Primer and Probe Sets for Group-Specific
Quantification of the Genera Nitrosomonas and
Nitrosospira Using Real-Time PCR
Juntaek Lim, Hyojin Do, Seung Gu Shin, Seokhwan Hwang
School of Environmental Science and Engineering, Pohang University of Science and
Technology, San 31, Hyoja-dong, Namgu, Pohang, Gyungbuk 790-784, South Korea;
telephone: þ82-54-279-2282; fax: þ82-54-279-8299; e-mail: email@example.com
Received 21 July 2007; revision received 12 October 2007; accepted 24 October 2007
Published online 19 November 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21715
ABSTRACT: Use of quantitative real-time PCR (QPCR)
with TaqMan probes is increasingly popular in various
environmental works to detect and quantify a specific
microorganism or a group of target microorganism.
Although many aspects of conducting a QPCR assay have
become very easy to perform, a proper design of oligonu-
cleotide sequences comprising primers and a probe is still
considered as one of the most important aspects of a QPCR
application. This work was conducted to design group
specific primer and probe sets for the detection of ammonia
oxidizing bacteria (AOB) using a real-time PCR with a
TaqMan system. The genera Nitrosomonas and Nitrosospira
were grouped into five clusters based on similarity of their
16S rRNA gene sequences. Five group-specific AOB primer
and probe sets were designed. These sets separately detect
four subgroups of Nitrosomonas (Nitrosomonas europaea-,
Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitroso-
monas cryotolerans-clusters) along with the genus Nitrosos-
pira. Target-group specificity of each primer and probe set
was initially investigated by analyzing potential false results
in silico, followed by a series of experimental tests for QPCR
efficiency and detection limit. In general, each primer and
probe set was very specific to the target group and sensitive
reaction mixture. QPCR efficiency, higher than 93.5%,
could be achieved for all primer and probe sets. The primer
and probe sets designed in this study can be used to detect
and quantify the b-proteobacterial AOB in biological nitri-
fication processes and various environments.
Biotechnol. Bioeng. 2008;99: 1374–1383.
? 2007 Wiley Periodicals, Inc.
KEYWORDS: ammonia oxidizing bacteria; microbial quan-
tification; nitrification; primer and probe set; quantitative
Biological nitrogen removal (BNR) processes have widely
been used for treating nitrogenous wastewater, which can
adversely impact the quality of the receiving water.
Significant pollution concerns related to the nitrogenous
wastewater include dissolved oxygen depletion, toxicity,
eutrophication, and methemoglobinemia (Gerardi, 2002).
Microbial nitrogen metabolism in an aquatic environment
involves a consortium of microorganisms and is based on a
series of reactions, the slowest of which determines the
overall efficiencyof theprocess.InBNR processes, ammonia
oxidizing bacteria (AOB) play a key role in oxidizing
ammonia by participating in the first step of nitrification.
Because ammonia oxidation is commonly the rate-limiting
given to investigating the most favorable conditions to
ensure efficient ammonia oxidation (Kowalchuk and
Stephen, 2001; Nicolaisen and Ramsing, 2002; Rhee et al.,
Although BNR relies on microbial activity, process
monitoring focuses mainly on physico-chemical parameters
rather than biological data in almost all engineered
nitrification systems. Measurable metabolic parameters
including residual ammonia concentration and rate of
oxygen uptake are determined as a function of the whole
microbial community. This is primarily because of
fastidious culture conditions and slow growth rates of the
AOB in vitro (Tchobanoglous and Burton, 1991). However,
it should be realized that an understanding of AOB
community structures as well as their dynamics is essential
to improve the prediction, and effective control of the
process operations related to microbial growth (Dionisi
et al., 2003; Robinson et al., 2003).
The study of AOB has benefited enormously from recent
advances in environmental molecular techniques. Various
polymerase chain reaction (PCR)-mediated methods such
Correspondence to: S. Hwang
Contract grant sponsor: Advanced Environmental Biotechnology Research Center
Contract grant number: R11-2003-006-04005-0
Biotechnology and Bioengineering, Vol. 99, No. 6, April 15, 2008
? 2007 Wiley Periodicals, Inc.
restriction fragment length polymorphism (T-RFLP), and
16S rRNA clone library analysis have shown the remarkable
possibility of analyzing AOB communities in a wide variety
of environments (Kindaichi et al., 2006; Kowalchuk et al.,
2000; Nicolaisen and Ramsing, 2002). Results of the
fingerprinting techniques, however, are often qualitative
rather than quantitative. Although it is useful to understand
to control an engineered process if the absolute quantity of
target AOB in the sample can be estimated. Recently,
molecular techniques employing a quantitative real-time
PCR (QPCR) with a TaqMan probe are widely used for
microbial quantification in a variety of environmental
research areas (Kindaichi et al., 2006; Limpiyakorn et al.,
2005; Yu et al., 2006; Zhang and Fang, 2006). Using the
QPCR assay, wide range quantification of seven to eight
logarithmic decades and high reliable quantification results
can be obtained. This assay is also faster and easier to
perform than the hybridization techniques. Although many
aspects of conducting a QPCR assay including instrumenta-
tion and reagents have become increasingly simple to
perform, one of the most important aspects, the design and
selection of primer and probe sets, is still left to the
investigators. For example, use of a fluorescence probe in a
TaqMan system makes the QPCR assay more specific than a
conventional PCR system employing two primers, whereas
the requirement of additional oligonucleotide makes it
difficult to design PCR primers and probe combinations for
a sequence of interest (Houghton and Cockerill, 2006).
