M IN I R E V I EW
Advantages and limitationsofquantitativePCR (Q-PCR)-based
approaches inmicrobial ecology
Cindy J. Smith & A. Mark Osborn
Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, UK
Correspondence: A. Mark Osborn,
Department of Animal and Plant Sciences,
University of Sheffield, Western Bank,
Sheffield S10 2TN, UK. Tel.: 144 114 222
4626; fax: 144 114 222 0002; e-mail:
a.m.osborn@sheffield.ac.uk
Received 20 June 2008; revised 10 October
2008; accepted 24 October 2008.
First published online December 2008.
DOI:10.1111/j.1574-6941.2008.00629.x
Editor: Michael Wagner
Keywords
Q-PCR; RT-Q-PCR; 16S rRNA gene; mRNA.
Abstract
Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied
in microbial ecology to quantify the abundance and expression of taxonomic and
functional gene markers within the environment. Q-PCR-based analyses combine
‘traditional’ end-point detection PCR with fluorescent detection technologies to
record the accumulation of amplicons in ‘real time’ during each cycle of the PCR
amplification. By detection of amplicons during the early exponential phase of the
PCR, this enables the quantification of gene (or transcript) numbers when these
are proportional to the starting template concentration. When Q-PCR is coupled
with a preceding reverse transcription reaction, it can be used to quantify gene
expression (RT-Q-PCR). This review firstly addresses the theoretical and practical
implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, high-
lighting key experimental considerations. Secondly, we review the applications of
(RT)-Q-PCR analyses in environmental microbiology and evaluate the contribu-
tion and advances gained from such approaches. Finally, we conclude by offering
future perspectives on the application of (RT)-Q-PCR in furthering understanding
in microbial ecology, in particular, when coupled with other molecular approaches
and more traditional investigations of environmental systems.
Introduction
The application of PCR in combination with the extraction
of nucleic acids (DNA and RNA) from environmental
matrices has been central to the development of culture-
independent approaches in microbial ecology. These meth-
ods, which have been applied since the early 1990s (e.g.
Giovannoni et al., 1990), enabling the analysis of the total
microbial communities present within environmental sys-
tems, have revolutionized our understanding of microbial
community structure and diversity within the environment.
Coupling environmental nucleic acid isolation to subse-
quent PCR amplification of both taxonomic (i.e. rRNA)
and functional gene markers and in combination with DNA
fingerprinting- and sequencing-based analyses has enabled
description of the hitherto uncharacterized majority of
environmental microorganisms (Head et al., 1998) driving
the discovery of new microbial lineages and enabling the
description of genetic diversity in a wealth of functional
gene markers (Larkin et al., 2005). Although recently devel-
oped ultra-high-throughput sequencing technologies such
as pyrosequencing (Margulies et al., 2005; Edwards et al.,
2006) now dwarf PCR-based sequence studies in terms of
sequence coverage, the ability of the PCR to specifically
target particular taxonomic or functional markers from
domain – down to strain – or phylotype levels means that
PCR will remain an invaluable method in the molecular
microbial ecologist’s toolbox. Nevertheless, PCR has inher-
ent limitations (von Wintzingerode et al., 1997), particularly
those that result in biases in the template to product ratios of
target sequences amplified during PCR from environmental
DNA (Suzuki & Giovannoni, 1996; Polz & Cavanaugh,
1998), with such amplification biases found to increase with
increasing numbers of PCR cycles. These limitations pre-
sented a significant challenge to microbial ecologists who
were interested in determining the abundance of individual
genes present in environmental samples. To circumvent such
challenges, an adaptation of the PCR method developed by
Holland et al. (1991) utilizing the so-called ‘50 nuclease
assay’ was applied to quantify target 16S rRNA genes
amplified from environmental DNA by PCR (Becker et al.,
2000; Suzuki et al., 2000; Takai & Horikoshi, 2000). This
FEMS Microbiol Ecol 67 (2009) 6–20c� 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
development had been facilitated by the earlier combination
of the 50 nuclease assay developed by Holland et al. (1991)
with fluorescence detection following cleavage of an internal
(TaqManTM) DNA probe (Livak et al., 1995), enabling the
accumulation of amplicons to be monitored after each cycle
(in real-time) and hence facilitating quantitative determina-
tion of the initial template gene (or transcript) numbers.
