Note: Descriptions are shown in the official language in which they were submitted.
CA 02479990 2004-09-20
Method for the identification of microorganisms by means of in situ
hybridization
and flow cytometry
The invention relates to a combined method for the specific detection of
microorganisms by
in situ hybridization and flow cytometry. The inventive method is particularly
characterized
by an improved specificity and a shorter process time as opposed to methods
known in the
prior art.
Traditionally, microorganisms are detected by cultivation. However, this
detection method
has a number of disadvantages. Particularly in the analysis of the biocoenosis
of
environmental samples the cultivation has been shown to be completely
unsuitable.
Cultivation-dependent methods provide only a very false view of the
composition and
dynamics of the microbial biocoenosis. For example, it could be shown that in
recording the
flora of the activated sludge by cultivation a cultivation shift occurs
(Wagner, M., R. Amann,
H. Lemmer and K.H. Schleifer, 1993, Probing activated sludge with
oligonucleotides specific
for proteobacteria: inadequacy of culture-dependent methods of describing
microbial
community structure, Appl. Environ. Microbiol. 59:1520-1525).
Because of this medium-dependent distortion of the real conditions within the
bacterial
population, the importance of bacteria which play only a minor role in
activated sludge, but
which are well adjusted to the cultivation conditions used, is dramatically
overestimated. It
could thus be shown that due to such cultivation artifacts the bacterial genus
Acinetobacter
was completely misjudged with respect to its role as biological phosphate
remover in the
purification of sewage. Such misconceptions result in the cost-intensive,
error-prone and
imprecise creation of plants. The efficiency and reproducibility of such
simulation
calculations is low.
But the cultivation has significant disadvantages also in the analysis of
foodstuffs or medical
samples. The methods used here are often very tedious, require a multiplicity
of successive
cultivation steps and produce results which are not infrequently unclear. The
testing of a
water sample for the presence or absence of faecal streptococci is described
here by way of
example. The detection methods recommended in the Drinking Water Ordinance are
based on
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the direct cultivation of the water sample or an membrane filtration and
subsequent
introduction of the filter in 50 ml azide-glucose-broth. The cultivation
should be carried out
for at least 24 hours, in the case of a negative result for 48 hours, at
36°C. If after 48 hours
clouding or sedimentation of the broth is still not detectable, the absence of
faecal
streptococci in the tested sample is deemed to have been proven. In the case
of clouding or
sedimentation, streaking of the culture on enterococci selective agar
according to Slanetz-
Barthley and re-incubation at 36°C for 24 hours takes place. If reddish-
brown or pink colonies
form, these will be examined in more detail. After transfer to a suitable
liquid medium and
cultivation for 24 hours at 36°C, faecal streptococci are deemed to
have been detected when
propagation in nutrient broth at a pH of 9.6 takes place and the propagation
in
6.5% NaCI-broth is possible as well as in the case of esculin degradation.
Esculin degradation
is checked by the addition of freshly prepared 7% aqueous solution of iron(II)
chloride to
esculin broth. In the case of degradation a brownish-black colour develops.
Frequently, a
Gram stain for differentiating bacteria from Gram-negative cocci is
additionally carried out as
well as a catalase test for differentiating from staphylococci. Faecal
streptococci react Gram-
positive and catalase-negative. The traditional detection procedure is thus
shown to be a
tedious (48-100 hours) and, in suspected cases, an extremely elaborate method.
Due to the disadvantages of the cultivation described, modern methods for the
identification
of bacteria all have a common aim: they attempt to get around the
disadvantages of
cultivation in that they no longer require the cultivation of the bacteria, or
at least reduce the
cultivation to a minimum.
In PCR, polymerase chain reaction, a characteristic piece of the respective
bacterial genome is
amplified with primers specific for bacteria. If the primer finds its target
site, a million-fold
amplification of a piece of the inherited material occurs. Upon the following
analysis by an
agarose gel separating DNA fragments, a qualitative evaluation can take place.
In the most
simple case this leads to the conclusion that the target sites are present in
the tested sample.
Further conclusions are not possible, because the target sites can originate
from a living
bacterium, a dead bacterium or from naked DNA. Differentiation is not possible
with this
method. A further refinement of this technique is the quantitative PCR, which
tries to
establish a correlation between the amount of bacteria present and the amount
of DNA
obtained and amplified. However, various substances contained in the analyzed
sample can
CA 02479990 2004-09-20
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lead to an inhibition of the DNA amplifying enzyme, the Taq polymerase. This a
common
cause of false negative results of the PCR. Advantages of PCR are its high
specificity, its ease
of application and its low expenditure of time. Its main disadvantages are its
high
susceptibility to contamination and therefore false positive results, as well
as the
aforementioned lack of possibility to discriminate between living and dead
cells or naked
DNA, respectively, and finally the danger of false positive results due to the
presence of
inhibitory substances.
But also biochemical parameters are used for the identification of bacteria.
Thus, the
establishment of bacterial profiles on the basis of quinone determinations
serves to render an
image of the bacterial population which is as distortion-free as possible
(Hiraishi, A. 1988.
Respiratory quinone profiles as tools for identifying different bacterial
populations in
activated sludge. J. Gen. Appl. Microbiol. 34:39-56). But also this method is
dependent on the
cultivation of individual bacteria, since the establishment of the reference
database requires
the quinone profiles of the bacteria in pure culture. Moreover, the
determination of the
quinone profiles of the bacteria cannot give a real impression of the actual
populations present
in the sample.
In contrast hereto, the detection of bacteria by antibodies is a more direct
method (Brigmon,
R.L., G. Bitton, S.G. Zam, and B. O'Brien. 1995. Development and application
of a
monoclonal antibody against Thiothrix spp. Appl. Environ. Microbiol. 61: 13-
20).
