Multi-Amplicon Nitrogen Cycling Gene Standard: An Innovative Approach for Quantifying N-transforming Soil Microbes in Terrestrial Ecosystems

Agriculture accounts for nearly three-fourths of all nitrous oxide (N2O) anthropogenic emissions, a potent greenhouse gas (GHG). Improvements in agricultural management practices are critical for improving plant nitrogen (N) use efficiency and reducing N2O emissions. While implementing "climate-smart" management can reduce N2O emissions, building a linkage between these emissions and the N-cycling microbes that drive them will require N-cycling gene assays that are cost-effective, practical, and accessible to climate-smart researchers. In a recent study, Han et al. (2023) demonstrated the suitability of synthetic oligonucleotides to serve as real-time quantitative PCR (qPCR) standards for quantifying microbial genes in soils. While this study is not based on the works of Han et al. (2023), our results complement their findings and offer fresh insights into this novel methodology. Here, we describe an innovative approach that provides precision in the absolute quantification of five N-transforming target genes (AOB amoA, AOA amoA, nirK, nirS, and nosZ) in soils. Similar to Han et al. (2023), our method obviates the cost-intensive aspects of traditional qPCR methods for preparing standard curves in soil microbial ecology (e.g., gel purification of PCR products and in-vivo cloning of plasmids) and is capable of being extended to any biochemical system, or environment of interest. Additionally, our qPCR approach provides several advantages: it utilizes a single multi-amplicon synthetic oligonucleotide, requires only a single dilution series and one stock solution to quantify all gene targets, and contains a non-edaphic gene (ZIKV) that can serve as a soil spike to determine DNA extraction efficiency and DNA recovery estimates. Furthermore, our protocol mitigates PCR inhibition from soil-derived substances using a modified DNA extraction protocol and inhibitor-tolerant polymerase. Ultimately, our work aims to reduce the technical burden on non-biologists/biochemists when conducting soil microbiological assays, support the development of climate mediation practices, and advance climate-smart agriculture goals.