Note: Descriptions are shown in the official language in which they were submitted.
CA 02474957 2004-07-30
Methods for specific rapid detection of pathogenic food-relevant bacteria
The invention relates to a method for the detection of pathogenic food-
relevant bacteria,
particularly to a method for the simultaneous specific detection of bacteria
of the genus
Listeria and the species Listeria monocytogenes by in situ-hybridization as
well as to a
method for the specific detection of bacteria of the species Staphylococcus
aureus by in situ-
hybridization as well as to a method for the specific detection of bacteria of
the genus
Campylobacter and the species C. coli and C. jejuni by in situ-hybridization
as well as the
corresponding oligonucleotide probes and kits, with which the inventive
methods may be
carried out.
Listeria are gram-positive short motile rods. Six species belong to the genus
Listeria (L.):
L. grayi, L, innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L.
welshimeri. The
worldwide distribution of these ubiquitous bacteria extends to both aquatic
areas as well as to
soil and vegetation.
Listeria gain special medicinal importance because of an infectious disease
known as
Listeriosis caused in humans as well as in domestic and wild animals. In
humans the
listeriosis, which has a highly variable incubation period from a few days up
to two months, is
caused by the species L. monocytogenes, but in some diseases also L. ivanovii,
L. seeligeri
and L. welshimeri were detected. A listeria infection can manifest itself in
severe diseases
such as sepsis, meningitis or encephalitis. Especially in newborn infants, who
can be infected
via the placenta or during delivery, as well as in elderly people listeriosis
carries a high health
risk. The fatality rate in the case of newborn listeriosis is up to 50%. An
infection prior to
birth can result in the fetus being aborted. The occurrence of a listeriosis
in elderly or
otherwise immuno-compromised people can be fatal in up to 30% of those
infected.
Transmission usually occurs from consuming contaminated foodstuffs. Especially
milk
products are a frequent source of infection. But also nearly all other
foodstuffs are potential
sources of listeria infections. Besides milk and various milk products such as
cheese, butter
or ice-cream, also other foodstuffs were identified as a source of listeriosis
in the past. These
include such diverse products as coleslaw, mussels, pork, chicken, fish,
cornmeal or rice
salad. In many cases outbreaks of listeriosis caused by consumption of the
mentioned
foodstuffs have proved fatal.
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Of particular importance in this connection is the fact that listeria are able
to multiply also at
4°C (in milk even at -0,3°C). This means that despite cool
storage of foodstuffs, listeria can
multiply and accumulate in the foodstuffs. Even after cooking, roasting or
smoking listeria
may accumulate in the relevant foodstuffs as a consequence of insufficient
treatment or a
secondary contamination.
Therefore a continuous monitoring of foodstuffs for the occurrence of listeria
is an important
part of both quality assurance in manufacturing companies as well as the daily
routine in
hygiene institutes.
The classic method for the detection of L. monocytogenes is very time-
consuming. In this
case, first an enrichment in a selective liquid medium, the so-called '/2
Fraser bouillon is
carned out 30°C for 24 hours. This is followed by a second enrichment
step, now in Fraser
bouillon, at 37°C for 48 hours. Both enrichments are then plated on
selective agar media
(Oxford-Agar and PALCAM-Agar) and these are incubated at 30°C or
37°C for 24 hours to
48 hours. To confirm that the colonies grown in this way are Listeria or L.
monocytogenes
further sub-cultivations are made (on Trypton Soya yeast agar or sheep's blood
agar) for a
period of at least 24 hours, at most five days. The overall period of the
classic detection
method is therefore five to ten days.
Staphylococcus intoxications belong to the worldwide most prevalent diseases
which are
caused by bacteria and transmitted by foodstuffs. These are especially caused
by strains of
Staphylococcus (S.) aureus. S. aureus is a gram-positive, immotile, coagulase-
positive
bacterium and occurs on the skin, the mucosa of the nasopharynx, in stool,
faeces, abscesses
and pustules. S. aureus is also widespread among the healthy population. S.
aureus can be
detected in the nasopharynx of half of all healthy people.
Food poisoning as a consequence of an infection with S. aureus is caused by
enterotoxins
produced by these bacteria in foodstuffs and is characterized by vomiting and
diarrhea.
Enterotoxin A has the strongest effect with an emetic dose of below 1 fig.
Even 0.1-0.2 ~m
Enterotoxin lead to food poisoning. Toxin F also deserves special mention,
which leads to a
shock syndrome and is therefore also called "Toxic Shock Syndrome Toxin" (TSST-
1).
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Characteristics of the shock syndrome caused by Toxin F are pulmonary edema,
endothelial
cell degenerations, renal failure and shock.
Transmission of S. aureus usually also occurs through the consumption of
contaminated
foodstuffs, the spectrum of potential sources of infection being quite wide.
The following
foodstuffs were involved among others in incidences of the disease: pre-cooked
convenience
foods containing meat, pies, ham, gammon, milk and milk products, egg-
containing dishes,
salads, creams, cake fillings, ice cream, pasta.
Routine detection nowadays is performed mostly by cultivation and confirmation
testing of
suspect colonies, because enterotoxin detection is quite complicated to
perform. For the
detection by cultivation the sample to be examined is first incubated for 48
hours on a suitable
selective medium (e.g. Baird) at 37°C. If this first cultivation step
was performed in liquid
medium, a second one (again for 48 hours) follows on a solid medium (e.g.