Fluorescence labelled antibodies are mixed with the sample and allow a highly
specific
attachment to the bacterial antigens. The thus labeled bacteria are then
detected in the epi-
fluorescence microscope based on their emitted fluorescence. In this way,
bacteria can be
identified up to the level of the strain. However, there are crucial
disadvantages which limit
the applicability of this method drastically. First of all, pure cultures of
the bacteria to be
detected are required for the production of the antibodies. This means of
course that
ultimately only those bacteria which are cultivatable at all are detectable by
antibodies.
However, the majority of bacteria is not cultivatable and can therefore not be
detected using
this method. Secondly, the often large and bulky antibody-fluorescence-
molecule-complex
has problems in entering the target cells. Thirdly, the application of
antibodies is limited to
certain samples which are present in a suitable form or appropriately
prepared. Especially
environmental samples, which often have a high percentage of particles, (e.g.
soil samples or
CA 02479990 2004-09-20
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sludge samples), can only be inadequately analyzed by antibodies. In these
samples,
unspecific adsorption of the antibodies to the particles contained
increasingly occurs. This can
lead to false positive results, when the fluorescent particles are confused
with the bacteria to
be detected. The evaluation of the analysis is at least made very difficult,
since unspecifically
glowing particles have to be distinguished from specifically glowing bacteria.
Fourthly, the
detection using antibodies is often too specific. The antibodies often detect
only a certain
bacterial strain of a bacterial species with high specificity, but leave other
strains of the same
bacterial species undetected. However, in most cases strain-specific detection
of bacteria is
not required, but rather the detection of all bacteria of a bacterial species
or an entire bacterial
group. For many bacterial species this has so far not been successful, namely
the development
of a detection method based on antibodies which detects not only individual
strains but all
bacteria of a species. Fifthly, the production of antibodies is a relatively
tedious and expensive
process.
As a novel approach, the method of in situ hybridization with fluorescence
labelled oligo-
nucleotide probes was developed at the beginning of the nineties, which is
known as
fluorescence in situ hybridization (FISH; Amann et al. (1990) J. Bacteriol.
172:762; Amann,
R.L, W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identification and in
situ detection
of individual microbial cells without cultivation: Microbiol. Rev. 59:143-
169). Using this
method, bacterial species, genera or groups may be identified and if
necessary, also visualized
or quantified directly in a sample with high specificity. This method is the
only one providing
a distortion-free representation of the actual in situ conditions of the
biocoenosis. Even
bacteria not cultivated up to now and thus not yet described can be
identified.
The FISH technique is based on the fact that in bacterial cells there are
certain molecules
which have only been mutated to a small extent in the course of evolution
because of their
essential function. These are the 165 and the 23S ribosomal ribonucleic acid
(rRNA). Both
are parts of the ribosomes, the sites of protein biosynthesis, and can serve
as specific markers
on account of their ubiquitous distribution, their size and their structural
and functional
constancy (Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51, p. 221-
271).
For the application of the FISH, so-called gene probes (usually small, 16-25
bases long,
single-stranded desoxyribonucleic acid pieces) are developed which are
complementary to a
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defined region of the rRNA. This defined region is selected in such a way that
it is specific for
a bacterial species, genus or group.
In FISH, labeled gene probes enter the cells present in the tested sample. If
a bacterium of the
species, genus or group for which the gene probes were developed is present in
the sample
tested, the gene probe binds to its target sequence in the bacterial cell and
the cells can be
detected thanks to the labeling of the gene probes.
The advantages of the FISH technique compared to the above described methods
for the
identification of bacteria (cultivation, PCR or antibodies) are manifold.
Firstly, using gene probes numerous bacteria can be detected which are not
detectable using
traditional cultivation. Whereas using cultivation a maximum of only 15% of
the bacterial
population of a sample can be visualized, the FISH technique allows detection
of up to 100%
of the total bacterial population in many samples. Secondly, detection of
bacteria using the
FISH technique is much faster than using cultivation. Whereas the
identification of bacteria
by cultivation often takes several days, using the FISH technique there is
only a few hours
between sampling and the bacteria identification, even on the species level.
Thirdly, in
contrast to a cultivation medium the specificity of the gene probes can be
almost freely
selected. Individual species can be detected with one probe just as well as
entire genera or
bacterial groups. Fourthly, bacterial species or entire bacterial populations
can be exactly
quantified directly in the sample.
In contrast to PCR, FISH can reliably detect only living bacteria. False
positive results by
naked DNA or dead bacteria as in the case of PCR are ruled out using FISH.
Furthermore,
false negative results due to the presence of inhibitory substances are
equally ruled out as are
false positive results due to contaminations.
In contrast to the antibody technology, the production of nucleic acid probe
molecules is
simple, fast and inexpensive. Further, the complex of nucleic acid probe
molecule and
fluorescence stain is by far smaller than the antibody-stain-complex and, in
contrast to the
latter, can easily enter the cells to be detected. As already described above,
also the specificity
of the nucleic acid probe molecules can be almost freely selected. Individual
species can be
CA 02479990 2004-09-20
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detected with a probe as can entire genera or bacterial groups. Finally, in
contrast to the
antibody technology, the FISH technique is suitable for the testing of many
different types of
samples.
The FISH technique is thus an outstanding tool for detecting bacteria fast and
highly
specifically directly in a sample. In contrast to the cultivation method, it
is a direct method
and moreover allows also a quantification.
