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Appl Environ Microbiol, January 1998, p. 34-37, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Toxin-Producing Cyanobacteria by Use
of Paramagnetic Beads for Cell Concentration and DNA
Purification
Knut
Rudi,1,*
Frank
Larsen,2 and
Kjetill S.
Jakobsen1
Division of General Genetics, Department of
Biology, University of Oslo, 0315 Oslo,1 and
Dynal A.S., 0212 Oslo,2 Norway
Received 13 August 1997/Accepted 17 October 1997
 |
ABSTRACT |
Early detection of water blooms caused by potential toxin-producing
cyanobacteria is important in environmental monitoring. We present a
new nucleic acid-based method for detection of cyanobacteria in water
that utilizes the same paramagnetic solid phase (beads) for both
bacterial cell concentration and subsequent DNA purification. In the
cell concentration step, the beads were attracted to a magnet after
cell adsorption (in an alcohol- and salt-containing solution), and the
supernatant was removed. For DNA purification, a buffer containing
guanidine thiocyanate and Sarkosyl lysed the concentrated cells. The
addition of alcohol precipitated the released DNA onto the same solid
phase as was used for the cell concentration. Finally, to remove PCR
inhibitors, the DNA was washed twice in alcohol while bound to the
beads. All of the bead-DNA complex was used in the subsequent PCR
amplification. The detection limit, as measured by 16S rDNA PCR
amplification, was 50 cells in a 0.5-ml water sample, which is
considerably lower than the limit (500 cells/ml) of toxic cyanobacteria
tolerated in drinking water (New South Wales Blue-Green Algae Task
Force, 1992). Testing of water from natural habitats showed a detection
limit in the same range as that for the defined samples. The detection
limits and the simplicity of the method (paramagnetic beads can be
handled in automated systems) suggest that our method is suitable for
routine environmental monitoring.
| |
INTRODUCTION |
PCR allows high-sensitivity
detection of bacteria in environmental water samples (11,
21). Isolation of bacteria and DNA purification are crucial steps
for reproducibility and high sensitivity. Commonly, bacteria are
isolated from water by centrifugation, filtration, or specific affinity
binding to antibodies attached to a solid phase (5, 10, 22).
Since many of the naturally occurring bacteria in natural waters
contain gas vesicles (20), additional steps (e.g., heating
to break the vesicles) are required for precipitation by
centrifugation. Filtration procedures usually require resuspension of
the cells on the filter for downstream applications. For antibody
affinity binding, specific antibodies for each of the bacteria of
interest have to be produced and tested. This limits the number of
different bacterial species that can be detected by a particular cell
concentration and DNA purification assay. All the above-mentioned cell
concentration approaches require subsequent DNA preparation by using
additional methods, often involving phenol-chloroform treatment
(16), or commercially available DNA purification kits.
However, for high-throughput and automated approaches, there is a need
for simple assays (adaptable for several target organisms) which
integrate cell concentration and DNA purification.
The aim of our work was to develop an integrated cell concentration and
DNA purification assay for early detection of potential toxic water
blooms formed by cyanobacteria in environmental water. We chose, as
model organisms, cyanobacteria belonging to the genera Microcystis, Planktothrix, and
Anabaena, which are commonly associated with toxic water
blooms in both drinking water and recreational water (6).
Previously, we showed that paramagnetic beads are suited for
purification of PCR-ready DNA from cyanobacterial cultures (13). Here, we have developed an assay involving nonspecific adsorption of bacteria (by lowering the water activity by the addition
of alcohol and salt) onto the beads. The DNA binding property of the
beads was then used for DNA purification. Since paramagnetic beads are
easy to manipulate both manually (13) and in automated
systems (8), this integrated cell concentration and DNA
purification method is suited for high-throughput assays. With the
optimized protocol (combined with PCR amplification), as few as 50 cells could be detected in a 0.5-ml water sample. Health authorities
(New South Wales Blue-Green Algae Task Force, 1992) have already
adopted a system where the authorities are alerted when cell counts
reach between 500 and 2,000 cells/ml for toxic cyanobacteria in
drinking water (4), suggesting that our method is suited for
early warning of potential toxic water blooms.
| |
MATERIALS AND METHODS |
Strains and strain cultivation.