Baird-Parker). In
the next step the suspect colonies are tested for the presence of coagulase.
For this, two
different methods are available. Usually first the so-called "tube test for
the presence of
clotting factor" is performed, which takes about six to eight hours. If this
test is negative, the
result has to be confirmed by the so-called "tube test using rabbit plasma".
This test takes up
to 24 hours. The overall period of the classic detection method is therefore
between 54 hours
and 5 days.
It has only been in the last 20-odd years that a previously underestimated
germ has been
playing a bigger role as food poisoner, namely Campylobacter. In contrast to,
for instance,
salmonella, it rarely propagates in food, however, for an infection with this
pathogen even a
few hundred bacterial cells are sufficient.
The genus Campylobacter (C.) comprises 20 species and sub-species. These
bacteria, which
have up to now been difficult to cultivate, are gram-negative, slender, curved
to spirally
curved rods, which require microaerophilic conditions for their growth.
Medically relevant are the species C. jejuni, C. coli and C. laris. They
populate the small
intestine and the colon and cause an acute gastroenteritis accompanied by the
following
symptoms: diarrhea, abdominal pain, fever, nausea, vomiting. These symptoms
are very
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difficult to distinguish from those of a gastric ulcer. A careful differential
diagnosis is thus
essential.
Presently the routine detection is performed via a multi-stage cultivation,
beginning with an
18-hour enrichment in selective liquid medium (Campylobacter selective medium
according
to Preston), followed by two periods of 48 hours each on two different solid
media (Karmali
agar, followed by Columbia blood agar). These five-day cultivations are
followed by the
biochemical or serological identification.
As a logical consequence of the difficulties, especially the lengthiness,
presented by the
above-mentioned methods for the detection of Listeria, S. aureus and
Campylobacter,
detection methods on the basis of nucleic acids seem to present an obvious
solution.
In PCR, polymerise chain reaction, a characteristic piece of the respective
bacterial genome is
amplified with specific primers. If the primer finds its target site, a
million-fold amplification
of a piece of the inherited material occurs. Upon the following analysis, for
example by an
agarose gel separating DNA fragments, a qualitative evaluation can take place.
In the most
simple case this leads to the conclusion that target sites for the primers
used were present in
the tested sample. Further conclusions are not possible; these target sites
can originate from
both a living bacterium and a dead bacterium or from naked DNA.
Differentiation is not
possible with this method. This often leads to false positive results, since
the PCR reaction is
positive also in the presence of a dead bacterium or naked DNA. 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 amplified DNA. 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.
A unique approach to combine the specificity of molecular biological methods
such as PCR
with the possibility of the visualization of bacteria, which is facilitated by
the antibody
methods, is the method of fluorescence in situ hybridization (FISH; R.I.
Amann, W. Ludwig
and K.-H. Schleifer, 1995. Phylogenetic identification and in situ detection
of individual
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microbial cells without cultivation. Microbiol. Rev. 59, p. 143-169). Using
this method
bacteria species, genera or groups can be identified and visualized with high
specificity.
The FISH technique is based on the fact that in bacteria 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 16S 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. S1, p. 221-
271). Based
on a comparative sequence analysis, phylogenetic relationships can be
established based on
these data alone. For this purpose, the sequence data have to be brought into
an alignment. In
the alignment, which is based on the knowledge about the secondary structure
and tertiary
structure of these macromolecules, the homologous positions of the ribosomal
nucleic acids
are brought into line with each other.
Based on these data, phylogenetic calculations can be made. The use of the
most modern
computer technology makes it possible to make even large-scale calculations
fast and
effectively, as well as to set up large databases which contain the alignment
sequences of the
16S rRNA and 23S rRNA. Because of the fast access to this data material, newly
acquired
sequences can be phylogenetically analyzed within a short time. These rRNA
databases can
be used to construct species-specific and genus-specific gene probes. Here all
available
rRNA sequences are compared with each other and probes are designed for
specific sequence
sites, which probes cover a specific species, genus or group of bacteria.
In the FISH (fluorescence in situ hybridization) technique, these gene probes,
which are
complementary to a certain region on the ribosomal target sequence, are
brought into the cell.
The gene probes are generally small, 16-20 bases long, single-stranded
desoxyribonucleic
acid pieces and are directed against a target region which is typical for a
bacterial species or a
bacterial group. If a fluorescence labeled gene probe fords its target
sequence in a bacterial
cell, it binds to it and the cells can be detected in the fluorescence
microscope because of their
fluorescence.
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The FISH analysis is always performed on a slide, because for the evaluation
the bacteria are
visualized by irradiation with a high-energy light. But herein lies one of the
disadvantages of
the classical FISH analysis: because naturally only relatively small volumina
can be analyzed
on the slide, the sensitivity of the method may be unsatisfactory and not
sufficient for a
reliable analysis. The present invention thus combines the advantages of the
classical FISH
analysis with those of cultivation. A comparatively short cultivation step
ensures that the
bacteria to be detected are present in sufficient number before the bacteria
are detected using
specific FISH.