Quantitative-PCR or Q-PCR (often referred to as real-
time PCR) is now widely used in microbial ecology to
determine gene and/or transcript numbers present within
environmental samples. The target specificity of any Q-PCR
assay is determined by the design of the primers (and in
some cases an internal probe), allowing quantification of
taxonomic or functional gene markers present within a
mixed community from the domain level down to the
quantification of individual species or phylotypes. Q-PCR
has been shown to be a robust, highly reproducible and
sensitive method to quantitatively track phylogenetic and
functional gene changes across temporal and spatial scales
under varying environmental or experimental conditions.
Moreover, the quantitative data generated can be used to
relate variation in gene abundances and/or levels of gene
expression (in terms of transcript numbers) in comparison
with variation in abiotic or biotic factors and/or biological
activities and process rates. The provision of Q-PCR data
sets that describe the abundance of specific bacteria or genes
to complement other quantitative environmental data sets
is of increasing importance in microbial ecology as it
furthers understanding of the roles and contributions of
particular microbial and functional groups within ecosys-
tem functioning. Furthermore, reverse transcription (RT)
analyses are now increasingly combined with Q-PCR
methods in RT-Q-PCR assays, offering a powerful tool for
quantifying gene expression (in terms of numbers of rRNA
and mRNA transcripts) and relating biological activity to
ecological function.
In this review, we firstly discuss the mechanistic aspects of
Q-PCR and RT-Q-PCR methodologies, hereafter defined
collectively as (RT)-Q-PCR, and highlight the key experi-
mental considerations in the design and implementation of
(RT)-Q-PCR protocols and the analysis of resultant data
sets. Secondly, we explore the application of (RT)-Q-PCR
approaches in microbial ecology, and finally we discuss how
these methods can be applied together with other molecular
and also conventional approaches to provide an increased
understanding of microorganisms within environmental
systems.
Advantages of Q-PCR over traditional end-
point PCR
Q-PCR approaches combine the detection of target template
with quantification by recording the amplification of a PCR
product via a corresponding increase in the fluorescent
signal associated with product formation during each cycle
in the PCR. Quantification of gene (or transcript) numbers
is determined during the exponential phase of the PCR
amplification when the numbers of amplicons detected are
directly proportional to the initial numbers of target
sequences present within the environment (discussed in
more detail in Target quantification). Quantification of the
target gene during exponential amplification avoids pro-
blems that are associated with so-called ‘end-point’ PCR
(in which amplicons are only analysed after completion of
the final PCR cycle). In end-point PCR, the proportions
of numerically dominant amplicons do not necessarily
reflect the actual abundances of sequences present within
the environment due to the inherent biases of PCR that
are associated with amplification of targets from mixed
template community DNA (Reysenbach et al., 1992; Suzuki
& Giovannoni, 1996; Polz & Cavanaugh, 1998). Moreover,
Q-PCR that uses fluorescence-based detection offers greater
sensitivity and enables discrimination of gene numbers
across a wider dynamic range than is found with end-point
PCR; for example twofold changes in target concentration
can be discriminated using Q-PCR. Before the development
of fluorescence-based Q-PCR-based methods, two alterna-
tive PCR-based methods for gene number quantification
had been developed, namely competitive PCR (Diviacco
et al., 1992) and limiting dilutions or most probable number
(MPN)-PCR (Skyes et al., 1992). However, these methods
are time- and resource-consuming, requiring post-PCR
analysis, and have now largely been replaced by fluores-
cence-based Q-PCR methods.
Fluorescence detection chemistries used
to detect template amplification during
Q-PCR
Quantitative real-time PCR works in essentially the same
manner as end-point PCR, i.e. multiple amplification cycles
in which template DNA is initially denatured, followed by
annealing of oligonucleotide primers targeting specific
sequences, followed by subsequent extension of a comple-
mentary strand from each annealed primer by a thermo-
stable DNA polymerase, resulting in an exponential increase
in amplicon numbers during the PCR. However, in contrast
to end-point PCR, the increase in amplicon numbers is
recorded in ‘real-time’ during the PCR via detection of a
fluorescent reporter that indicates amplicon accumulation
during every cycle. Two reporter systems are commonly
used, namely, the intercalating SYBR green assay (Wittwer
et al., 1997) and the TaqMan probe system (Holland et al.,
1991; Livak et al., 1995).