Routine FISH analysis is performed on a suitable solid, optically transparent
substrate, as e.g.
a slide or a microtiter plate. Evaluation is then performed in a microscope,
the bacteria being
visualized by irradiation with a high-energy light.
The conventional FISH method for the detection of microorganisms using a solid
substrate
has however also got its limitations. It cannot be automated, or at least only
with difficulty,
and is thus comparatively protracted and moreover not always reproducible with
the same
quality. Furthermore, another possible source of error for quantitative
analysis in a
microscope is the subjectivity of the observer, which can never entirely be
eliminated.
Above all, the lack of automation and the thus comparatively laborious and
protracted
handling as well as the quantification which is subject to the subjective
impression of the
observer have led to the fact that the FISH analysis has up to now only been
used in industry
for single and multiple analysis, but not for high-throughput analysis and is
only rarely used
for exact quantification. However, since the analysis of bacteria using this
method still has
important advantages compared to all other microbiological analysis methods
presently used
in industry, as described, there is a need for a combined method which
combines the
advantages of the FISH analysis with those of a fast, simple and objective
method which can
be automated and which therefore allows the simple, fast and reliable analysis
of samples for
the identification and quantification of microorganisms.
In the past few years flow cytometry has acquired a strong influence as
"automated
microscopy"especially in biological and medical research as well as in
diagnostics. It offers
the advantages of automation, objectivity and high evaluation speed (several
thousand cells
can be measured per second).
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The flow cytometry allows the counting and the analysis of physical and
molecular
characteristics of particles (such as e.g. cells) in a liquid stream. Flow
cytometry allows the
documentation of certain characteristics of cells or cell populations on the
single cell level.
Flow cytometry is principally consisting of a liquid system and an optical
system. The basis
for a successful test is the hydrodynamic focussing of the sample by the
liquid system. Here,
the cells contained in the sample are thinned out and arranged with a very
high degree of
accuracy (deviation 1 Vim) linearly on the measuring point. The optical system
generally
consists of an excitation light source (e.g. diverse lasers or a mercury
pressure lamp), various
optical mirrors and detectors for the forwards scattered light, the sideways
scattered light and
the fluorescence light. Flow cytometers are already available from several
suppliers, such as
e.g. the product Microcyte from Optoflow, Norway, or the BD FACSCalibur
apparatus from
BD Biosciences, Becton, Dickinson and Company. In addition, companies and
institutes offer
the performance of corresponding flow cytometry analysis and/or use times for
flow
cytometers.
At the beginning of the analysis the sample is fed into the centre of the
transport liquid. The
separating and centering of the cells is achieved by the coat stream
(Mantelstrom), which is
generated by a higher flow speed of the transport liquid compared to the
sample stream. By
means of the liquid system which is under pressure, the single cells are now
passed by the
excitation light source of the optical system continuously and with constant
speed.
The laser light impacting the cells is first scattered in two directions: the
scattered light
directed "forward" at an angle of 2-15° (FSC for forward scatter) and
"sideways" at an angle
of 15-90° (SSC for sideways scatter) is a measure of the size and
granularity of the cell. In
addition, various fluorochromes can be excited for the emission of light
quants via diverse
lasers (e.g. argon or helium-neon laser) or a mercury pressure lamp with
suitable optical
filters. 'The light quants are then detected by suitable sensors. The measured
values obtained
are visualized in the form of histograms or dot plots on the computer.
The flow cytometry has however hitherto only been used on a very small scale
for micro-
biological tests. First attempts to combine the FISH and the flow cytometry
were performed
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by Wallner (Wallner, G. et al. (1993) Cytometry 14(2):136-143; Wanner G. et
al. (1995)
Appl. Environ. Microbiol. 61(5):1859-1866; Wallner, G. et al. (1997) Appl.
Environ.
Microbiol. 63(11 ):4223-4231 ).
Disadvantages of the method described in the prior art are again the
relatively tedious
procedure (hybridization time of 3 hours, centrifugation step between
hybridization and
washing, washing time of 0.5 hours), the uncertain specificity of the method
as a result of this
tedious procedure as well as the unsuitability of this method for the
detection of gram-positive
bacteria.
It is the object of the present invention to overcome the above-described
disadvantages of the
prior art and to provide a method by which microorganisms can be detected
specifically,
simply, reproducibly, reliably, fast and objectively.
According to the invention the above-mentioned object is solved by the
features of the
independent claim. Further embodiments will become clear from the features of
the sub-
claims.
The implementation of the method according to the invention for the specific
detection of
microorganisms in a sample comprises the following steps:
a) fixing the microorganisms contained in the sample,
b) incubating the fixed microorganisms with nucleic acid probe molecules
contained in a
hybridization solution in order to achieve hybridization (= hybridization
step),
c) adding a washing solution to the fixed microorganisms incubated with the
nucleic acid
probe molecules (= washing step),
d) detecting the microorganisms with hybridized nucleic acid probe molecules
by flow
cytometry,
wherein the hybridization solution is not removed between the hybridization
step b) and the
washing step c).
In a preferred embodiment the method further comprises between the fixing step
a) and the
hybridization step b) the step i) drying the probe and removing the fixing
agent.
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In a further preferred embodiment the method according to the invention
further comprises
between the fixing step a) and the hybridization step b), or between the
drying step i) and the
hybridization step b) the step ii) lysing the fixed microorganisms.
~A particularly preferred embodiment of the method for the specific detection
of micro-
organisms in a sample therefore provides the following steps:
a) fixing the cells contained in the sample,
i) drying the sample and removing the fixing agent,
ii) complete lysis of the cells contained in the sample,
b) incubating the fixed and lysed cells with nucleic acid probe molecules in
order to
achieve hybridization,
c) adding a washing solution,
d) detecting the cells with hybridized nucleic acid probe molecules in the
flow cytometer,
wherein between step b) and step c) the hybridization solution containing the
nucleic acid
probe molecules is not removed.