Realization of the methods described in the present application for the
simultaneous and
specific detection of bacteria of the genus Listeria as well as the species L.
monocytogenes or
for the specific detection of bacteria of the species S. aureus or for the
simultaneous and
specific detection of bacteria of the genus Campylobacter as well as the
species C. coli and C.
jejuni therefore comprises the following steps:
- cultivating the bacteria present in the sample to be tested
- fixing the bacteria present in the sample
- incubating the fixed bacteria with nucleic acid probe molecules, in order to
achieve
hybridization,
- removing or washing off the non-hybridized nucleic acid probe molecules and
- detecting the bacteria hybridized with the nucleic acid probe molecules.
Within the scope of the present invention "cultivating" is understood to mean
the propagation
of the bacteria present in the sample in a suitable cultivation medium. For
the detection of
Listeria the cultivation may occur, for example, in %i Fraser bouillon for 24
hours at 30°C.
For the detection of S. aureus the cultivation may occur, for example, as
blood culture (e.g.
BACTEC 9240, Becton Dickinson Instruments) for 8 hours to 48 hours at
35°C. For the
detection of Campylobacter the cultivation may occur, for example, in
selective medium
according to Preston for 24 hours at 42°C. In any case, the expert can
find suitable cultivation
methods in the prior art.
Within the scope of the present invention "fixing" of the bacteria 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
CA 02474957 2004-07-30
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,
low percentage paraformaldehyde solution or a diluted formaldehyde solution,
enzymatic
treatments or the like.
Within the scope of the present invention the fixed bacteria are incubated
with fluorescence
labeled nucleic acid probes for the "hybridization". These nucleic acid
probes, 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 in the cell.
Binding is to be
understood as formation of hydrogen bonds between complementary nucleic acid
pieces.
The nucleic acid probe 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 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 a target site, since the ribosomes as sites of protein
biosynthesis are
present many thousandfold in each active cell.
The nucleic acid probe within the meaning of the invention may be a DNA or RNA
probe
comprising usually between 12 and 1,000 nucleotides, preferably between 12 and
500, more
preferably between 12 and 200, especially preferably between 12 and SO and
between 15 and
40, and most preferably between 17 and 25 nucleotides. The selection of the
nucleic acid
probes is done according to criteria of whether a complementary sequence is
present in the
microorganism to be detected. By selecting a defined sequence, a bacterial
species, a
bacterial genus or an entire bacterial group may be detected. In a probe
consisting of 15
nucleotides, the sequences should be 100% complementary. In oligonucleotides
of more than
15 nucleotides, one or more mismatches are allowed.
Within the scope of the methods according to the invention for the
simultaneous specific
detection of bacteria of the genus Listeria and the species L. monocytogenes
the nucleic acid
probe molecules of the present invention have the following lengths and
sequences:
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SEQ ID No. 5'- ggc ttg cac cgg
1: cag tca ct
SEQ ID No. 5'- cgg ctt aca ccg
2: gca gtc act
SEQ ID No. 5'- ccc ttt gta cta
3: tcc att gta
SEQ ID No. 5'- ccc ttt gta cca
4: tcc att gta
SEQ ID No. 5'- ccc ttt gta tta
5: tcc att gta g
SEQ ID No. 5'- ccc ttt gta ctg
6: tcc att gta
For example, the detection method for Listeria and L. monocytogenes is
performed as
follows: the oligonucleotide SEQ ID No. 1 is specifically labeled, for example
with a green
fluorescent dye, and serves for the specific detection of all bacteria of the
genus Listeria. The
oligonucleotide SEQ ID No. 2 remains unlabeled and inhibits as competitor the
binding of the
labeled oligonucleotide SEQ ID No. 1 to bacteria which do not belong to the
genus Listeria.
The oligonucleotide of the SEQ ID No. 3 is also labeled specifically, but
differently from the
oligonucleotide SEQ ID No. 1, for example with a red fluorescent dye, and
serves for the
specific detection of all bacteria of the species Listeria monocytogenes. The
oligonucleotides
SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 again remain unlabeled and inhibit
as
competitors the binding of the labeled oligonucleotide SEQ ID No. 3 to
bacteria which do not
belong to the species L. monocytogenes. In this way, the simultaneous and
highly specific
detection of bacteria belonging to the genus Listeria or to the species L.
monocytogenes,
respectively, is possible. The different markers, e.g. a green fluorescent dye
on the one hand
and a red fluorescent dye on the other hand, are easy to distinguish from each
other, e.g. by
using different filters in the fluorescence microscopy.
Within the scope of the method of the present invention for the specific
detection of bacteria
of the species S. aureus, the nucleic acid probe molecules of the present
invention have the
following lengths and sequences:
SEQ ID No. 7: 5'- GAA GCA AGC TTC TCG TCC G
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SEQ ID No. 5'- GGA GCA AGC TCC TCG TCC
8: G
SEQ ID No. 5'- GAA GCA AGC TTC TCG TCA
9: TT
SEQ ID No. 5'- CTA ATG CAG CGC GGA TCC
10:
SEQ ID No. 5'- CTA ATG CAC CGC GGA TCC
11:
SEQ ID No. 5'- CTA ATG CGG CGC GGA TCC
12:
SEQ ID No. 5'- CTA ATG CAG CGC GGG TCC
13:
For example, the detection method for S. aureus takes place as follows: The
oligonucleotides
SEQ ID No. 7 and SEQ ID No. 10 are labeled specifically, for example with a
red fluorescent
dye, and serve for the specific detection of all bacteria of the species
Staphylococcus aureus.
