Steps in the global nitrogen cycle are mainly catalyzed by microorganisms. Accordingly, the activities of these microorganisms affect the health and productivity of ecosystems. Their activities are also used in wastewater treatment systems to remove reactive nitrogen compounds and prevent eutrophication events triggered by nutrient discharges. Therefore, tracking the activities of these microorganisms can provide insights into the functioning of these systems. The presence and abundance of genes encoding nitrogen-metabolizing enzymes can be traced via polymerase chain reaction (PCR); however, this requires primers that are sensitive to a heterogenous gene pool yet specific enough to the target biomarker. The ever-expanding diversity of sequences available from databases includes many sequences relevant to nitrogen metabolism that match poorly with primers previously designed to track their presence and/or abundance. This includes genes encoding ammonia monooxygenase (AMO) of ammonia oxidizing microorganisms, nitrite oxidoreductase (NXR) of nitrite oxidizing bacteria, and nitrous oxide reductase (NOS) of denitrifying bacteria. Some primers are also not designed to generate the short (~200 nucleotides) amplicons required for real-time quantitative PCR (qPCR) and reverse-transcriptase qPCR (qRT-PCR). In this study, genes collected from the Integrated Microbial Genomes database (IMG) were aligned to design PCR primers that could capture more sequence diversity than is possible using existing primers. Primers were designed to target three clades of AMO (Betaproteobacteria, Chrenarchaeota, and complete ammonia oxidizing Nitrospira), periplasmic NXR and two clades of NOS (Proteobacteria and Bacteroidetes/Firmicutes). These primers successfully amplified target sequences from two wastewater treatment plants with biological nitrogen removal (one with simultaneous nitrification/denitrification and one with distinct anoxic/oxic zones) and estuary sediment. Nucleotide sequences of the amplicons retrieved homologs when used to query GenBank by BLAST. While convincingly identified as target sequences for these primer pairs, these amplicons were divergent from each other, and quite divergent (as low as 73%) from those present in GenBank, suggesting these primers are capable of capturing a diverse range of sequences. A direct comparison showed that primers designed here are better suited to environmental samples, such as wastewater treatment facilities, by producing a greater number of amplicons from the same sample than primers currently established in literature.

全球氮循环中的步骤主要由微生物催化。因此，这些微生物的活动影响生态系统的健康和生产力。它们的活动还用于废水处理系统，以去除活性氮化合物并防止由营养物排放引发的富营养化事件。因此，跟踪这些微生物的活动可以深入了解这些系统的功能。可以通过聚合酶链式反应（PCR）追踪编码氮代谢酶的基因的存在和丰度；然而，这需要对异源基因库敏感但对目标生物标志物足够特异的引物。数据库中可用的序列不断扩大的多样性包括许多与氮代谢相关的序列，这些序列与以前设计用于跟踪它们的存在和/或丰度的引物匹配不佳。这包括编码氨氧化微生物的氨单加氧酶 (AMO)、亚硝酸盐氧化细菌的亚硝酸盐氧化还原酶 (NXR) 和反硝化细菌的一氧化二氮还原酶 (NOS) 的基因。一些引物也不是为生成实时定量 PCR (qPCR) 和逆转录酶 qPCR (qRT-PCR) 所需的短（约 200 个核苷酸）扩增子而设计的。在这项研究中，从综合微生物基因组数据库 (IMG) 收集的基因被比对以设计 PCR 引物，这些引物可以捕获比使用现有引物可能的更多的序列多样性。Betaproteobacteria、Chrenarchaeota和完全氨氧化Nitrospira）、周质 NXR 和 NOS 的两个进化枝（Proteobacteria和Bacteroidetes/Firmicutes）。这些引物成功地扩增了来自两个污水处理厂的目标序列，这些污水处理厂具有生物脱氮功能（一个具有同时硝化/反硝化作用，一个具有不同的缺氧/好氧区）和河口沉积物。当用于通过 BLAST 查询 GenBank 时，扩增子的核苷酸序列检索到同源物。虽然令人信服地确定为这些引物对的靶序列，但这些扩增子彼此不同，并且与 GenBank 中存在的那些有很大不同（低至 73%），这表明这些引物能够捕获多种序列。直接比较表明，此处设计的引物更适合环境样品，例如废水处理设施，与目前文献中建立的引物相比，从同一样品中产生更多数量的扩增子。