Strains were cultivated in
glass flasks containing 50 ml of medium Z8 in a constant-temperature
room at 17 ± 2°C (15). Dilution series from
107 to 100 cells of the cyanobacteria
Microcystis aeruginosa NIVA-CYA 43, Planktothrix
agardhii NIVA-CYA 29 and Anabaena lemmermannii NIVA-CYA 83/1 per ml (18) were used in the development and
optimization of the assays described in this work. The cells were
counted by microscopy in a Fuchst-Rosenthal counting chamber (Karl
Hecht, Sondheim, Germany).
Standard (reference) cell concentration and DNA purification
protocols.
Bacterial cells were concentrated by centrifugation in
a microcentrifuge (model 2231 M; Hermle GmbH, Goshe, Germany) at
3,000 × g for 10 min. Bacteria with gas vesicles were
heated to 65°C for 2 min to break the vesicles before being pelleted.
We tested two different filter types
a glass microfiber filter (GF/C;
Whatman International Ltd.) and cellulose nitrate membrane (Sartorius Corp., Edgewood, N.Y.)commonly used for concentration of
cyanobacteria by filtration (17). For each water sample, 65 ml was filtered onto a 12.5-cm2 membrane. A
0.1-cm2 piece, corresponding to the filtration of 0.5 ml of
water, was subsequently excised with a scalpel. The cells on the filter
or on centrifugation pellets were resuspended in the respective lysis buffers by brief vortexing before DNA purification.
For reference DNA purification strategies, we used both a standard
phenol-chloroform method (13, 16) and a solid-phase purification strategy (Dynabeads DNA DIRECT; Dynal A.S, Oslo, Norway)
involving heating to 65°C (13). In the phenol-chloroform method, the cells were resuspended in 200 µl of buffer containing 10 mM EDTA (pH 8.0) and 12.5 mM TrisHCl (pH 8.0) and then homogenized for
5 min with 30 mg of alumina type A-5 (Sigma Chemical Co., St. Louis,
Mo.) and a pestle (Kontes Scientific Instruments, Vineland, N.J.) to
break the bacterial cell walls. Then 0.3 mg of lysozyme (Sigma Chemical
Co.) and 0.1 mg of RNase A (Sigma Chemical Co.) were added, and the
mixture was incubated at 37°C for 30 min. Sodium dodecyl sulfate was
added to a final concentration of 0.5%, together with 0.1 mg of
proteinase K (Boehringer GmbH, Mannheim, Germany), and the mixture was
incubated at 65°C for 1 h. Cell debris and alumina were pelleted
by a brief centrifugation, and the supernatant was transferred to a
fresh tube. The supernatant was extracted twice with 200 µl of
phenol-chloroform-isoamyl alcohol (25:24:1) and once with 100 µl of
chloroform-isoamyl alcohol (24:1) with brief vortexing between the
extractions. The DNA was precipitated at 
20°C for 2 h with 2 volumes of ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2) and
pelleted by centrifugation at 15,000 × g in a
microcentrifuge (model 2231 M) at 4°C for 30 min. The pellet was
rinsed once with ice-cold 70% ethanol and dried in a vacuum
centrifuge. Finally, the DNA was rehydrated in 40 µl of water with
agitation for 2 h at room temperature. In the reference solid-phase method, 200 µl of Dynabeads DNA DIRECT (1 U) was added to
the cell sample, and the mixture was incubated at 65°C for 15 min and
stored at room temperature for 5 min. The beads bound to DNA were drawn
to the side of a microcentrifuge tube with an MPC-E magnet (Dynal A.S).