The oligonucleotides SEQ ID No. 8 and 9 as well as SEQ ID No. 11, 12 and 13
remain
unlabeled and inhibit as competitors the binding of the labeled
oligonucleotides to bacteria
which do not belong to the species S. aureus. In this way, highly specific
detection of
bacteria belonging to the species S. aureus is possible.
In a preferred embodiment the intensity of the signals obtained may be
enhanced by using so-
called "helper probes". These helper probes are unlabeled oligonucleotides
having the
following sequence:
SEQ ID No. 14: TCG CTC GAC TTG CAT GTA TTA GGC A
SEQ ID No. 15: ACC CGT CCG CCG CTA ACA TCA G
The use of the helper probes is not necessary but optional. The helper probes
facilitate the
binding of the labeled probes to their target sites and thus enhance the
signal intensity. The
detection method however also functions very well without these helper probes.
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Within the scope of the methods according to the present invention for the
simultaneous
specific detection of bacteria of the genus Campylobacter and the species C.
coli and
C. jejuni, the nucleic acid probe molecules of the present invention have the
following lengths
and sequences:
SEQ ID No. 16 S' CTG CCT CTC CCT CAC TCT AG
SEQ ID No. 17 S' CTG CCT CTC CCT TAC TCT AG
SEQ ID No. 18 5' CTG CCT CTC CCC TAC TCT AG
SEQ ID No. 19 5' CTG CCT CTC CCC CAC TCT AG
SEQ ID No. 20 5' CCT ACC TCT CCC ATA CTC TAG A
SEQ ID No. 21 S' CCA TCC TCT CCC ATA CTC TAG C
SEQ ID No. 22 5' CCT ACC TCT CCA GTA CTC TAG T
SEQ ID No. 23 5' CCT GCC TCT CCC ACA CTC TAG A
SEQ ID No. 24 5' CGC TCC GAA AAG TGT CAT CCT C
SEQ ID No. 25 5' CTA AAT ACG TGG GTT GCG
SEQ ID No. 26 5' CTA AAC ACG TGG GTT GCG
SEQ ID No. 27 5' AGC AGA TCG CCT TCG CAA T
SEQ ID No. 28 5' AGC AGA TCG CTT TCG CAA T
SEQ ID No. 29 S' AGT AGA TCG CCT TCG CAA T
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SEQ ID No. 30 5' TCG AGT GAA ATC AAC TCC
C
SEQ ID No. 31 5' TCG GGT GAA ATC AAC TCC
C
SEQ ID No. 32 5' CGT AGC ATG GCT GAT CTA
C
SEQ B7 No. 33 5' CGT AGC ATA GCT GAT CTA
C
SEQ ID No. 34 5' CGT AGC ATT GCT GAT CTA
C
SEQ ID No. 35 5' GCC CTG ACT AGC AGA GCA
A
SEQ ID No. 36 5' TTC TTG GTG ATC TCT ACG
G
SEQ ID No. 37 5' TTC CTG GTG ATC TCT ACG
G
SEQ ID No. 38 5' TTC TTG GTG ATA TCT ACG
G
SEQ ID No. 39 5' TTG AGT TCT AGC AGA TCG
C
SEQ ID No. 40 5' TTG AGT TCC AGC AGA TCG
C
SEQ ID No. 41 5' TTG AGT TCT AGC AGA TAG
C
SEQ ID No. 42 5' TTG AGT TCC AGC AGA TAG
C
SEQ ID No. 43 5' CGC GCC TTA GCG TCA GTT
GAG
SEQ ID No. 44 S' CAC GCC TTA GCG TCA GTT
GAG
SEQ ID No. 45 5' CGC GCC TTA GCG TCA GTT
AAG
SEQ ID No. 46 5' CAC GCA TTA GCG TCA GTT
GAG
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SEQ >D No. 47 5' CGA GCA TTA GCG TCA GTT GAG
SEQ ID NO. 48 5' TAC ACT AGT TGT TGG GGT GG
SEQ ID NO. 49 5' TTC GCG CCT CAG CGT CAG TTA CAG
The detection method for the genus Campylobacter or the species C. coli and C.
jejuni,
respectively, is performed as follows: The oligonucleotides SEQ ID No. 16 to
SEQ ID No. 19
as well as the oligonucleotides SEQ >D No. 24 to SEQ ID No. 28 as well as the
oligonucleotide SEQ ID No. 30 as well as the oligonucleotides SEQ ID No. 32 to
34 are
specifically labeled, for example with a green fluorescent dye, and serve for
the specific
detection of all bacteria of the genus Campylobacter. The oligonucleotides SEQ
ID No. 20 to
23 as well as the oligonucleotide SEQ ID No. 29 as well as the oligonucleotide
SEQ >D No.
31 remain unlabeled and inhibit as competitors the binding of the
aforementioned labeled
oligonucleotides which are specific for the genus Campylobacter to bacteria
which do not
belong to the genus Campylobacter.
