Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR.

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 oligonucleotide 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 Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. 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 to detect target DNA as low as two 16S rRNA gene copies per 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 beta-proteobacterial AOB in biological nitrification processes and various environments.

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ARTICLE
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: shwang@postech.ac.kr
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
todetecttargetDNAaslowastwo16SrRNA genecopiesper
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
real-time PCR
Introduction
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
stepinmostnitrificationprocesses,majorattentionhasbeen
given to investigating the most favorable conditions to
ensure efficient ammonia oxidation (Kowalchuk and
Stephen, 2001; Nicolaisen and Ramsing, 2002; Rhee et al.,
1997).
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
asdenaturing gradientgelelectrophoresis(DGGE),terminal
Correspondence to: S. Hwang
Contract grant sponsor: Advanced Environmental Biotechnology Research Center
(AEBRC)
Contract grant number: R11-2003-006-04005-0
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? 2007 Wiley Periodicals, Inc.

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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
theAOBdiversityinanaturalecosystem,itisfarmoreuseful
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
AOBoutlinedinBergey’sManualofSystematicBacteriology
(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
microorganisms,asearchforthefalseresultsusingtherRNA
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
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Figure 1.
sequence divergence.
Neighbor joining tree inferred from comparison of 16S rRNA gene sequences belonging to b-proteobacterial AOB from RDP-II. The bar indicates 5% estimated
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decreases. Contrary to false-positive detection, the possibi-
lity of underestimation (i.e., false-negative result) increases
as the degree of mismatches in a target group increases.
Target strains with one mismatch may not cause significant
underestimation, while target strains with two or more
mismatches would cause greater false-negative results than
those with one mismatch. Therefore, a primer and probe set
must be designed as much as possible not to detect non-
target organisms.
Overall, a sequential procedure of clustering (or group-
ing) the AOB, designing the group-specific primer and
probe sets, and evaluating the matching efficiency followed
by false detection analyses was used. AOB was regrouped to
repeat this procedure until the probability of the false-
positive and false-negative detection was minimal.
Sources and Extraction of DNA
A total of eight strains of nitrifying bacteria were purchased
from American Type Culture Collection (ATCC), Deutsche
Sammlung von Mikroorganismen
(DSMZ), and Korean Collection for Type Cultures (KCTC),
and used to test the specificity of each primer and probe set.
After in silico specificity analysis, eight non-nitrifying
bacteria including ones frequently found in wastewater
treatment plants (Amann et al., 1998; Manefield et al., 2005;
Nsabimana et al., 1996; Tchobanoglous and Burton, 1991)
were also purchased to test the potential false positive
detection. The bacteria were Acinetobacter calcoaceticus
KCTC 2357T
(A. cal), Aeromonas hydrophila subsp.
hydrophila KCTC 2358T(A. hyd), Enterococcus faecalis
KCTC 3206T(E. fae), Escherichia coli KCTC 2443 (E. coli),
Nitrobacter hamburgensis DSM 10229 (N. ham), Nitrobacter
vulgaris DSM 10236 (N. vul), Nitrobacter winogradskyi DSM
10237T
(N. win), Nitrococcus mobilis ATCC 25380T
(N. mob), Nitrosococcus oceani ATCC 19707 (N. oce),
Nitrosomonas cryotolerans ATCC 49181 (N. cry), Nitroso-
monas europaea ATCC 19718 (N. eur), Nitrosospira
multiformis ATCC 25196T(N. mul), Pseudomonas carbox-
ydohydrogena DSM 1083 (P. car), Pseudomonas stutzeri
KCTC 1066 (P. stu), Rhodopseudomonas palustris DSM 123T
(R. pal), and Thiobacillus plumbophilus DSM 6690 (T. plu).
DuetothelimitednumberoftargetAOBavailablefromthe
culture collections, seven environmental clones, isolated from
a domestic and an industrial wastewater treatment plant, were
included for the specificity test. The clones were identified as
Nitrosomonas halophila (N. hal), Nitrosococcus mobilis
und Zellkulturen
(N. mob), Nitrosomonas nitrosa (N. nit), Nitrosomonas sp.
Is343(N.Is343),Nitrospirasp.RC14(N.RC14),Nitrospirasp.
RC19 (N. RC19), and Thiobacillus denitrificans (T. den).