Optionally, the first step is preceded by a short cultivation for the
enrichment of the cells
contained in the sample to be tested.
In a further embodiment the method can be performed without centrifugation
after the
washing step. By dispensing completely with centrifugation the method
according to the
invention can be performed even faster and more simply.
Within the scope of the present invention "fixing" of the microorganisms is
understood to
mean a treatment with which the bacterial envelope is made permeable for
nucleic acid
probes. For fixation, usually ethanol is used. If the cell wall cannot be
penetrated by the
nucleic acid probes using these techniques, the expert will know a sufficient
number of other
techniques which lead to the same result. These include, for example,
methanol, mixtures of
alcohols, a low percentage paraformaldehyde solution or a diluted formaldehyde
solution or
the like.
Within the scope of the present invention "drying" is understood to mean an
evaporation of
the sample at elevated temperature, until the fixation solution is completely
evaporated.
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Within the scope of the present invention, "complete lysis of the cells" is
understood to mean
an enzymatic treatment of the cells. By this treatment, the cell wall of gram-
positive bacteria
is made permeable for nucleic acid probe molecules. For this purpose, for
example lysozyme
in a concentration of 0,1 -10 mg/ml H20 is suitable. But also other enzymes,
such as for
instance mutanolysine or proteinase K can be used alone or in combination.
Suitable
concentrations and solvents are well known to the expert. It goes without
saying that the
method according to the invention is also suitable for the analysis of gram-
negative bacteria;
the enzymatic treatment for complete cell lysis is then adapted accordingly,
it can then also be
completely dispensed with.
Within the scope of the present invention the fixed bacteria are incubated
with fluorescence
labeled nucleic acid probe molecules for the "hybridization". These nucleic
acid probe
molecules, which consist of an oligonucleotide and a marker linked thereto can
then penetrate
the cell wall and bind to the target sequence corresponding to the nucleic
acid probe molecule
within the cell. Binding is to be understood as formation of hydrogen bonds
between
complementary nucleic acid pieces.
The nucleic acid probe molecule here can be complementary to a chromosomal or
episomal
DNA, but also to an mRNA or rRNA of the microorganism to be detected. It is
advantageous
to select a nucleic acid probe molecule which is complementary to a region
present in copies
of more than 1 in the microorganism to be detected. The sequence to be
detected is preferably
present in 500-100,000 copies per cell, especially preferred 1,000-50,000
copies. For this
reason the rRNA is preferably used as target site, since the ribosomes as
sites of protein
biosynthesis are present many thousandfold in each active cell.
The nucleic acid probe molecule within the meaning of the invention may be a
DNA or RNA
probe usually comprising between 12 and 1,000 nucleotides, preferably between
12 and 500,
more preferably between 12 and 200 and between 12 and 100, especially
preferably between
12 and 50 and between 14 and 40 and between 15 and 30, and most preferably
between 16
and 25 nucleotides. The selection of the nucleic acid probe molecules is done
according to
criteria of whether a suitable complementary sequence is present in the
microorganism to be
detected. By selecting a defined sequence, a bacterial species, a bacterial
genus or an entire
CA 02479990 2004-09-20
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bacterial group may be detected. In a probe consisting of 1 S nucleotides, the
sequences should
be 100% complementary. In oligonucleotides of more than 15 nucleotides, one or
more
mismatches are allowed.
A sequence is suitable if it is on the one hand specific for the microorganism
to be detected
and on the other hand accessible for the entering nucleic acid probe molecule,
i.e. not masked
by ribosomal proteins or the secondary structure of the rRNA.
Within the scope of the present invention the nucleic acid probe molecules are
used with
suitable hybridization solutions. Suitable compositions of this solution are
well known to the
expert. Such a hybridization solution contains organic solvents, in particular
formamide, in a
concentration of between 0% and 80% and has a salt concentration (preferably
NaCI) between
0,1 mol/1 and 1.5 mol/1. Also contained is a detergent (usually SDS) in a
concentration of
between 0% and 0.2% as well as a buffer substance suitable for the buffering
of the solution
(e.g. Tris-HCI, Na-citrate, HEPES, PIPES or similar), usually in a
concentration of between
0.01 mol/1 and 0.1 moll. Usually, the hybridization solution has a pH of
between 6.0 and 9Ø
The concentration of the nucleic acid probe in the hybridization solution
depends on the kind
of its label and on the number of target structures. In order to allow rapid
and efficient
hybridization, the number of nucleic acid probe molecules should exceed the
number of target
structures by several orders of magnitude. However, it has to be noted that
too high levels of
fluorescence labeled nucleic acid probe molecules result in increased
background
fluorescence. The concentration of the nucleic acid probe molecules should
therefore be in the
range between 0.5 and 500 ng/~1. Within the scope of the method of the present
invention the
preferred concentration is 1-10 ng for each nucleic acid probe molecule used
per ~1
hybridization solution. The volume of the hybridization solution used should
be between 8 ~1
and 100 ml, in a preferred embodiment of the method of the present invention
it is between
~l and 1000 ml, especially preferred it is between 20 ~1 and 200 ~l.
It is characteristic for the method according to the invention that the
concentration and the
volume of the hybridization solution used are adjusted to the volume of the
enzyme solution
used in the preceding step, if enzymatic lysis takes place. Immediately after
mixing the
enzyme and the hybridization solution, the chemicals contained in the
hybridization solution
CA 02479990 2004-09-20
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are present in the concentration required for the specificity of the detection
reaction. At the
same time, the hybridization solution is composed in such a way that the
enzyme reaction for
the cell lysis is stopped by the addition of the hybridization solution. In
this way the duration
of the enzymatic treatment of the tested probe can be controlled very
precisely, without a
separate working step for removing the enzyme solution being necessary.