Failure of amplification reactions or amplification of
nonspecific target microbe(s) is likely attributable to
improperly designed primer and probe sets (Yu et al.,
2005). Thus,a proper designof theQPCRprimeralong with
the TaqMan probe is a critical but challenging task in
applying a QPCR assay. For subsequent discussions, a
‘primer and probe set’ denotes the three oligonucleotides of
two primers, a forward and reverse, and a dually labeled
fluorescent TaqMan probe.
Oligonucleotide sequencesoftheprimerandprobe setsto
detect entire populations of (i.e., universal) and/or species
levels of AOB can be found in the literature (Harms et al.,
2003; Hermansson and Lindgren, 2001; Kindaichi et al.,
2006; Layton et al., 2005). While it would be ideal to have
complete sets of primer and probe targeting each species of
AOB, the design and application of such sets to a biological
system is not feasible; nor is it possible, by observing the
entire population, to determine which of the species
comprising a mixed microbial population is responsible
for the biochemical reaction of interest. In this study, we
hypothesized if primers and probe sets to detect and
quantify a mixed populations of all AOB at a narrower
taxonomic level than the universal ones are available, this
would allow to study their interactions or simply to
determine the collective properties of the mixture of species
selected by the conditions imposed. Therefore, this study
focused on the design and characterization of genus-specific
primer and probe sets using 16S rRNA gene sequences of
b-proteobacterial AOB, which are likely to be found in most
engineered BNR processes (Prosser and Embley, 2002;
Schramm, 2003). 16S rRNA gene based phylogeny was used
to select the different target AOB groups for the develop-
ment of primer and probe sets. Five separate primer and
probe sets to quantify 16S rRNA gene of b-proteobacterial
AOB belonging to the Nitrosomonas europaea-, Nitrosococ-
cus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas
cryotolerans-clusters, and the genus Nitrosospira were
designed for a QPCR assay. Specificities along with QPCR
efficiencies of the designed primer and probe sets for the
corresponding targetAOB were verified with DNA extracted
from pure strains and environmental samples.
Materials and Methods
Clustering AOB and Designing Primer and Probe Sets
Phylogenetic relationships based on 16S rRNA sequence
comparisons from the Ribosomal Database Project (RDP-II;
Cole et al., 2007) were used to define the target groups of
(Staley et al., 2005; Fig. 1). Complete or partial 16S rRNA
gene sequences of 145 b-proteobacterial AOB (Fig. 1) were
selected and aligned. The regions of identity (i.e., signature
sequences) within the multiple alignments of the 16S rRNA
gene were investigated to find the candidate sequences to be
used as either primers or probes. As described previously for
the design of primers and probes (Yu et al., 2005), amplicon
size, melting temperature (Tm), percentage of GþC bases,
possibility of self-complementarity of the sequence, possi-
bility of forming primer–primer or primer–probe dimers,
and the degree of degeneracy were investigated.
For each AOB group, we tested different combinations of
primers and probes that could detect the corresponding
groups. Among several possible combination sets which
satisfy the design criteria, a combination of two primers and
a probe with the highest matching efficiency was selected for
the target groups. The matching efficiency of a candidate
sequence is defined as the ratio of the number of organisms
that are complementary to the sequence to all organisms in
the target group and was analyzed by PRIMROSE.
Specificity of each primer and probe set was examined in
silico using a PROBE MATCH program on the RDP-II.
In a QPCR application to detect a target group of
gene sequence database is also essential to testing an overall
specificity of the primer and probe set. The 16S rRNA gene
sequences of non-target organisms can be detected by a
primer and probe set specific to a target microbial group if
only one or two nucleotide bases in rRNA gene sequences of
the non-target organism are mismatched with each of three
oligonucleotide sequences comprising the primer and probe
set. This non-target organism potentially causes false-
positive results (i.e., overestimation), where the probability
of overestimation increases as the number of mismatches
Lim et al.: Ammonia Oxidizing Bacterial Primer and Probe Sets for Real-Time PCR
Biotechnology and Bioengineering
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Lim et al.: Ammonia Oxidizing Bacterial Primer and Probe Sets for Real-Time PCR
Biotechnology and Bioengineering