As described previously (Yu et al., 2005), a fully
automated nucleic acid extractor (Magtration System
6GC, PSS Co., Chiba, Japan) employing magnetic bead
separation technique (Obata et al., 2001) was used to extract
and purify genomic DNA from the various sources (i.e., 16
purchased strains, 7 environmental clones). Purified DNA
was stored at ?208C until used for subsequent experiments.
Plasmid DNA Construction
In this study, we used plasmid DNA asQPCRstandards. Itis
precedented that real-time QPCR efficiency with genomic
DNAdecreasescomparedtothatwithplasmidDNAbecause
of inhibitory effects of complexgenomicDNA (Harmsetal.,
2003; Lozada et al., 2006). Recently, plasmid DNA has been
much more widely used as reference templates for QPCR
assays, including AOB quantification (Layton et al., 2005;
Limpiyakorn et al., 2005; Okano et al., 2004).
Different PCR primers, of which sequences are shown in
Table I, were used to amplify the 16S rRNA gene from the
genomic DNA extracted from both the strains purchased
and the environmental samples to construct plasmids
containing the 16S rRNA gene. The amplification program
included an initial denaturation step at 948C for 10 min
followed by 40 cycles of denaturation at 948C for 1 min, and
annealing at 558C for 1 min. This was followed by an
extension at 728C for 1 min, with a final extension step at
728C for 7 min. PCR products were cloned with a pGEM-T
Easy Vector cloning kit (Promega, Madison, WI). Plasmids
were extracted using a plasmid DNA purification kit
(Plasmid Quick, General Biosystem, Seoul, Korea), and the
cloned 16S rRNA genes were sequenced through a
commercial sequencing service (Macrogen, Seoul, Korea).
Clone sequences were compared with those deposited in the
GenBank database using the BLASTn program.
The constructed plasmids, normalized to be 103copies of
corresponding 16S rRNA genes/mL, were used as templates
to test specificity of the designed primer and probe sets. A
fluorometer (TD-700, Turner Designs, Sunnyvale, CA)
using PicoGreen dsDNA Quantitation Kits (Molecular
Probes, Eugene, OR) was used to measure concentration of
the template DNA. For convenience, a P was prefixed to the
microbial abbreviations described previously to denote
the plasmids constructed using the corresponding strains.
Table I.
Primers used for plasmid DNA construction in this study.
Target Primer name Sequence (50!30)
AGAGTTTGATYMTGGCTCAG
ACGGGCGGTGTGTRC
TGGGGRATAACGCAYCGAAAG
AGACTCCGATCCGGACTACG
GCTCATGTCCTATCAGCTTG
AGGCATAAAGGCCATGCTG
E. coli numbering References
Bacterial 16S rRNA gene 8f
1392r
bAMOf
bAMOr
NTS-232f
NTS-1200r
8–27Juretschko et al. (1998)
McHugh et al. (2004)
McCaig et al. (1994)
McCaig et al. (1994)
This study
This study
1,392–1,406
143–163
1,296–1,315
232–251
1,200–1,218
AOB 16S rRNA gene
Nitrospira 16S rRNA gene
Lim et al.: Ammonia Oxidizing Bacterial Primer and Probe Sets for Real-Time PCR
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The plasmid constructed using the strain A. calcoaceticus
(A. cal), for example, is a pAcal.
Real-Time QPCR
TaqMan probes were labeled with a fluorescent reporter dye
FAM (6-carboxyfluorescein) and a fluorescent quencher dye
TAMRA (6-carboxytetramethylrhodamine), which were
attached to the 50and the 30ends, respectively. All QPCR
assays were performed using a LightCycler 1.2 (Roche
Diagnostics, Mannheim, Germany) with 20 mL reaction
capillary tubes. Each capillary tube was separately loaded
with 2 mL of a template DNA, followed by an addition of
1 mL (final concentration, 500 nM) of the forward and
reverse primers along with 1 mL (final concentration,
100 nM) of the TaqMan probe corresponding to each
primer and probe set, 4 mL of the LightCycler TaqMan
Master mix (Roche Diagnostics), and PCR-grade sterile
water to a final volume of 20 mL. A control without the
corresponding template DNA was included in every QPCR
assay for each primer and probe set. All experiments were
duplicated.