The hybridization usually lasts between 10 minutes and 12 hours, preferably
the hybridization
lasts for about 1.5 hours. The hybridization temperature is preferably between
44°C and 48°C,
especially preferred 46°C, wherein the parameter of the hybridization
temperature as well as
the concentration of salts and detergents in the hybridization solution may be
optimized
depending on the nucleic acid probes, especially their lengths and the degree
to which they
are complementary to the target sequence in the cell to be detected. The
expert is familiar with
the appropriate calculations.
According to the invention it is further preferred that the nucleic acid probe
molecule is
covalently linked with a detectable marker. This detectable marker is
preferably selected from
the group of the following markers:
- fluorescence marker,
- chemoluminescence marker,
- radioactive marker,
- enzymatically active group,
- haptene,
- nucleic acid detectable by hybridization.
The detectable marker is preferably a fluorescence marker.
Within the scope of the present invention "removing" or "displacing" of the
non-bound
nucleic acid probe molecules is achieved by the addition of a washing
solution. That means,
in contrast to the prior art, the hybridization solution is not removed prior
to the washing step,
e.g. by a centrifugation step. Suitable compositions of this solution axe well
known to the
expert. If desired, this washing solution can contain 0.001-0.1% of a
detergent such as SDS,
as well as Tris-HCl in a concentration of 0.001-0.1 moll, wherein the pH of
Tris-HCl is in the
range of 6.0 to 9Ø The detergent can be included, but is not absolutely
necessary.
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Furthermore, the washing solution usually contains NaCI, the concentration
being from 0.003
mol/1 to 0.9 moll, preferably from 0.01 mol/1 to 0.9 mol/1, depending on the
required
stringency. Also, the washing solution can contain EDTA, wherein the
concentration is
preferably 0.005 moll. Further, the washing solution can also contain
preservatives in
suitable amounts which are known to the expert.
It is characteristic for the method according to the invention that the
concentration and the
volume of the washing solution used are adjusted to the volume of the
hybridization solution
used in the preceding step. Immediately after mixing the hybridization
solution and the
washing solution, the chemicals contained in the washing solution are present
in the
concentration required for the specificity of the detection reaction. In
contrast to the method
according to the invention, in the prior art the hybridization solution is
first removed (e.g. by a
centrifugation step) and then the washing solution is added. In this process
the temperature of
the reaction mixture drops to roam temperature, resulting in unspecific false
positive results
of the detection reaction. In contrast, using the method according to the
invention ensures that
the temperature can be kept constant during the entire hybridization and
washing procedure,
thus for the first time guaranteeing the specificity of the detection methods.
The superior specificity of the method according to the invention compared to
the prior art
could be proven by using different probe molecules and different samples, i.e.
different
microorganisms. The improved specificity is mainly due to the fact that the
hybridization
solution is not removed between the hybridization step and the washing step,
but that the
washing solution is added to the cells to be detected and the hybridization
solution.
Very good results were achieved when the volume of the hybridization solution
was
50-150 ul, especially preferred 80-120 ~l, and when the solution was
concentrated I to 3-fold,
especially preferred 1 to 1.5-fold and when the volume of the washing solution
was 20-50 pl,
especially preferred 30-40 ul and when the washing solution was concentrated 3
to 6-fold,
especially preferred 4 to 5-fold.
The non-bound nucleic acid probe molecules are usually "washed off' at a
temperature in the
range of 44°C to 52°C, preferably of 44°C to 50°C
and especially preferred at 46°C for 10-40
minutes, preferably for 15 minutes.
CA 02479990 2004-09-20
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The specifically hybridized nucleic acid probe molecules are then detected in
the respective
cells, provided that the nucleic acid probe molecule is detectable, e.g. by
linking the nucleic
acid probe molecule to a marker by covalent binding. As detectable markers,
for example,
fluorescent groups, such as for example CY2 (available from Amersham Life
Sciences, Inc.,
Arlington Heights, USA), CY3 (also available from Amersham Life Sciences), CYS
(also
obtainable from Amersham Life Sciences), FITC (Molecular Probes Inc., Eugene,
USA),
FLUOS (available from Roche Diagnostics GmbH, Mannheim, Germany), TRITC
(available
from Molecular Probes Inc., Eugene, USA), 6-FAM or FLUOS-PRIME are used, which
are
well known to the person skilled in the art. Also chemical markers,
radioactive markers or
enzymatic markers, such as horseradish peroxidase, acid phosphatase, alkaline
phosphatase,
peroxidase may be used. For each of these enzymes a number of chromogens is
known which
may be converted instead of the natural substrate and may be transformed to
either coloured
or fluorescent products. Examples of such chromogens are listed in the
following table:
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Table
Enzyme Chromogen
1. Alkaline phosphatase and 4-methylumbelliferyl phosphate (*), bis(4-
acid phosphatase methylumbelliferyl phosphate, (*) 3-O-methylfluorescein,
flavone-3-diphosphate triammonium salt (*), p-
nitrophenylphosphate disodium salt
2. Peroxidase tyramine hydrochloride (*), 3-(p-hydroxyphenyl)-
propionate (*), p-hydroxyphenethyl alcohol (*), 2,2'-
azino-di-3-ethylbenzothiazoline sulfonic acid (ABTS),
ortho-phenylendiamine dihydrochloride, o-dianisidine, 5-
aminosalicylic acid, p-ucresol (*), 3,3'-dimethyloxy
benzidine, 3-methyl-2-benzothiazoline hydrazone,
tetramethylbenzidine
3. Horseradish peroxidase HZOZ + diammonium benzidine
H2O2 + tetramethylbenzidine
4. (3-D-galactosidase o-nitrophenyl-(3-D-galactopyranoside, 4-
methylumbelliferyl-~i-D-galactoside
5. Glucose oxidase ABTS, glucose and thiazolyl blue
* fluorescence
Finally, it is possible to generate the nucleic acid probe molecules in such a
way that another
nucleic acid sequence suitable for hybridization is present at their 5' or 3'
ends. This nucleic
acid sequence in turn comprises about 15 to 1,000, preferably 15-50
nucleotides. This second
nucleic acid region may in turn be detected by a nucleic acid probe molecule,
which is
detectable by one of the above-mentioned agents.
