1. Introduction

Soil microorganisms are a critical component of the Earth system by contributing significantly to global elemental cycles through a complex network of biogeochemical reactions (Schimel & Schaeffer, 2012). In many ecosystems, microorganisms gain energy for growth and survival through breaking down organic matter (OM), using carbon (C) and nitrogen (N) to build up biomass and releasing the greenhouse gases carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) to the atmosphere (Canfield et al., 2005; Hutchins & Capone, 2022; Oertel et al., 2016). Therefore, quantifying microbial abundance (as a proxy for biomass) is crucial to assess the importance of the microorganisms and understand their role or functions in ecosystems.
Over the past few decades, numerous techniques have been employed to quantify the population size of specific microorganisms or groups of microorganisms in environmental samples or synthetic communities in microbial ecology. These include, but are not limited to, direct epifluorescence microscopy (EFM) (Caron, 1983; Kepner & Pratt, 1994), flow cytometry (FCM) (Deng et al., 2019; Frossard et al., 2012; Frossard et al., 2016), fluorescence in situ hybridization (FISH) (Bouvier & del Giorgio, 2003), catalyzed reporter deposition-FISH (CARD-FISH, (Eickhorst & Tippkotter, 2008; Schippers et al., 2005), phospholipid quantification (Phospholipid-derived fatty acids, PLFAs) (Frostegard et al., 1991; White et al., 1979), and real-time quantitative polymerase chain reaction (qPCR) (Brankatschk et al., 2012; Han et al., 2020; Han et al., 2016; Hartmann et al., 2014; Smith & Osborn, 2009).
Among these approaches, qPCR has been widely used in molecular biology, as this method has proved to be relatively cheap, straightforward and efficient with a high sensitivity, covering a linear range over 7-8 orders of magnitude, and high throughput. qPCR relies on optical reporter systems, either using a double-stranded DNA-binding fluorescent dye such as SYBR® Green or DNA probes dual-labeled with reporter dyes and quenchers, such as TaqManTMprobes (Arya et al., 2005; Orlando et al., 1998; VanGuilder et al., 2008). Alongside measuring the abundance of the bacterial, archaeal and fungal communities (using general bacterial, archaeal or universal primers for the 16S rRNA gene (Takai & Horikoshi, 2000) or of the ITS region for fungi (Fierer et al., 2005)), qPCR has been applied for detecting and quantifying copy numbers of microbial functional genes involved in C and N cycling. Among the functions frequently studied in diverse environments using qPCR are CH4 production (methyl coenzyme M reductase A:mcr A) and oxidation (particulate methane monooxygenase:pmo A), nitrogen fixation (nitrogenase: nif H), ammonia oxidation (archaeal and bacterial ammonia monooxygenase: amo A), nitrite reduction (nitrite reductase: nir S and nir K), nitrite oxidation (beta subunit of nitrite oxidoreductase: nxr B), N2O production (nitric oxide reductase: nor B) and reduction (nitrous oxide reductase: nos Z), and organic phosphorus hydrolysis (alkaline phosphatase D: pho D) (Church et al., 2005; Han et al., 2020; Han et al., 2016; Henry et al., 2006; Leininger et al., 2006; Luo et al., 2017; Perez-Mon et al., 2022).
In spite of the advantage of being a straightforward method not including too many steps, qPCR has a major drawback. To quantify a specific gene, qPCR assays require the corresponding standard for calibration under the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009). Classically, standards have been produced by cloning a target sequence into a plasmid, amplifying genes via PCR, using genomic DNA directly, or acquiring commercially approved biological standards (Dhanasekaran et al., 2010; Goodwin et al., 2018). However, these approaches often incur significant costs, in terms of time and money, and potentially generate contaminations, particularly when preparing multiple plasmid standards targeting different microbial genes in parallel. For instance, both PCR amplicons and plasmids need be purified before being used, procedure which is often causing contaminations (Cimino et al., 1991). Moreover, the quantification of plasmid copies per cell was shown to be unreliable (Conte et al., 2018; May et al., 2015). In recent years, there has been a growing interest to use artificially synthesized DNA and RNA sequences as qPCR standards. Synthesizing such sequences to produce standards is considerably faster, cleaner (low contamination risk) and also less expensive (following considerable reduction of the cost of custom DNA synthesis in recent years) compared to traditional plasmid standards. The synthetic gene fragments can be purchased in a length of 125 to 3000 base pair (bp) with none degenerate nucleotides of A, T, C and G (Conte et al., 2018; May et al., 2015). Up to now, most of the artificially synthesized standards have been used for medical purpose, focusing on viral or infectious microorganisms (Bandeira et al., 2020; Bivins et al., 2021; Fesolovich & Tobe, 2017; Lima et al., 2017; Magee et al., 2017; Munoz-Calderon et al., 2021; Tourinho et al., 2015), very few in environmental samples. The few studies using synthesized gene fragments as qPCR standards assessed bacterial 16S rRNA genes in hydrocarbon-contaminated soils (Gunawardana et al., 2014), 16S rRNA genes and mcr A in a biogas digester (May et al., 2015), and antibiotic resistance genes in environmental water, soil and faeces samples (Xu et al., 2019), and none in microbial ecology. We propose that, given the advantages, synthetic qPCR standards can and should be widely adopted for qPCR analysis of functional genes in environmental microbiology and microbial ecology. However, this new methodological approach should be thoroughly evaluated and compared to previous practice before being adopted.
Here, we designed qPCR standards for a number of frequently studied functional genes of the C, N and P cycle, and the ITS region and the 16S rRNA gene by synthesizing double-stranded DNA fragments obtained by generation of consensus sequences from alignments of microbial gene sequences. To provide a thorough evaluation of the effectiveness and reliability of synthetic DNA fragments as qPCR standards, we compared these newly synthesized qPCR standards with standards produced via plasmids in different qPCR assays, targeting several different taxonomic and functional genes of soil microorganisms.