Takenaka Ryota, Aoi Yoshiteru, Ozaki Noriatsu, Ohashi Akiyoshi, Kindaichi Tomonori
Department of Civil and Environmental Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashihiroshima 739-8527, Japan.
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima 739-8530, Japan.
Materials (Basel). 2018 Jul 3;11(7):1129. doi: 10.3390/ma11071129.
Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Saccharibacteria in activated sludge.
糖菌门是一个已被充分描述但尚未成功分离的候选菌门。尽管如此，16S rRNA基因测序数据表明了它的存在，并且它在自然环境和活性污泥中经常被检测到。由于缺乏糖菌门的纯培养代表菌株，用于测定其丰度和多样性的引物特异性应仔细评估。在本研究中，从先前的研究中选择了8种糖菌门特异性引物，并评估了它们对公共数据库的覆盖范围、引物组合的退火温度，以及通过克隆分析确定检测频率和系统发育多样性，以及通过定量聚合酶链反应（PCR）进行定量时的应用情况。在这8种引物中，有4种引物（TM7314F、TM7580F、TM7 - 910R和TM7 - 1177R）显示出高覆盖范围。克隆分析表明，4种引物组合（TM7314F和TM7 - 910R；TM7314F和TM7 - 1177R；TM7580F和TM7 - 910R；TM7580F和TM7 - 1177R）对日本东广岛市一家污水处理厂活性污泥中的糖菌门检测频率较高。定量PCR结果表明，包含TM7314F和TM7 - 910R的引物组合在活性污泥中对糖菌门的特异性检测方面表现更优。