While bound to the beads, the DNA was washed twice with the washing
buffer supplied with the kit. Finally, the bead-DNA complex was broken
up by thorough resuspension in 40 µl of water. DNA for PCR
amplification was also prepared by modifying a previously described
protocol involving direct lysis of the cells without subsequent
purification (9). In the modified protocol, the bacterial
cells were resuspended in water, frozen at 70°C, and heated to
95°C for 5 min (to lyse the cells and to denature the DNases). The
degree of cell lysis was determined by microscopic examination and
agarose gel electrophoresis.
Combined solid-phase cell concentration and DNA purification
protocols.
The cell detection limit with the standard Dynabeads
DNA DIRECT buffer system is too high for the purpose of environmental monitoring (see Results and Discussion). An additional step involving ethanol and salt DNA precipitation of the DNA onto the beads was included to lower the detection limit in the optimized protocol. Incubation of the cells at 65°C in 4 M guanidine thiocyanate-1% Sarkosyl for 10 min resulted in complete cell lysis (determined by
microscopic examination). Subsequent precipitation of the DNA onto 1 U
of the beads (i.e., the beads in 200 µl of lysis buffer) in 2 volumes
of ethanol led to reproducible detection of <50 cells (as determined
by PCR amplification). One unit of beads was also the maximum amount
tolerated in a 50-µl PCR mixture without enzyme inhibition
(determined by titration experiments). The cell isolation conditions
were accordingly optimized with 1 U of beads per sample test.
The bacteria tested have low affinity for the beads in water. To
increase the this affinity, the water activity was lowered by the
addition of alcohol and salt. Addition of 1 volume of ethanol increased
the cell recovery considerably, but not at low cell concentrations
(<104 cells/ml). This was improved by the addition of 1 volume of isopropanol instead, which increased the recovery about
10-fold at low cell concentrations. However, the optimal cell
concentration protocol involved 1 volume of isopropanol and 0.1 volume
of 7.5 M ammonium acetate for the cell adsorption step (the complex
formed by P. agardhii NIVA-CYA 29 and the beads is shown in
Fig. 1). Based on the optimized
conditions, both large-scale and small-scale protocols (for 25 and 0.5 ml of water, respectively) were developed.
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FIG. 1.
Micrograph showing adsorption of the filamentous
cyanobacterium P. agardhii NIVA-CYA 29 to magnetic beads.
Approximately 105 bacterial cells were mixed with the cell
binding buffer, incubated at room temperature for 20 min, and
concentrated with the MPC-E magnet for 2 min. Magnification, ×400,
with interference contrast.
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|
The protocols (large-scale values are indicated in brackets) involve
mixing of 0.5-ml [25-ml] water sample with 1 U of beads
(the beads in
200 µl of Dynabeads DNA DIRECT lysis buffer, prepared
by removing the
supernatant and washing the beads once with 200
µl of water), 50 µl
[2.5 ml] of 7.5 M ammonium acetate, and 0.5
ml [25 ml] of
isopropanol in a microcentrifuge tube [50-ml tube].
The samples were
incubated at room temperature for 20 min and
placed in an MPC-E
[MPC-1] magnet for 2 min [5 min], and the supernatant
was removed
carefully. Then, 20 µl of 4 M GTC-1% Sarkosyl was
added to the
beads, and the mixture was incubated at 65°C for
10 min. To
precipitate DNA onto the beads, 40 µl of 96% ethanol
was added, and
incubation was continued at room temperature for
an additional 5 min.
The beads were attracted to the tube wall
with the magnet, the
supernatant was removed, and the bead-DNA
complex was washed twice with
200 µl of 70% ethanol. Finally,
the complex was dried at 65°C for
5 min (it should be completely
dried) and resuspended in 5 µl water.
Maximum sensitivity was
obtained by using all of the bead-DNA complex
in the PCR.