The oligonucleotides SEQ ID No. 35 and 36 as well as the oligonucleotide SEQ
>D No. 39 are
also specifically labeled, but differently from the oligonucleotides SEQ ID
No. 16 to 19, 24 to
28, 30 as well as 32 to 34, i.e. distinguishably labeled from them, e.g. with
a blue fluorescent
dye, and serve for the specific detection of all bacteria of the species
Campylobacter coli. The
oligonucleotides SEQ ID No. 37, 38 as well as 40 to 42 again remain unlabeled
and inhibit as
competitors the binding of the labeled oligonucleotides specific for C. coli
to bacteria which
do not belong to the species C. coli.
The oligonucleotides SEQ ID No. 43 and 48 are also specifically labeled, but
again differently
from the aforementioned oligonucleotides, i.e. again distinguishably labeled
from them, e.g.
with a red fluorescent dye, and serve for the specific detection of all
bacteria of the species
Campylobacter jejuni. The oligonucleotides SEQ ID No. 44 to 47 and 49 again
remain
unlabeled and inhibit as competitors the binding of the labeled
oligonucleotides specific for C.
jejuni to bacteria which do not belong to the species C. jejuni.
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In this way, the simultaneous and highly specific detection of bacteria
belonging to the genus
Campylobacter or to the species C. coli or C. jejuni, respectively, is
possible.
The intensity of the signals obtained may optionally be enhanced by using so-
called helper
probes. The helper probes are also unlabeled, but facilitate the binding of
the labeled to their
target sites and thus enhance the signal intensity. This is just an
enhancement of the signal
intensity, the detection method of course also functions without these helper
probes.
In this way, the intensity of the signals obtained with the oligonucleotide
SEQ ID No. 24 may
be enhanced by using the unlabeled oligonucleotides mentioned below as helper
probes:
SEQ ID No. 50: 5' CAC GCG GCG TTG CTG CTG/T C
SEQ ID No. 51: 5' TCT TTT [C/T]CC [A/C/T][G/A]A [A/C/T]AA AAG GAG
TTA CG
Within the scope of the present invention, competitors are understood to mean
in particular
oligonucleotides which have a higher specificity for genera or species not to
be detected than
the labeled oligonucleotides which are specific for the genera or species to
be detected.
A further object of the invention are modifications of the above
oligonucleotide sequences,
demonstrating specific hybridization with target nucleic acid sequences of the
respective
bacterium despite variations in sequence and/or length, and which are
therefore suitable for
use in a method according to the invention. These especially include:
a) nucleic acid molecules (i) being identical to one of the above
oligonucleotide
sequences (SEQ ID No. 1 to SEQ ID No. S 1 ) to at least 60%, 65%, preferably
to at
least 70%, 75%, more preferably to at least 80%, 84%, 87% and particularly
preferred
to at least 90%, 94%, 96% of the bases (wherein the sequence region of the
nucleic
acid molecule is to be considered which corresponds to the sequence region of
one of
the above oligonucleotides (SEQ ID No. l to SEQ ID No. 51 ) and not the entire
sequence of a nucleic acid molecule, which possibly may be extended by one or
multiple bases compared to the above-mentioned oligonucleotides (SEQ ID No. 1
to
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SEQ ID No. 51), or (ii) differs from the above oligonucleotide sequences (SEQ
ID No.
1 to SEQ ID No. 51) by one or more deletions and/or additions and which render
possible a specific hybridization with nucleic acid sequences of bacteria of
the genus
Listeria and the species L. monocytogenes, of bacteria of the species S.
aureus or of
bacteria of the genus Campylobacter and the species C. coli and C. jejuni. In
this
context "specific hybridization" means that under the hybridization conditions
described here or those known to the person skilled in the art in relation to
in situ
hybridization techniques, only the ribosomal RNA of the target organisms binds
to the
oligonucleotide, but not the rRNA of non-target organisms.
b) Nucleic acid molecules which specifically hybridize to a sequence
complementary to
the nucleic acid molecules mentioned in a) or to one of the probes SEQ ID No.
1 to
SEQ ID No. 51 under stringent conditions.
c) Nucleic acid molecules comprising an oligonucleotide sequence of SEQ ID No.
1 to
SEQ ID No. 51 or the sequence of a nucleic acid molecule according to a) or b)
and
having at least one further nucleotide in addition to the mentioned sequences
or their
modifications according to a) or b) and allowing specific hybridization with
nucleic
acid sequences of target organisms.
The degree of sequence identity of a nucleic acid molecule to the probes SEQ
ID No. 1 to
SEQ ID No. 51 can be determined using the usual algorithms. In this respect,
for example,
the program for determining the sequence identity available under
http://www.ncbi.nlm.nih.gov/BLAST (on this page for example the link "Standard
nucleotide-nucleotide BLAST [blastn]") is suitable.
In the case of the detection of bacteria of the genus Listeria or the species
L. monocytogenes
the specific oligonucleotide probes preferably correspond to oligonucleotides
SEQ ID No. 1
or SEQ ID No. 3. But also modifications are possible, as long as there is
still specific
hybridization between probe and target sequence. It can be sufficient that the
oligonucleotide
probe used is identical in 15, preferably 16 and 17 and particularly preferred
18 and 19
successive nucleotides to SEQ ID No. 1 or SEQ ID No. 3. The same is true for
the
oligonucleotides serving as competitors with respect to the sequences SEQ ID
No. 2, 4, 5 and
6.
