Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR
Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR
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DOI：
10.1080/01490450490275307
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发表时间：
2004-04-01
影响因子：
2.3
通讯作者：
Silva, SD
中科院分区：
文献类型：
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作者：
Kondo, R;Nedwell, DB;Silva, SD
通过使用定量竞争性 PCR，研究了英国埃塞克斯郡科恩河口沉积物中硫酸盐还原菌 (SRB) 的分布，覆盖了不同盐水浓度的沉积物孔隙水。在这里，我们证明了新的 PCR 引物组和使用 PCR 的新定量方法是自然环境中 SRB 检测和计数的有用工具。设计了针对SRB异化亚硫酸盐还原酶基因(dsr)选择性的PCR引物组。使用单组 dsr 特异性引物进行 PCR 扩增，得到所有 27 种测试的 SRB 菌株（包括革兰氏阴性和阳性物种）的预期大小的 PCR 产物。使用该引物从沉积物 DNA 中获得的 60 个克隆进行了测序，所有克隆都与预测的 SRB dsr 密切相关。这些结果表明，使用新设计的引物组的 PCR 可用于从天然样品中选择性检测 SRB。该引物组用于通过使用竞争者的 dsr 选择性竞争 PCR 来估计细胞数，该竞争者比 dsr 的目标区域短约 20%。该程序适用于来自英国埃塞克斯郡科恩河口的沉积物样品，并同时测量硫酸盐还原率的原位。通过竞争性PCR估计SRB的高密度范围为0.2-5.7 x 10(8)细胞ml(-1)湿沉积物，假设所有SRB都具有单个dsr拷贝。使用这些估计值，计算出SO42-细胞(-1)天(-1)的10(-17)至10(-15)摩尔的细胞特异性硫酸盐还原率，该还原率在之前报告的SRB纯培养物的范围内或更低。我们的结果表明，新开发的针对 dsr 的竞争性 PCR 技术是一种快速、可重复地原位估计 SRB 数量的强大工具，并且优于使用依赖于培养的技术。
The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of 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 all 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 samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2-5.7 x 10(8) cells ml(-1) wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10(-17) to 10(-15) mol of SO42- cell(-1) day(-1) were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. 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.