PCR amplification.
For PCR amplification, the following 16S
rDNA PCR primer pairs were used: 5'-AGCCAAGTCTGCCGTCAAATCA-3'
(CH) and 5'-ACCGCTACACTGGGAATTCCTG-3' (CI) for
amplifying M. aeruginosa, 5'-AAGGGTCCGCAGGTGGCAT-3'
(CL) and 5'-GCACAGCTCGGGTCGATACG-3' (CM) for
amplifying A. lemmermannii, and finally
5'-GGAAGGTTCTTGGATTGTCAACCC-3' (CN) and
5'-TGCCTTTGCGAGGTTAAGCCT-3' (CO) for amplifying P. agardhii. These primer sets are based on sequence information
given by Rudi et al. (15). Amplifications were done with the
GeneAmp 2400 PCR system (Perkin Elmer, Norwalk, Conn.) in 50-µl
volumes containing 10 pmol of primers, 200 µM each deoxynucleoside
triphosphate, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mM
KCl, 0.1% Triton X-100, 1 U DynaZyme thermostable DNA polymerase
(Finnzymes Oy, Espoo, Finland), and 5 µl of bead-DNA complex. The PCR
was initiated with a 4-min denaturation step at 94°C; this was
followed by 40 cycles with the following denaturation, annealing, and
synthesis parameters: 94, 58, and 72°C for 30 s each. An
extension step for 7 min at 72°C was included at the end of the PCR.
| |
RESULTS AND DISCUSSION |
Testing of the combined solid-phase cell concentration and DNA
purification protocols.
Based on the PCR results obtained, as
visualized on ethidium bromide-stained agarose gels, we estimated a
detection limit of 100 cells/ml by the small-scale protocol (Fig.
2A) and 10 cells/ml by the large-scale
protocol (Fig. 2B). For the highest cell concentrations tested
(106 and 107 cells/ml), we experienced
difficulties in attracting the beads to the magnet in the cell
concentration step. The purified DNA, however, gave reproducible PCR
amplifications without enzyme inhibition (results not shown). Samples
of water from natural habitats (containing Anabaena sp.)
gave a detection limit of 250 cells/ml (Fig.
3), indicating that complex samples do
not interfere with the analysis. The difference in the detection limit
between diluted cultures and natural samples could be because the
fast-growing organisms in culture contain more copies of the genome per
cell than do the slow-growing organisms in nature. There were no
significant differences in the sensitivity and specificity of the cell
concentration and DNA purification assay between M. aeruginosa NIVA-CYA 43 and 228/1 (from 105 to
100 cells/ml) diluted in pure water, in water containing
105 cells of P. agardhii NIVA-CYA 29 per ml, in
water containing 105 cells of A. lemmermannii
NIVA-CYA 83/1 per ml, and in environmental water sampled from Lake
Akersvatnet, Norway (results not shown). This indicates that the
composition of the sample is not important for cell recovery, probably
because there is an excess of bead surface for bacterial adsorption.
The small-scale protocol has also successfully been used for detection
of the gram-negative Escherichia coli NovaBlue and the
gram-positive bacterium Bacillus cereus AH75. Although the
protocol is not optimized for these species, the detection limits were
in the same range as those of PCR directly on diluted cultures.
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FIG. 2.
Detection limits for the small-scale (A) and large-scale
(B) protocols. Cells and DNA were isolated from 0.5-ml (A) and 25-ml
(B) cultures from the following dilution series: lane 1, 105 cells/ml; lane 2, 104 cells/ml; lane 3, 103 cells/ml; lane 4, 102 cells/ml; lane 5, 101 cells/ml; lane 6, 100 cells/ml. The agarose
gel displays PCR products of 16S rDNA from the three species M. aeruginosa NIVA-CYA 43 amplified with primers CH and CI, A. lemmermannii NIVA-CYA 83/1 amplified with primers CL and CM, and
P. agardhii NIVA-CYA 29 amplified with primers CN and CO.
Twenty percent of the amplification products were loaded in each lane.
mw, molecular weight standards. nt, fragment size (in nucleotides).