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The same is true for the detection of S. aureus. In this case, the specific
oligonucleotide
probes preferably have a sequence which is identical to the one of SEQ ID No.
7 or SEQ ID
No. 10 in 13 and 14 and preferably 15, 16 or 17 successive nucleotides. The
same is true for
the oligonucleotides serving as competitors with respect to the sequences SEQ
ID No. 8, 9
and 11 to 13.
The same is true for the detection of bacteria of the genus Campylobacter and
the species
Campylobacter coli and Campylobacter jejuni. Also in this case the specific
oligonucleotide
probes preferably have a sequence which is identical to SEQ ID No. 16 to 19,
24 to 28, 30, 32
to 36, 39, 43 and 48 in 13 or 14, preferably 15 or 16 and particularly
preferred 17 or 18
successive nucleotides. The same is true for the oligonucleotides serving as
competitors with
respect to the sequences SEQ ID No. 20 to 23, 29, 31, 37, 38, 40 to 42, 44 to
47 and 49.
The nucleic acid probe molecules according to the invention may be used within
the scope of
the detection method with various hybridization solutions. Various organic
solvents may be
used in concentrations of 0-80%. By keeping stringent hybridization
conditions, it is
guaranteed that the nucleic acid probe molecule indeed hybridizes to the
target sequence.
Moderate conditions within the meaning of the invention are e.g. 0% formamide
in a
hybridization buffer as described below. Stringent conditions within the
meaning of the
invention are for example 20-80% formamide in the hybridization buffer.
Within the scope of the method according to the invention for simultaneous
specific detection
of bacteria of the genus Listeria and the species L. monocytogenes a typical
hybridization
solution contains 0%-80% formamide, preferably 20%-60% formamide, especially
preferred
40% formamide. In addition, it has a salt concentration of 0.1 mol/1- 1.5
mol/1, preferably of
0.5 mol/1- 1.0 moll, more preferred of 0.7 mol/1- 0.9 mol/1 and especially
preferred of 0.9
mol/1, the salt preferably being sodium chloride. Further, the hybridization
solution usually
comprises a detergent, such as for instance sodium dodecyl sulfate (SDS) in a
concentration
of 0.001% - 0.2%, preferably in a concentration of 0.005 - 0.05%, more
preferred of 0.01 -
0.03%, especially preferred in a concentration of 0.01%. For buffering of the
hybridization
solution, various compounds such as Tris-HC1, sodium citrate, PIPES or HEPES
may be used,
which are usually used in concentrations of 0.01 - 0.1 mol/1, preferably of
0.01 to 0.05 moll,
CA 02474957 2004-07-30
-16-
in a pH range of 6.0 - 9.0, preferably 7.0 to 8Ø The particularly preferred
inventive
embodiment of the hybridization solution contains 0.02 mol/1 Tris-HCI, pH 8Ø
Within the scope of the method according to the invention for the specific
detection of
bacteria of the species S. aureus, a typical hybridization solution contains
0% - 80%
formamide, preferably 20% - 60% formamide, particularly preferred 20%
formamide. In
addition it has a salt concentration of 0.1 mol/1- 1.5 mol/1, preferably of
0.7 mol/1 to 0.9
mol/1, particularly preferred of 0.9 mol/1, the salt preferably being sodium
chloride. Further,
the hybridization solution usually comprises a detergent, such as for example
sodium dodecyl
sulfate (SDS), in a concentration of 0.001% - 0.2%, preferably in a
concentration of 0.005 -
0.05%, more preferably 0.01 - 0.03%, especially preferred in a concentration
of 0.01%. For
buffering of the hybridization solution, various compounds such as Tris-HCI,
sodium citrate,
PIPES or HEPES may be used, which are usually used in concentrations of 0.01 -
0.1 mol/1,
preferably of 0.01 to 0.05 mol/1, in a pH range of 6.0 - 9.0, preferably 7.0
to 8Ø The
particularly preferred inventive embodiment of the hybridization solution
contains 0.02 mol/1
Tris-HC1, pH 8Ø
Within the scope of the method of the present invention for the specific
detection of bacteria
of the genus Campylobacter and the species C. coli and C. jejuni, a typical
hybridization
solution contains 0% - 80% formamide, preferably 20% - 60% formamide,
especially
preferred 20% formamide. In addition it has a salt concentration of 0.1 mol/1-
1.5 mol/1,
preferably of 0.7 mol/1- 0.9 mol/1, especially preferred of 0.9 mol/1, the
salt preferably being
sodium chloride. Further, the hybridization solution usually comprises a
detergent such as for
example sodium dodecyl sulfate (SDS), in a concentration of 0.001 - 0.2%,
preferably in a
concentration of 0.005 - 0.05%, more preferably 0.01 - 0.03%, especially
preferred in a
concentration of 0.01%. For buffering of the hybridization solution, various
compounds, such
as Tris-HCI, sodium citrate, PIPES or HEPES may be used, which are usually
used in
concentrations of 0.01 - 0.1 mol/1, preferably of 0.01 to 0.05 mol/1, in a pH
range of 6.0 - 9.0,
preferably 7.0 to 8Ø The particularly preferred inventive embodiment of the
hybridization
solutions contains 0.02 mol/1 Tris-HCI, pH 8Ø
It shall be understood that the expert can choose the given concentrations of
the constituents
of the hybridization buffer in such a way that the desired stringency of the
hybridization
CA 02474957 2004-07-30
-17-
reaction is achieved. Especially preferred embodiments reflect stringent to
particularly
stringent hybridization conditions. Using these stringent conditions the
expert can determine
whether a particular nucleic acid molecule enables the specific detection of
nucleic acid
sequences of target organisms and may thus be reliably used within the scope
of the
invention. The expert is able to increase or decrease the stringency by
variation of the
parameters of the hybridization buffer if needed or depending on the probe or
the target
organism.