Another possibility is the coupling of the detectable nucleic acid probe
molecules to a haptene
which may subsequently be brought into contact with a haptene-recognising
antibody.
Digoxigenin may be mentioned as an example of such a haptene. Other examples
in addition
to those mentioned are well known to the expert.
CA 02479990 2004-09-20
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The final detection of the cells labelled as described above takes place in a
flow cytometer.
The values obtained from this measurement are visualized in the form of
histograms or dot
plots on the computer and permit reliable statements about the kind and amount
of the
bacteria contained.
Furthermore, a kit for carrying out the method according to the invention is
provided which
contains at least one nucleic acid probe molecule for the specific detection
of a micro-
organism, preferably already in the suitable hybridization solution.
Preferably, also the
suitable washing solution, the fixation solution as well as the solution for
the cell lysis and
optionally reaction vessels are included.
Important advantages of the method according to the invention are thus the
very easy
handling as well as speed, reproducibility, reliability and objectivity with
which the specific
detection of microorganisms in a sample is possible.
A further advantage is that the advantageous method of in-situ hybridization
in solution can
now for the first time also be performed for gram-positive organisms. Thus,
the combined
advantages of the FISH and the flow cytometry can for the first time be used
for the analysis
of gram-positive organisms.
A further advantage is the hybridization time, which, compared to the prior
art, is reduced
from 3 hours to preferably 1.5 hours.
A further advantage is the specificity of the method. Here it is crucial that
the concentration
and the volume of the washing solution used is adjusted to the volume of the
hybridization
solution used in the preceding step. Immediately after mixing the
hybridization solution and
the washing solution the chemicals contained in the washing solution are
present in the
concentration required for the specificity of the detection reaction.
According to the
techniques of prior art, the hybridization solution has first to be removed
(e.g. through a
centrifugation step) before the washing solution can be added. In this process
the temperature
of the reaction mixture drops down to room temperature. At this low
temperature the nucleic
acid probe molecules used in the hybridization reaction bind unspecifically
also in those cells
CA 02479990 2004-09-20
- 17-
which do not contain the exact target sites for the nucleic acid probe
molecules but only
similar sequences. In the final detection step also these non-target cells,
which are labelled
due to the unspecific binding of the nucleic acid probe molecules, are
detected. A false
positive result is the consequence. In contrast, using the method according to
the invention
ensures that the temperature remains constant during the whole hybridization
and washing
procedure, as a result of which the specificity of the detection method is for
the first time
guaranteed.
A further advantage is the washing time, which is reduced compared to the
prior art from 30
minutes to preferably 15 minutes.
The microorganism to be detected by the method according to the invention can
be a
prokaryotic or a eukaryotic microorganism. In most cases it will be desired to
detect
unicellular microorganisms. These unicellular microorganisms can also be
present in larger
aggregates, so-called filaments. Relevant microorganisms are especially yeast,
algae, bacteria
or fungi.
The method according to the invention may be used in various ways.
For example, environmental samples may be tested for the presence of
microorganisms.
These samples may be collected from air, water or may be taken from the soil.
Another field of application of the method according to the invention is the
control of
foodstuffs. In preferred embodiments the food samples are obtained from milk
or milk
products (yogurt, cheese, sweet cheese, butter, buttermilk), drinking water,
beverages
(lemonades, beer, juices), bakery products or meat products.
The method according to the invention may further be used for testing
medicinal samples. It
is suitable for the analysis of tissue samples, e.g. biopsy material from the
Lung, tumour tissue
or inflamed tissue, from secretions such as sweat, saliva, semen and
discharges from the nose,
uretha or vagina as well as for urine and stool samples.
CA 02479990 2004-09-20
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A further field of application for the present method is the testing of
sewage, e.g. activated
sludge, sludge or anaerobic sludge. Apart from this, it is also suitable for
the analysis of
biofilms in industrial plants, as well as for testing of naturally forming
biofilms or biofilms
forming in the purification of sewage. Also the testing of pharmaceutical and
cosmetic
products, e.g. ointments, creams, tinctures, juices, etc. is possible with the
method according
to the invention.
The following example is intended to illustrate the invention without limiting
it.
Example:
Combined method for the specific detection of microorganisms taking as an
example the
detection of lactobacilli harmful to beer.
The sample to be tested is cultivated for 24-48 hours in a suitable manner.
Various suitable
methods and cultivation media are well known to the expert. An aliquot of the
culture (e.g.
2 ml) is transferred into a suitable reaction vessel and the cells contained
are pelleted by
centrifugation (4000 x g, 5 min, room temperature).
Then a suitable volume (preferably 20 ~1) of the fixation solution is added
and the open
reaction vessel is incubated at > 37°C until the fixation solution is
completely evaporated.