SYBR green binds to all double-stranded DNA via inter-
calation between adjacent base pairs. When bound to DNA,
FEMS Microbiol Ecol 67 (2009) 6–20 c� 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
7Application of Q-PCR in microbial ecology
a fluorescent signal is emitted following light excitation (Fig.
1a). As amplicon numbers accumulate after each PCR cycle,
there is a corresponding increase in fluorescence. Because
SYBR green binds to all double-stranded DNA, it is essential
to use primer pairs that are highly specific to their target
sequence to avoid generation of nonspecific products that
would contribute to the fluorescent signal, resulting in an
overestimation of the target. Extensive optimization of
primer concentrations used in SYBR green Q-PCR assays
may be required to ensure that only the targeted product is
formed. Primer pairs that exhibit self-complementarity
should also be avoided to prevent primer–dimer formation.
A post-PCR dissociation (melting) curve analysis should be
carried out to confirm that the fluorescence signal is
generated only from target templates and not from the
formation of nonspecific PCR products. During a dissocia-
tion curve, the double-stranded template is heated over a
temperature gradient and fluorescence levels are measured
at each discrete temperature point. As the double-stranded
template is heated, it denatures, resulting in a corresponding
decline in fluorescence due to SYBR green dissociation from
the double-stranded product (Giglio et al., 2003; Gonzalez-
Escalona et al., 2006). The temperature at which 50% of the
double-stranded template is denatured can be used to
confirm that the template being targeted is present, along
with the presence of other nonspecific template and primer
dimers in much the same way as agarose gel electrophoresis
of an end-point PCR product is used.
The TaqMan probe method utilizes a fluorescently
labelled probe that hybridizes to an additional conserved
region that lies within the target amplicon sequence. The
TaqMan probe is fluorescently labelled at the 50 end and
contains a quencher molecule at the 30 end (Livak et al.,
1995). The close proximity on the probe of the quencher
molecule to the fluorophore prevents it from fluorescing
due to fluorescent resonance energy transfer. During the
annealing step of each cycle of the PCR, primers and the
intact probe bind to their target sequences. During subse-
quent template extension, the 50 exonuclease activity of the
Taq polymerase enzyme cleaves the fluorophore from
the TaqMan probe and a fluorescent signal is detected as
the fluorophore is no longer in close proximity to the
quencher (Fig. 1b). Amplification of the template is thus
measured by the release and accumulation of the fluoro-
phore during the extension stage of each PCR cycle. The
additional specificity afforded by the presence of the
TaqMan probe ensures that the fluorescent signal generated
during Q-PCR is derived only from amplification of the
target sequence. Multiple TaqMan probes and primer sets
can be used in different Q-PCR assays to differentiate
between closely related sequences (Smith et al., 2007), or
alternatively, probes can be labelled with different fluoro-
phores, facilitating the development of multiplex Q-PCR
protocols whereby different targets can be coamplified and
quantified within a single reaction (Neretin et al., 2003;
Baldwin et al., 2003, 2008). For example, Baldwin et al.
(2003) developed a multiplex Q-PCR assay targeting a
number of different aromatic oxygenase genes using bacter-
ial strains and then subsequently applied the assay to
simultaneously quantify aromatic oxygenase genes in con-
taminated groundwater (Baldwin et al., 2008). TaqMan
probes are, however, a more expensive option than using
SYBR green chemistry and the former requires the presence
of an additional conserved site within the short amplicon
sequence to be present. Identification of three conserved
regions within a short region (typically c.o 100 bp) may not
Fig. 1. Real-time PCR chemistries: (a) SYBR
green detection. SYBR green binds to all
double-stranded DNA and emits a fluorescent
signal. In its unbound state, SYBR green does not
fluoresce. Template amplification is therefore
measured in each cycle by the corresponding
increase in fluorescence. (b) TaqMan (50 nuclease)
assay using TaqMans probes. During annealing,
the TaqMan probe and primers bind to the
template. When the TaqMan probe is intact,
energy is transferred between the quencher and
the reporter; as a result, no fluorescent signal is
detected. As the new strand is synthesized by Taq
polymerase, the 50 exonuclease activity of the
enzyme cleaves the labelled 50 nucleotide of the
probe, releasing the reporter from the probe.