The concentration of the nucleic acid probe in the hybridization buffer
depends on the kind of
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 in
fluorescence in
situ-hybridization (FISH) 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,
preferably between
1.0 and 100 ng/~1, and especially preferred between 1.0 - 50 ng/gl.
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 ~l hybridization solution. The
volume of the
hybridization solution used should be between 8 ~1 and 100 ml, in an
especially preferred
embodiment of the method of the present invention it is 30 pl.
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.
After hybridization the non-hybridized and excess nucleic acid probe molecules
should be
removed or washed off, which is usually achieved by a conventional washing
solution. This
washing solution may, if desired, contain 0.001-0.1%, preferably 0.005-0.05%,
especially
CA 02474957 2004-07-30
-18-
preferred 0.01%, of a detergent such as SDS, as well as Tris-HC1 in a
concentration of 0.001-
0.1 mol/1, preferably 0.01-0.05 mol/1, especially preferred 0.02 mol/1,
wherein the pH value of
Tris-HCl is within the range of 6.0 to 9.0, preferably of 7.0 to 8.0,
especially preferred 8Ø A
detergent may be contained, although this is not absolutely necessary.
Furthermore, the
washing solution usually contains NaCI, wherein the concentration is 0.003
mol/1 to 0.9 mol/1,
preferably 0.01 mol/1 to 0.9 mol/1, depending on the stringency required. An
NaCI
concentration of 0.07 mol/1 (method for the simultaneous specific detection of
bacteria of the
genus Listeria and the species L. monocytogenes) or 0.215 mol/1 (method for
the specific
detection of bacteria of the species S. aureus) or 0.215 mol/1 (method for the
simultaneous
specific detection of bacteria of the genus Campylobacter and of the species
C. coli and C.
jejuni) is especially preferred. Moreover, the washing solution may contain
EDTA, wherein
the concentration is preferably 0.005 moll. The washing solution may further
contain
suitable amounts of preservatives known to the expert.
Generally, buffer solutions are used in the washing step, which can in
principle be very
similar to the hybridization buffer (buffered sodium chloride solution),
except that the
washing step is usually performed in a buffer with a lower salt concentration
or at a higher
temperature. For theoretical estimation of the hybridization conditions, the
following formula
may be used:
Td = 81.5 + 16.6 lg[Na+] + 0.4 x (% GC) - 820/n - 0.5 X (%FA)
Td = dissociation temperature in °C
[Na+] = molarity of the sodium ions
GC = percentage of guanine and cytosine nucleotides relative to the total
number of bases
n = hybrid length
FA = percentage of formamide
Using this formula, the formamide content (which should be as low as possible
due to the
toxicity of the formamide) of the washing buffer may for example be replaced
by a
correspondingly lower sodium chloride content. However, the person skilled in
the art knows
from the extensive literature concerning in situ hybridization methods the
fact that, and in
CA 02474957 2004-07-30
-19-
which way, the mentioned contents can be varied. Concerning the stringency of
the
hybridization conditions, the same applies as outlined above for the
hybridization buffer.
The "washing off ' of the non-bound nucleic acid probe molecules is usually
performed 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 to 40 minutes, preferably for 15 minutes.
In an alternative embodiment of the method according to the invention, the
nucleic acid
molecules according to the invention are used in the so-called Fast-FISH
method for the
specific detection of the mentioned target organisms. The Fast-FISH method is
known to the
expert and is, for example, described in the applications DE 199 36 875 and WO
99/18234.
Reference is herewith expressly made to the disclosure contained in these
documents
regarding the performance of the detection methods described therein.
The specifically hybridized nucleic acid probe molecules can then be 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:
CA 02474957 2004-07-30
-20-
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 H202 + diammonium benzidine
H202 + tetramethylbenzidine
4. (3-D-galactosidase o-nitrophenyl-/3-D-galactopyranoside, 4-
methylumbelliferyl-(3-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 02474957 2004-07-30
-21-
The final evaluation depends on the kind of labelling of the probe used and is
possible with an
optical microscope, epifluorescence microscope, chemoluminometer, fluorometer,
etc.
An important advantage of the methods described in this application for the
simultaneous
specific detection of bacteria of the genus Listeria and the species L.
monocytogenes or for
the specific detection of bacteria of the species S. aureus, or for the
specific detection of
bacteria of the genus Campylobacter and the species C. coli and C. jejuni
compared to the
detection methods described above is the exceptional speed. In comparison to
conventional
cultivation methods which need up to 10 days, the result is obtained within 24
to 48 hours
when the methods according to the invention are used.