Then a suitable volume of the enzyme solution (preferably 30-40 ~1 lysozyme [1
mg/ml
H20]) is added and the sample is incubated for 7 minutes at room temperature.
Then a suitable volume (preferably 90-120 pl) of 1.33-fold concentrated
hybridization
solution containing the labelled nucleic acid probe molecules for the specific
detection of
lactobacilli harmful to beer is added and the sample is incubated
(46°C, 1.5 hours).
Then a suitable volume of 5-fold concentrated washing solution (preferably 30-
40 ~1) is
added and the sample is incubated for another 15 minutes at 46°C.
- CA 02479990 2004-09-20
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Then the sample is centrifuged (4000 x g, 5 min, room temperature). The
supernatant is
discarded and the pellet is dissolved in a suitable volume of buffered
phosphate solution
(preferably 100-200 ~1).
The sample thus prepared is now analysed on a flow cytometer (e.g. Microcyte,
Optoflow,
Norway).
Example:
Combined method for the specific detection of microorganisms taking as example
the
detection of lactobacilli.
1. Material
1.1 Microorganisms used
Organism Name of Cultivation conditions
the strain
Lactobacillus WSB L32 M11/30C/standing - micro-aerophilic
brevis
Escherichia coliDSM 30083 M1/37C/agitated 100 rpm-
aerobic
Pediococcus damnosusTUM 618 M231/30C/standing - micro-aerophilic
Salmonella cholerasuisDSM 554 M1/37C/agitated 100 rpm -
aerobic
ssp. cholerasuis
Staphylococcus DSM 1104 M1/37C/agitated 100 rpm -
aureus aerobic
ssp. aureus
The bacteria strains designated DSM are available from the DSMZ (German
Collection of
Microorganisms and Cell Cultures GmbH, Braunschweig, Germany). The strains WSB
L32
and TUM 618 are strains from the laboratory collection of the WSB (Faculty of
Technology
of Brewery I, Freising-Weihenstephan, Germany) and of the Technical University
Munich
TUM (Faculty of Microbiology, Freising-Weihenstephan, Germany).
CA 02479990 2004-09-20
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1.2 Media used
Medium 11: MRS MEDIUM
Casein-Pepton, tryptic digest...............10.00 g
Meat-Extract........................................10.00 g
Yeast-Extract.......................................5.00 g
Glucose................................................20.00 g
Tween 80 .............................................1.00 g
KZHP04 ...............................................2.00 g
Na-Acetate...........................................5.00 g
(NH4)z Citrate......................................2.00 g
MgS04 x 7 H20..................................Ø20 g
MnS04 x HZO.....................................Ø05 g
distilled water ..................................1000.00
ad ml
Adjust the pH to 6.2-6.5.
Medium 231: PEDIOCOCCUS DAMNOSUS MEDIUM
Adjust the pH of Medium 11 (MRS-Medium) to pH 5.2.
Medium 1: NUTRIENT Medium
Peptone ..........................................5.0 g
Meat-Extract..................................3.0 g
Distilled water .................... ad 1000.0 ml
Adjust the pH to 7Ø
All aforementioned media used for the cultivation of bacteria are commercially
available from
the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH,
Braunschweig,
Germany).
CA 02479990 2004-09-20
-21 -
1.3 Solutions used
~ n~~ _1__a:..~ l1 C r la nn.wnnwtrA/'Pl~~
11 Ua a111~ts~avu
vvavavam. s Amount Final ConcentrationFinal Concentration
Ingredient 1.5-fold 1-fold
NaCI solution (5 2,7 ml 1350 mmol/1 900 mmol/1
mol/1)
Tris-HCl Buffer 300 ~1 30 mmol/1 20 mmol/I
(1 mol/1)
Water 1,7 ml -- --
SDS Solution {10%)7,5 ~l 0,015 % 0,01%
Formamide 5,3 ml 52,5 % 35%
Final volume' ~ 10 ~ --- I -
ml
_.._~__ i~ e..m .. .,t...,to.~l
vv a~mu wau.a.~aa
z Amount Final ConcentrationFinal Concentration
Ingredient
4-fold 1-fold
Tris Buffer (1 4 ml 80 mmol/1 20 mmol/1
Mol/1)
NaCI solution (5 2,8 ml 280 mmol/1 70 mmol/1
mol/1)
EDTA solution (0,52 ml 20 mmol/1 5 mmolll
Molll)
Water 41,0 -- --
ml
Final volume: 50,0 -- --
ml
Z. Implementation
The enrichment of the bacterial cultures to be tested was carried out as
described under item
"1.1 Microorganisms used". Then an aliquot of the culture (1-2 ml) was
transferred to a
reaction vessel and the cells contained were pelleted by centrifugation (4000
x g, 5 min, room
temperature).
The supernatant was discarded and 15 ~1 of the fixation solution {99.8% EtOH)
were added to
the cell pellet and the open reaction vessel was incubated at 46°C
until the fixation solution
was completely evaporated.
Then 40 ~l of the enzyme solution (Lysozyme [ 1 mg/ml HZO]) were added and the
sample
was incubated for 7 minutes at room temperature.
CA 02479990 2004-09-20
-22-
Then 80 ~l 1.5-fold concentrated hybridization solution containing a Cy5-
labelled nucleic
acid probe molecule (Lgc-354a 5' - TGGAAGATTCCCTACTGC - 3') was added and the
sample was incubated (46°C, 1.5 hours).
Then 40 X14-fold concentrated washing solution was added and the sample was
incubated for
a further 15 minutes at 46°C.
