株（包括革兰氏阴性和阳性菌种）中获得预期大小的PCR产物。用该引物从沉积物DNA中扩增出60个克隆，测序结果均与预测的硫酸盐还原菌dsr高度相关。这些结果表明，使用新设计的引物组的PCR是有用的选择性检测SRB从自然样品。该引物组用于通过dsr选择性竞争性PCR使用竞争物估计细胞数量，所述竞争物比dsr的靶区域短约20%。该程序适用于厌氧水生环境的沉积物和水样。假设所有SRB都有一个dsr拷贝，通过竞争性PCR检测到高密度的SRB。我们的研究结果表明，新开发的竞争性PCR技术针对dsr是一个强有力的工具，快速和重复性的SRB数量原位估计，并上级使用依赖于培养的技术。我们还建立了竞争性RT-PCR方法的定量dsr在脱硫脱硫弧菌的mRNA。在不同生长条件下测定dsr mRNA的量，以发现每个细胞的dsr mRNA含量与代谢速率（细胞比硫酸盐还原速率，csSRR）之间的关系。硫酸盐还原菌细胞内dsr mRNA的最大含量与csSRR相关，表明dsr mRNA可作为自然界中测定细胞硫酸盐还原速率的分子指标。