Real-time
PCR
assays
using
TaqMan
or
Molecular
Beacon
probes
were
developed
and
optimized
for
the
quantification
of
total
bacteria
,
the
nitrite-oxidizing
bacteria
Nitrospira
,
and
Nitrosomonas
oligotropha-like
ammonia
oxidizing
bacteria
(
AOB
)
in
mixed
liquor
suspended
solids
(
MLSS
)
from
a
municipal
wastewater
treatment
plant
(
WWTP
)
using
a
single-sludge
nitrification
process
.
The
targets
for
the
real-time
PCR
assays
were
the
16S
rRNA
genes
(
16S
rDNA
)
for
bacteria
and
Nitrospira
spp.
and
the
amoA
gene
for
N.
oligotropha
.
A
previously
reported
assay
for
AOB
16S
rDNA
was
also
tested
for
its
application
to
activated
sludge
.
The
Nitrospira
16S
rDNA
,
AOB
16S
rDNA
,
and
N.
oligotropha-like
amoA
assays
were
loglinear
over
6
orders
of
magnitude
and
the
bacterial
16S
rDNA
real-time
PCR
assay
was
log-linear
over
4
orders
of
magnitude
with
DNA
standards
.
When
these
real-time
PCR
assays
were
applied
to
DNA
extracted
from
MLSS
,
dilution
of
the
DNA
extracts
was
necessary
to
prevent
PCR
inhibition
.
The
optimal
DNA
dilution
range
was
broad
for
the
bacterial
16S
rDNA
(
1000-fold
)
and
Nitrospira
16S
rDNA
assays
(
2500-fold
)
but
narrow
for
the
AOB
16S
rDNA
assay
(
10-fold
)
and
N.
oligotrophalike
amoA
real-time
PCR
assay
(
5-fold
)
.
In
twelve
MLSS
samples
collected
over
one
year
,
mean
cell
per
L
values
were
4.3
(
2.0
×
1011
for
bacteria
,
3.7
(
3.2
×
1010
for
Nitrospira
,
1.2
(
0.9
×
1010
for
all
AOB
,
and
7.5
(
6.0
×
109
for
N.
oligotropha-like
AOB
.
The
percent
of
the
nitrifying
population
was
1.7
%
N.
oligotropha-like
AOB
based
on
the
N.
oligotropha
amoA
assay
,
2.9
%
total
AOB
based
on
the
AOB
16S
rDNA
assay
,
and
8.6
%
nitriteoxidizing
bacteria
based
on
the
Nitrospira
16S
rDNA
assay
.
Ammonia-oxidizing
bacteria
in
the
wastewater
treatment
plant
were
estimated
to
oxidize
7.7
(
6.8
fmol
/
hr
/
cell
based
on
the
AOB
16S
rDNA
assay
and
12.4
(
7.3
fmol
/
hr
/
cell
based
on
the
N.
oligotropha
amoA
assay
.
*
Corresponding
author
phone
:
(
865
)
974-8080
;
fax
:
(
865
)
974-8086
;
e-mail
:
sayler
@
utk.edu.
†
Department
of
Microbiology
.
‡
Department
of
Civil
and
Environmental
Engineering
.
§
Center
for
Environmental
Biotechnology
.
10.1021
/
es0257164
CCC
:
$
25.00
Published
on
Web
12
/
04
/
2002

©
2003
American
Chemical
Society

1
.
Introduction