Yuan Jing, Deng Xuhan, Xie Xiaojing, Chen Liping, Wei Chaohai, Feng Chunhua, Qiu Guanglei
School of Environment and Energy, South China University of Technology, 382 Waihuandong Road, University Town, Guangzhou, Guangdong 510006, China.
Guangdong Provincial Key Laboratory of Solid Wastes Pollution Control and Recycling, 382 Waihuandong Road, University Town, Guangzhou, Guangdong 510006, China.
ISME Commun. 2024 Jan 23;4(1):ycae011. doi: 10.1093/ismeco/ycae011. eCollection 2024 Jan.
Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. . Competibacter probes showed promising coverage and specificity. Those for . Accumulibacter are desirable in coverage but targeted out-group bacteria, including . Competibacter, , , and some polyphosphate-accumulating Cyanobacteria. probes are good in specificity but poor in coverage. Probes targeting or showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for covered . Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of . Most species-specific probes for . Accumulibacter, . Lutibacillus, . Phosphoribacter, and are highly specific. Overall, 1.4% . Accumulibacter, 9.6% . Competibacter, 43.3% , and 54.0% in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of (<36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.
荧光原位杂交（FISH）和16S rRNA基因扩增子测序常用于生物强化除磷（EBPR）系统的微生物生态分析，其成功应用取决于所使用的寡核苷酸。我们对已知的聚磷菌（PAOs）和糖原积累菌（GAOs）常用的探针/引物进行了系统评估。大多数FISH探针存在盲点，覆盖了非目标细菌群。竞争杆菌属探针显示出良好的覆盖范围和特异性。聚磷菌属的探针在覆盖范围上是理想的，但靶向了外群细菌，包括竞争杆菌属、、和一些聚磷蓝细菌。探针特异性良好，但覆盖范围较差。靶向或的探针显示出低覆盖范围和特异性。具体而言，针对聚磷菌属的DEMEF455、Bet135和Dech453覆盖了聚磷菌属。使用这些探针来解析聚磷菌/糖原积累菌的表型时需要特别注意。大多数针对聚磷菌属、嗜泥杆菌属、磷杆菌属和的种特异性探针具有高度特异性。总体而言，MiDAS数据库中1.4%的聚磷菌属、9.6%的竞争杆菌属、43.3%的和54.0%的未被现有FISH探针覆盖。不同的16S rRNA扩增子引物组对已知的聚磷菌和糖原积累菌显示出不同的覆盖范围。它们都没有覆盖所有成员。总体而言，520F - 802R和515F - 926R显示出最平衡的覆盖范围。所有引物对的覆盖范围极低（<36.0%），这意味着它们在EBPR系统中的作用可能被忽视。清楚了解每个探针和引物组的优缺点是合理评估和解释所获得的群落结果的前提。
