The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil.

中文翻译：

---

使用用于经典和多重PCR阵列应用的新引物组，分层检测各种Clade II（非典型）nosZ基因。

一氧化二氮（N2O）还原为N2是规范反硝化过程中的关键步骤。一氧化二氮还原酶（NosZ）是与此生物学步骤相关的酶，但并不总是与反硝化微生物相关。与更常研究的反硝化装置中所携带的Clade I（典型）nosZ相反，最近发现此类生物具有Clade II（非典型）nosZ基因。随后的系统发育分析表明，与Clade I NosZ的微生物相比，Clade II NosZ的微生物多样性更高，前者包括非脱硝剂和反硝化剂。大多数尝试使用基于DNA的PCR方法来表征nosZ基因多样性的研究都只关注于Clade I nosZ，尽管最近进行的宏基因组测序研究表明，Clade II nosZ基因在许多生态系统（尤其是土壤）中占主导地位。结果，这些研究大大低估了生态系统中存在的减少N2O的遗传潜力。由于Clade II NosZ的高度多样性使得不可能设计出能够有效扩增该进化枝中所有nosZ基因的通用引物组，因此我们开发了一套引物组，专门针对10个Clade II nosZ基因指定子簇中的七个。新的引物组可为配对末端扩增子测序和qPCR产生合适的产物大小，这在常规单反应和多重阵列平台的使用中均得到了证明。此外，我们显示了这些引物在从土壤中提取的mRNA中检测nosZ基因转录本的实用性。