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FIG. 3.
Water sample from Lake Fløtjønni, Smøla, Norway. The
sample collected on 29 May 1996 contained approximately 2.5 × 104 cells of Anabaena sp. per ml (as determined
by microscopic examination). Cells and DNA were isolated from 0.5 ml of
water from the following dilution series: lane 1, 2.5 × 104 cells/ml; lane 2, 2.5 × 103 cells/ml;
lane 3, 2.5 × 102 cells/ml; lane 4, 2.5 × 101 cells/ml; lane 5, 2.5 × 100 cells/ml.
16S rDNA was PCR amplified with primers CL and CM. The amplified bands
were verified as Anabaena sp. by sequencing (98% sequence
identity to A. lemmermannii NIVA-CYA 83/1 16S rDNA). Twenty
percent of the amplification products were loaded in each lane.
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|
Bacteria have been kept in the cell binding buffer for 1 week at room
temperature with no detectable loss of sensitivity,
as measured by PCR
amplification. For field experiments, such
stability may be crucial.
Normally, the water sample has to be
processed within a few hours after
sampling because the cell composition
can be altered with time and
because DNA is unstable (with a half-life
of 4 to 6 h) in
environmental water (
12).
Comparison of the combined solid-phase cell concentration and DNA
purification protocols to standard (reference) protocols.
Commonly, nonspecific concentration of bacteria involves centrifugation
or filtration. DNA is then purified in a second step, either by
commercially available DNA purification kits or by
phenol-chloroform-based methods (1-3, 19).
We evaluated the cell concentration step developed in this work by
comparing the results with those of cell concentration
by
centrifugation or filtration. The centrifugation step resulted
in a
floating-cell fraction for organisms containing gas vesicles,
e.g.,
A. lemmermannii NIVA-CYA 83/1. Pretreatment by heating to
65°C for 2 min was required to break these vesicles before cell
pelleting. This caused precipitation of most of the cells in the
centrifugation step. With the filtration approach, the cells on
the
filter were resuspended in the lysis buffers before being
subjected to
further processing. Both these approaches usually
gave higher detection
limits (for 0.5-ml water samples) and were
less reproducible than the
cell adsorption step developed in this
work (Table
1). The lower reproducibility and higher
detection
limits for cell concentration by centrifugation or filtration
compared than by the solid-phase strategy may partially be due
to cell
disruption (caused by mechanical shearing) and cell loss
in the
centrifugation and the filtration steps (
7). For parameters
which are not easily measured, such as the simplicity of the method
and
the risk of cross-contamination, the solid-phase cell concentration
method also performed better than cell concentration by centrifugation
or filtration. For example, the cell pellet formed by the filamentous
cyanobacterium
P. agardhii NIVA-CYA 29 was loose and
difficult
to handle, even after centrifugation at 15,000 ×
g for 30 min.
Manual handling of the filters was difficult
and increased the
risk of cross-contamination. In the cell
concentration protocol
developed in this work, the cells were simply
concentrated by
being attracted to a magnet. The supernatant was then
easily removed
from the bead-cell complex (without extensive manual
handling).