Two-step amplification of the target DNA, combining the
annealing and the extension steps, was performed applying
the following conditions: an initial 10 min incubation at
948C for Taq DNA polymerase activation, 45 cycles of
denaturation at 948C for 10 s, and simultaneous annealing
and extension at 608C for 30 s. The transition rate was
208C/s for all segments in the two-step cycling. The
fluorescence response data were obtained during the
annealing and extension period in the ‘‘Single’’ mode with
the channel setting at F1. The fluorescent signal data were
processed using the LightCycler Software (version 4.0).
pNeur, pNhal, pNmob, pNnit, pNcry, pN343, and pNmul
were used as target AOB templates to construct the standard
curves (Table II). Different initial 16S rRNA gene copy
numberscoveringsixordersofmagnitudeforeachtargetwere
amplified using the QPCR with the corresponding sets.
Logarithmic values of the different 16S rRNA gene amounts
were plotted against the threshold cycle (CT) numbers from
each QPCR assay. The linear ranges of the plots were selected
based on the r2of the slope greater than 0.95. At each linear
range,theQPCRefficiency(E)ofeachsetwascalculatedusing
the equation, E¼[10(?1/slope)?1] (Labrenz et al., 2004).
Results and Discussion
Group-Specific Primer and Probe Sets
Two separate sets targeting each genus of Nitrosomonas and
Nitrosospira could not be designed mainly due to the high
similarityof16SrRNAgenesequencesbetween thetwoAOB
genus(datanot shown). Thisresultisin accordancewith the
previous study indicating that 16S rRNA gene sequences of
Nitrosospira multiformis C71 is higher than 92%, close to
Table II.
Characteristics of the group-specific primer and probe sets targeting 16S rRNA of the genus Nitrosomonas and Nitrosospira.
Name
Functiona
Target group
(target DNA)
Sequence
(50!30)
E. coli
numbering
Matching
efficiencyb(%)
Tmc(8C)
GC (%)
Amplicon
size (bp)
NSMeur-828F
F primer
Nitrosomonas europaea-cluster (pNeur and pNhal)
GTTGT CGGAT CTAAT TAAG
828–846
94.6
52.0
36.8
217
NSMeur-984T
TM probe
CCTAC CCTTG ACATG CTTGG AATC
984–1,007
94.6
63.8
50.0
NSMeur-1028R
R primer
TGTCT TGGCT CCCTT TC
1,028–1,044
70.3
57.4
52.9
NSMmob-988F
F primer
Nitrosococcus mobilis-cluster (pNmob)
GCTTG GAATT TTACG GAGAC
988–1,017
87.5
58.6
45.0
313
NSMmob-1243T
TM probe
AGTGT ACAGA GGGTA GCCAA CCC
1,243–1,265
100
65.7
56.5
NSMmob-1282R
R primer
CTACG AAGTG CTTTG TGAG
1,282–1,300
100
58.1
47.4
NSMnit-438F
F primer
Nitrosomonas nitrosa-cluster (pNnit)
TTCGG TCGGG AAGAW ATAG
438–456
100
58.1
47.4
217
NSMnit-483T
TM probe
CGGTA CCGAC ATAAG AAGCA CCGG
483–506
100
67.7
58.3
NSMnit-633R
R primer
CTAGT YATAT AGTTT CAAAC GC
633–654
100
54.6
31.8
NSMcry-211F
F primer
Nitrosomonas cryotolerans-cluster (pNcry and pN343)
AGACC TTRTG CTTTT GGAG
211–229
73.3
56.0
42.1
244
NSMcry-270T
TM probe
CCAAC TACTG ATCGT YGCCT TGGT
270–293
93.3
65.4
50.0
NSMcry-434R
R primer
TTTTC TTCTC RACTG AAAGA G
434–454
86.7
54.5
33.3
NSS-209F
F primer
Nitrosospira genus (pNmul)
CAAGA CCTTG CGCTY TT
209–225
92.2
55.8
47.1
287
NSS-432T
TM probe
TTTCG TTCCG GCTGA AAGAG CT
432–453
94.8
64.4
50.0
NSS-478R
R primer
TCTTC CGGTA CCGTC AKT
478–495
96.1
58.7
50.0
aTM, TaqMan.
bRatio (%) of number of organisms matched with the corresponding primer (or probe) of the target group to number of all organisms of the group. The ratios were analyzed by the PRIMROSE program.
cCalculated with the nearest-neighbor thermodynamic values method using the DNAMAN program (500 nM of primer, 100 nM of probe, and 100 mM of salt concentration).
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