Once it is no longer in close proximity, the
fluorescent signal from the probe is detected and
template amplification is recorded by the
corresponding increase in fluorescence.
FEMS Microbiol Ecol 67 (2009) 6–20c� 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
8 C.J. Smith & A.M. Osborn
always be possible, especially when primer/probe combina-
tions are being designed to target divergent gene sequences.
More recent advances in TaqMan probe technology have
involved the introduction of the minor groove binder
(MGB) probe (Kutyavin et al., 2000). The MGB molecule is
attached to the 30 end of the probe and essentially folds back
onto the probe. This not only increases the stability of the
probe, but allows the design of shorter probes (13–20 bp)
than are required for traditional TaqMan probes (20–40 bp),
while at the same time, maintaining the required hybridiza-
tion annealing temperature.
Target quantification using the cycle
threshold (C t) method
Irrespective of the fluorescence chemistry used, quantifica-
tion of the target template DNA is carried out in essentially
the same manner. There are a number of different commer-
cially available instruments to carry out Q-PCR, each with
its own associated software. Currently, there is considerable
debate as to which algorithms are the best used to analyse
Q-PCR data (reviewed in Rebrikov & Trofimov, 2006). All
the Q-PCR platforms collect fluorescent data from every
amplification cycle and the increase in fluorescence is
plotted against the cycle number, resulting in the typical
amplification curve shown in Fig. 2. The Q-PCR amplifica-
tion curve can be subdivided into four stages, namely
background noise, where the background fluorescence still
exceeds that derived from initial exponential template
accumulation, exponential amplification, linear amplifica-
tion and a plateau stage. During the exponential phase of the
amplification, the amount of target amplified is propor-
tional to the starting template and it is during these cycles
that gene numbers are quantified using the Ct method. The
Ct is reached when the accumulation of fluorescence (tem-
plate) is significantly greater than the background level
Fig. 2. Q-PCR amplification from known concentrations of template DNA to construct standard curves for quantification of unknown environmental
samples. (a) Log plot of the increase in fluorescence vs. cycle number of DNA standards ranging from 1� 104 to 1�108 16S rRNA gene amplicons mL�1.
(b) Linear plot indicating the three phases of a PCR amplification, the corresponding Ct values for each of the amplified standards and for the NTC. (c)
Simple linear regression of the Ct values (from b) vs. log of the initial rRNA gene number. Q-PCR descriptors are shown (boxed).
FEMS Microbiol Ecol 67 (2009) 6–20 c� 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
9Application of Q-PCR in microbial ecology
(Heid et al., 1996). During the initial cycles, the fluorescence
signal due to background noise is greater than that derived
from the amplification of the target template. Once the Ct
value is exceeded, the exponential accumulation of product
can be measured. When the initial concentration of the
target template is higher, the Ct will be reached at an earlier
amplification cycle.
Quantification of the initial target sequences of an
unknown concentration is determined from the Ct values
and can be described either in relative or in absolute terms.
In relative quantification, changes in the unknown target are
expressed relative to a coamplified steady state (typically a
housekeeping) gene. Any variation in the presence (or
expression) of the housekeeping gene can potentially mask
real changes or indicate artificial changes in the abundance
of the gene of interest. While this approach is commonly
applied for studying eukaryotic gene expression (reviewed
in Bustin, 2002), it is more difficult to apply this method for
studying prokaryotic genes where the identification of a
valid steady-state reference gene is problematic. Burgmann
et al. (2007) nevertheless successfully utilized such an
approach when confirming microarray-based determination
of the transcriptional responses of Silicibacter pomeroyi to
dimethylsulphoniopropionate additions. From microarray
experiments, they identified a gene whose expression was
not altered by experimental conditions and used the expres-
sion of this gene to normalize levels of expression of the
target genes of interest in RT-Q-PCR assays. In a number of
other studies, gene and transcript numbers of the target gene
of interest have been normalized to the numbers of 16S
rRNA gene or transcripts (Neretin et al., 2003; Treusch et al.,
2005; Kandeler et al., 2006). For example, Treusch et al.
(2005) normalized the number of amoA transcripts to
numbers of 16S rRNA gene transcripts
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