Then the sample was centrifuged (4000 x g, 5 min, room temperature). The
supernatant was
discarded and the pellet was dissolved in a suitable volume of buffered
phosphate solution
(preferably 100-200 ~l). This last centrifugation step is optional;
alternatively, the sample can
also be measured without any centrifugation step directly after the washing
step.
The sample prepared in this way was analysed on a flow cytometer (Microcyte,
Optoflow,
Norway) using the MC2200 software (Optoflow, Norway). Further, the software
WinMDI
2.8 Windows Multiple Document Interface for Flow Cytometry), a program freely
available
under http://facs.scripps.edu/software.html, was used for the graphic post-
editing of the
readings.
Alternative:
Alternatively, the supernatant was discarded after centrifuging the sample
aliquot and 5 ~,l of
the enzyme solution (Lysozyme [1 mg/ml H20]) was added to the cell pellet and
the sample
was incubated for 7 minutes at room temperature.
Then 10 ~1 of the fixation solution (99.8% EtOH) was added and the open
reaction vessel was
incubated at 46°C until the fixation solution was completely
evaporated.
In this case, the subsequent hybridization step was performed by adding 120 ~1
1-fold
concentrated hybridization solution (instead of 80 ~1 1.5-fold concentrated
solution). All
other steps remained unchanged. _ _
CA 02479990 2004-09-20
-23-
3. Results
In contrast to the visual inspection on a microscope, the possibility of
distinguishing between
unspecific binding or artefacts and a specific signal is very limited on the
flow cytometer, if
these events occur in a similar size range.
It is therefore essential to set a threshold or a detection limit. Readings
below this limit are
interpreted as background; readings above this limit are evaluated as a
positive result.
This detection limit was determined by measuring pure water, 1 x PBS, cells
hybridized
without probe and cells hybridized with a non-binding oligonucleotide probe
and was at
9 x 103 counts/ml.
3.1 Negative findings
Fig. 1 shows the results obtained with non-target organisms of the probe used.
The values
obtained were between 1.0 x 103 and 3.1 x 103 counts/ml (with a centrifugation
step after
washing, Fig. lA to C) or between 4.5 x 103 and 6.7 x 103 counts/ml (without a
centrifugation
step after washing, Fig. 1D to F), respectively, and were thus clearly below
the detection
limit. The values were lower with the final centrifugation step than without
this step, but also
without the final centrifugation step the analysis could be successfully
performed.
3.2 Positive findings
Fig. 2 shows the results obtained with target organisms of the probe used. The
values
obtained were all clearly above the detection limit. T'he readings obtained
with the analysis of
pure and mixed cultures (Fig. 2G-L) were stable and comparable with each
other. Also the
readings for different amounts of cells (processing of 1 ml or 2 ml of a
culture, respectively)
showed a good correlation both for Lactobacillus brevis as well as for
Pediococcus damnosus.
The measurement of Lactobacillus brevis (see Fig. 2I, J and L) and Pediococcus
damnosus
(see Fig. 2G and H) cells produced not only reproducible readings, but also
different
distributions of the single events depending on the morphology of the cells.
CA 02479990 2004-09-20
-24-
The different shape of the plots obtained can primarily be explained by the
different
morphology (P. damnosus = cocci and L. brevis = rods). Additionally, the
homogeneity or
the heterogeneity of a culture, respectively, is made clear in the different
way of presentation.
In this way the culture of P. damnosus consisting of cells of essentially the
same size and the
same shape is presented comically (see Fig. 2G and H). The distribution of the
single readings
of the very heterogeneous culture of L. brevis consisting of cells with very
different morphol-
ogy and size (short, long, rods with partially filamentous structures) is
presented in the shape
similar to a triangle (see Fig. 2I, J and L). The distribution of the single
measuring events of a
mixed culture of L. brevis and P. damnosus shown in Fig. K shows both
different distribution
forms in one reading.
Comments on the Figures:
Figure 1: Results of some experiments with negative findings
A: Density plot of Staphylococcus aureus cells stained with the probe Lgc-
354a;
reading: 1.3 x 10z counts/ml B: density plot of Escherichia coli cells stained
with the probe
Lgc-354a; reading: 1.0 x 103 counts/ml C: density plot of Salmonella
cholerasuis cells stained
with the probe Lgc-354a; reading: 3.1 x 103 counts/ml D: density plot of
Staphylococcus
aureus cells stained with the probe Lgc-354a; reading: 4.5 x 103 counts/ml E:
density plot of
Escherichia coli cells stained with the probe Lgc-354a; reading: 5.2 x 103
counts/ml F:
density plot of Salmonella cholerasuis cells stained with the probe Lgc-354a;
reading:
6.7 x 103 counts/ml.
Figure 2: Results of some experiments with positive findings
G: Density plot of 1 ml Pediococcus damnosus cells hybridized with the probe
Lgc-354a;
reading: 5,2 x 105 counts/ml H: Density plot of 2 ml P. damnosus cells
hybridized with the
probe Lgc-354a~ reading -9,8 x __105 c ounts/ml I: Density plot of lml
Lactobacillus brevis
cells detected with the probe Lgc-354a; reading: 6,16 x 105 counts/ml J:
Density plot of 2 ml
L. brevis cells detected with the probe Lgc-354a; reading:: 1,27 x 106
counts/ml K: Density
plot of a mixture of 1 ml P. damnosus cells and 1 ml L. brevis cells stained
with the probe
CA 02479990 2004-09-20
-25-
Lgc-354a; reading: 1,6 x 106 L: Density plot of 1 ml L. brevis cells detected
with the probe
Lgc-354a; reading: 6,34 x 105 counts/ml. G to K: with centrifugation after the
washing step;
L: without centrifugation after the washing step.
