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.