3. Results and
Discussion
3.1. Performance of synthetic and
plasmid DNA
standards
For the six targeted genes which were tested with synthetic DNA and
plasmid DNA standards (Table 1), both standards for qPCR quantifications
yielded significantly (P < 0.001) linear calibration curves
featuring a coefficient value (R2) of >
0.99 (Fig. 1), together with similar high R2 derived
from the genes tested with synthetic DNA standards (Supplementary Fig.
S1). The dilution series of both synthetic DNA and plasmid DNA standards
exhibited smooth and exponential amplification curves (Fig. 2).
Coefficients of variation of Cq values among the replicates for the
standards in the range from 101 to
108 copies per μl were between 0.03% and 4.15%,
which indicated good repeatability and reproducibility. Additionally,
the slopes of the synthetic standards were similar to those of the
plasmid standards, with only minor differences (Fig. 1). These, all
together, provided compelling evidence that, similar to traditional
plasmid standards, synthetic DNA standards of serial dilutions can be
amplified effectively and produce high-quality and consistent standard
curves. Comparable standard curves between synthetic DNA standards and
traditional standards (PCR amplicons and plasmids of cloning) have been
reported in previous studies targeting human mitochondrial gene
(Conte et al., 2018), antibiotic
resistance gene (Xu et al., 2019), human
T-cell leukaemia virus type 1 (HTLV-1)
(Bandeira et al., 2020) and HBV virus
(Portilho et al., 2018). Yet, to the best
of our knowledge, this is the first study to use synthetically designed
and produced DNA fragments as qPCR standards targeting a broad range of
genes involved in C and N cycling employed in microbial ecology.
Standard curves from both synthetic and plasmid standards showed high
and similar amplification efficiency (E) values, confirming the
reliability of synthetic gene fragments as qPCR standards (Fig. 1 and
2). PCR efficiency of the standard curves for 16S rRNA gene reached 0.84
for synthetic DNA and 0.96 for plasmid standards. It attained 0.98 and
0.90 for fungal ITS region for the synthetic and plasmid standard
respectively. Efficiency values of the remaining four genes tested
(mcr A, pmo A, nif H and nos Z), were all lower
than 0.90, irrespective of plasmid or synthetic standards (Fig. 1).
Ideally, an efficiency value over 0.90 is considered a well amplified
standard and a qualified standard curve
(Svec et al., 2015). However, often times
due to the potential PCR inhibition, such as self-inhibition, polymerase
and protein inhibition, primer specificity and contamination, E values
can be as low as 0.70 (Luby et al., 2016;
Xu et al., 2019). The slightly lower E
value for synthetic 16S rRNA gene standard during PCR might be caused by
a small peak (PCR byproduct) right before the main PCR product peak of
16S rRNA gene, especially the least diluted ones, implied by the melting
curves (Supplementary Fig. S2A), which caused the differences in the
standard curves from synthetic and plasmid standards. Lower E values of
the genes mcr A, pmo A, nif H and nos Z from
both synthetic and plasmid standards were likely also related to PCR
inhibitions, in particular to the less diluted standards. Usually, 3.3
(a slope of – 3.3) cycles apart of the 10-fold dilutions were
considered as an indicator of 100% PCR efficiency
(Svec et al., 2015)). However, much
higher Cq value differences between the dilution series were found for
these four genes from both standards (Fig. 1), indicating an inhibition
effect. Additionally, different instruments and volume for standard
dilution also made huge differences in PCR efficiency, which turned out
a larger volume transferred during dilution (10 µL) could increase the
efficiency (Svec et al., 2015), while 2
µL was used in this study.
In spite of the similarity of qPCR standard curves between synthetic and
plasmid standards, there were slight differences in the slopes and E
values of standard curves between these two standards (Fig. 1). Formcr A, pmo A, nif H and nos Z, standard curves
from synthetic standards were always steeper (higher absolute slopes)
than those from plasmid standards, with Cq values of the least diluted
standards from synthetic standards lower than those of the least diluted
plasmid standards, even when the copy numbers of the least diluted
standards from synthetic standards were lower than those from plasmid
standards for mcr A and pmo A (Supplementary Table S4). This
indicated a self-inhibition of the least diluted plasmid standards,
which took more cycles (higher Cq values) to get fully amplified. For
ITS region, standard curve from plasmid standard was slightly steeper
than that from synthetic standard, which also indicated an effect of
inhibition. In addition to self-inhibition, there might be also a
conformation effect of non-linear plasmid standards, which could also
impact PCR efficiency as well (Hou et al.,
2010).
3.2. Microbial gene abundances in
soils based on synthetic and plasmid DNA
standards
In order to validate the reliability of our synthetic DNA standards, the
abundances of the tested genes were quantified in eDNA extracts from
soils by qPCR assays and the gene copy abundance calculated using
standards from synthetic and plasmid DNA (Fig. 3). No amplification was
observed in negative control reactions, confirming the absence of
contamination during the reaction preparation steps. Overall, gene
copies calculated with either standard curves (i.e. from synthetic or
plasmid DNA) were not significantly different for all the genes studied
across all soil samples except for few assays (marked with asterisks in
Supplementary Fig. S3). These significant differences reflect the
described differences of the standards curves thus that gene copy
numbers calculated from synthetic standards were on average lower than
those from plasmid standards (difference of 5.1±4.4% for mcr A,
15.4±1.2% for pmo A, 23.8±4.1% for nif H and 6.9±6.2% fornos Z) or higher for 16S rRNA gene (17.7±3.2%) and ITS region
(41.6±16.4% higher). Gene copies varying within one log (10 times) were
widely reported for qPCR quantifications of viruses with synthetic and
plasmid standards, and such results have been considered good agreement
of the two methods (Bandeira et al., 2020;
Lima et al., 2017;
Portilho et al., 2018;
Tourinho et al., 2015).
Despite the described deviation, correlation of gene copies in the soil
samples using the synthetic standard and copies by using plasmid
standard, was significant with a squared coefficient
(R2) of over 0.99 (Linear correlations, all
significant P < 0.001) for all the six tested genes (Fig. 3),
which showed highly identical results with both standards. Similarly,
high R2 (0.83) based on Linear correlations were also
observed in a study on human virus by qPCR when comparing synthetic DNA
and plasmid DNA standards (Bandeira et al.,
2020). Furthermore, when comparing the relative differences in gene
copy numbers to the highest observed soil value within each gene, there
were no any significant differences in the relative differences across
all sites of all the six tested genes between synthetic and plasmid
standards (Supplementary Fig. S4).
Therefore, considering the sensitivity and efficiency of qPCR,
inhibition and anthropogenic interference (i.e. pipetting errors),
differences in copy numbers within 50% variation in this study are very
much acceptable, especially with gene concentrations reaching up to more
than 1010 copies per dry gram soil. All taken
together, our results demonstrated that the synthetic DNA standards are
reliable for qPCR quantification of various taxonomic and functional
genes in soils.