Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas, ES-08003, Barcelona, Catalunya, Spain.
Microb Ecol. 2012 Nov;64(4):1047-55. doi: 10.1007/s00248-012-0087-x. Epub 2012 Jul 18.
Assumptions on the matching specificity of group-specific bacterial primers may bias the interpretation of environmental microbial studies. As available sequence data continue growing, the performance of primers and probes needs to be reevaluated. Here, we present an evaluation of several commonly used and one newly designed Bacteroidetes-specific primer (CF418). First, we revised the in silico primer coverage and specificity with the current SILVA and RDP databases. We found minor differences with previous studies, which could be explained by the chosen databases, taxonomies, and matching criteria. We selected eight commonly used Bacteroidetes primers and tested them with a collection of assorted marine bacterial isolates. We also used the denaturing gradient gel electrophoresis (DGGE) approach in environmental samples to evaluate their ability to yield clear and diverse band patterns corresponding to Bacteroidetes phylotypes. Among the primers tested, CF968R did not provide satisfactory results in DGGE, although it exhibited the highest in silico coverage for Flavobacteria. Primers CFB560 and CFB555 presented undesirable features, such as requiring nested protocols or presence of degeneracies. Finally, the new primer CF418 and primer CF319a were used to explore the Bacteroidetes dynamics throughout a 1-year cycle in Mediterranean coastal waters (Blanes Bay Microbial Observatory). Both primers provided clear and diverse banding patterns, but the low specificity of CF319a was evidenced by 83.3 % of the bands sequenced corresponding to nontarget taxa. The satisfactory DGGE banding patterns and the wide diversity of sequences retrieved from DGGE bands with primer CF418 prove it to be a valuable alternative for the study of Bacteroidetes communities, recovering a wide range of phylotypes within the group.
假设针对特定菌群的细菌引物具有匹配特异性，这可能会影响环境微生物研究的结果解释。随着现有序列数据的不断增加，需要重新评估引物和探针的性能。在这里，我们评估了几种常用的和一种新设计的拟杆菌属特异性引物（CF418）。首先，我们使用当前的 SILVA 和 RDP 数据库对计算机模拟引物的覆盖范围和特异性进行了修正。我们发现与之前的研究存在一些微小差异，这可以通过所选择的数据库、分类学和匹配标准来解释。我们选择了 8 种常用的拟杆菌属引物，并对各种海洋细菌分离株进行了测试。我们还使用变性梯度凝胶电泳（DGGE）方法对环境样本进行了测试，以评估它们产生与拟杆菌属生物型相对应的清晰多样条带模式的能力。在所测试的引物中，CF968R 在 DGGE 中没有提供令人满意的结果，尽管它在丝状菌门的计算机模拟覆盖度最高。引物 CFB560 和 CFB555 表现出不理想的特征，例如需要嵌套协议或存在简并性。最后，新引物 CF418 和引物 CF319a 用于探索地中海沿海水域（布拉内斯湾微生物观测站）一年周期内的拟杆菌属动态。这两个引物都提供了清晰多样的条带模式，但 CF319a 的低特异性表现在 83.3%测序的条带对应非目标分类群。引物 CF418 提供了令人满意的 DGGE 条带模式和从 DGGE 条带中回收的广泛多样的序列，证明它是研究拟杆菌属群落的一种有价值的替代方法，能够恢复该属内的广泛生物型。