Our DNA purification step was compared to DNA purification by the
phenol-chloroform method or the commercially available Dynabeads
DNA
DIRECT method (the manufacturer recommends not to use more
than 20% of
the beads-DNA complex in each PCR for this buffer
system). For the
phenol-chloroform and the Dynabeads DNA DIRECT
purification strategies,
12.5% of the purified DNA was used in
the subsequent 50-µl PCR
mixtures. Relative to the number of cells
used in each PCR, the
detection limits were about 500 cells for
both reference methods. This
is considerably higher than the detection
limit of less than 50 cells
obtained for the strategy developed
in this work. As for most other DNA
purification strategies, the
phenol-chloroform and the Dynabeads DNA
DIRECT strategies are
designed for DNA purification from cell cultures
and tissues (where
the amount of material is not limited). At low cell
concentrations,
the DNA was probably lost during the many steps
required for the
phenol-chloroform method of DNA purification (see
Materials and
Methods). The Dynabeads DNA DIRECT strategy is based on
coaggregation
of beads and DNA (
13). This aggregate was not
formed at low
cell concentrations, which might have resulted in the
poor DNA
recovery. However, with ethanol precipitation, the association
of the beads and the DNA is tighter, probably leading to the high
recovery at low DNA concentrations for our method.
Finally, the combined cell concentration and DNA purification protocol
was compared to direct lysis of the cells without subsequent
DNA
purification (
9). Direct lysis of cultures of known density
and subsequent PCR directly on the lysate (5 µl of lysed culture
in a
50-µl PCR mixture) gave a detection limit of 1,000 cells/ml,
which is
10 and 100 times higher than for the small-scale and
large-scale
protocols, respectively. The number of directly lysed
cells tolerated
in a 50-µl PCR volume varied considerably, i.e.
from more than
10
5 to less than 10
4 cells, for 19 different
cyanobacterial strains tested. Thus,
direct lysis of the cells can be
used only in some cases and is
not suitable for methods intended for
several types of environmental
samples. Our DNA purification strategy,
however, gave reproducible
amplification for more than 10
5
cells per sample test for all these strains, indicating removal
of the
PCR-inhibitory substances (results not shown).
Most of the previously developed methods for processing environmental
water samples have been designed with the aim of detecting
specific
pathogenic microorganisms (e.g.,
Legionella,
Salmonella,
and
Shigella) (
1). For
research purposes, methods have been
developed for qualitative DNA
purification from large volumes
of water processed by filtration
through a plankton net (
23)
or by tangential-flow filtration
of 1,000 to 8,000 liters of water
(
7). None of these methods
can be universally applied to different
types of environmental samples
(
2). In most cases, detection
and quantitation of naturally
occurring bacteria (e.g., cyanobacteria)
in water require higher
detection limits than for pathogenic bacteria.
Thus, simpler protocols
can be used for detection and quantitation
of naturally occurring
bacteria than of pathogenic bacteria. We
conclude that our combined
cell concentration and DNA purification
protocol is favorable for the
detection and quantitation of samples
containing more than 10 cells of
target organisms per ml. For
lower detection limits, however, a
separate cell concentration
step may be required.
Complete assay for routine monitoring.
The simplicity of the
methodwithout filtration or centrifugation stepsallows for high
throughput of samples and makes it adaptable for automation.
Furthermore, the detection limits obtained suggest that the method is
suitable for early detection of water blooms formed by potential toxic
cyanobacteria (4). However, the versatility of the method
has to be further tested empirically by large-scale screenings, since
both the biological and chemical compositions of environmental water
can be diverse and difficult to define. In this regard, we are
currently developing competitive PCR strategies and colorimetric
detection assays for complete high-throughput systems suitable for such
screenings (14).
| |
ACKNOWLEDGMENTS |
This work has been supported by the Norwegian Research Council
(NFR) to K.S.J. (grant 107622/420).
We are grateful to Olav M. and Randi Skulberg for preparing and
cultivating the cyanobacterial species used in this work, to Anne-Brit
Kolstø for the Bacillus cereus strain, and to Heidi Rudi
and John Stacy for critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division
of General Genetics, Department of Biology, University of Oslo, P.O.
Box 1031 Blindern, 0315 Oslo, Norway. Phone:
(47.)22.85.45.73. Fax: (47.)22.85.46.05. E-mail:
knut.rudi{at}bio.uio.no.
| |
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Appl Environ Microbiol, January 1998, p. 34-37, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