Another advantage is the simultaneous detection of bacteria of the genus
Listeria and the
species L. monocytogenes. With the methods common up to now only bacteria of
the species
L. monocytogenes are detected more or less reliably. Epidemiological
investigations have
however shown that besides L. monocytogenes also other species of the genus
Listeria can
cause the dangerous listeriosis. According to the information presently
available, the
detection of L. monocytogenes alone is thus not sufficient.
Another advantage is the possibility to discriminate between bacteria of the
genus Listeria
and those of the species L. monocytogenes. This is easily and reliably
possible by using
different labels for the nucleic acid probe molecules specific for the
corresponding genus or
species.
Another advantage is the specificity of these methods. With the nucleic acid
probe molecules
used, both all species of the genus Listeria, and only the species L.
monocytogenes can be
specifically detected and visualized. Equally reliably, the species S. aureus
and all species of
the genus Campylobacter, but also only the species C. coli or C. jejuni are
detected with high
specificity.
Another advantage is the possibility to discriminate between bacteria of the
genus
Campylobacter and those of the species C. coli or C. jejuni. This is possible
easily and
CA 02474957 2004-07-30
-22-
reliably by using different labels for the nucleic acid probe molecules
specific for the
corresponding genus or species.
By visualization of the bacteria a visual control may be performed at the same
time. False-
positive results, such as the ones often occurring in polymerase chain
reactions, are therefore
ruled out.
A further advantage of the methods according to the invention is their ease of
use. For
example, using this methods, large amounts of samples can easily be tested for
the presence
of the mentioned bacteria.
The methods according to the invention may be used in various ways.
For example, food samples (e.g. poultry, fresh meat, milk, cheese, vegetables,
fruit, fish, etc.)
may be tested for the presence of the bacteria to be detected.
For example, also environmental samples may be tested for the presence of
bacteria to be
detected. These probes may be, for example, collected from soil or be parts of
plants.
The method according to the invention may further be used for testing of
sewage samples or
silage samples.
The method according to the invention may further be used for testing
medicinal samples, e.g.
stool samples, blood cultures, sputum, tissue samples (also cuts), wound
material, urine,
samples from the respiratory tract, implants and catheter surfaces.
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.
CA 02474957 2004-07-30
-23-
A further field of application of the method according to the invention is the
analysis of
pharmaceutical and cosmetic products, e.g. ointments, creams, tinctures,
juices, solutions,
drops, etc.
Furthermore, according to the invention, kits for performing the respective
methods are
provided. The hybridization arrangement contained in these kits is described
for example in
German patent application 100 61 655Ø Express reference is herewith made to
the
disclosure contained in this document with respect to the in situ
hybridization arrangement.
Besides the described hybridization arrangement (referred to as VIT reactor),
the most
important component of the kits is the respective hybridization solution
(referred to as VIT
solution) with the nucleic acid probe molecules specific for the
microorganisms to be
detected, which are described above (VIT solution). Further contained are the
respective
hybridization buffer (Solution C) and a concentrate of the respective washing
solution
(Solution D). Also contained are optionally fixation solutions (Solution A
(50% ethanol) and
Solution B (absolute ethanol)) as well as optionally an embedding solution
(finisher).
Finishers are commercially available, they prevent, among other things, the
rapid bleaching of
fluorescent probes under the fluorescence microscope. Optionally, solutions
for parallel
carrying out of a positive control as well as of a negative control are
contained.
The following example is intended to illustrate the invention without limiting
it. The buffers
and solutions used have the compositions given above.
Example
Specific rapid detection of pathogenic food-relevant bacteria in a sample.
A sample is cultivated for 20 to 44 hours in a suitable manner. For the
detection of Listeria
cultivation may be performed for example in %2 Fraser bouillon for 24 hours at
30°C. For the
detection of S. aureus the cultivation may be performed for example as blood
culture (e.g.
BACTEC 9240, Becton Dickinson Instruments) for 8 hours to 48 hours at
35°C. For the
detection of Campylobacter the cultivation may be performed, for example, in
selective
medium according to Preston for 24 hours at 42°C.
CA 02474957 2004-07-30
-24-
To an aliquot of the culture the same volume of fixation solution (Solution B)
is added.
Alternatively, an aliquot of the culture may be centrifuged (4000 g, S min,
room temperature)
and, after discarding the supernatant, the pellet may be dissolved in 4 drops
of fixation
solution.
For performing the hybridization a suitable aliquot of the fixed cells
(preferably 40 pl) is
applied onto a slide and dried (46°C, 30 min, or until completely dry).
Alternatively, the cells
may also be applied to other Garner materials (e.g. a microtiter plate or a
filter). The dried
cells are then completely dehydrated by again adding the fixation solution
(Solution B,
preferably 40 ~l). The slide is again dried (room temperature, 3 min, or until
completely dry).
Then the hybridization solution (VIT solution) containing the above described
nucleic acid
probe molecules specific for the microorganisms to be detected is applied to
the fixed,
dehydrated cells. The preferred volume is 40 ~l. The slide is then incubated
in a chamber
humidified with hybridization buffer (Solution C, corresponding to the
hybridization solution
without probe molecules), preferably the VIT reactor (46°C, 90 min).
Then the slide is removed from the chamber, the chamber is filled with washing
solution
(Solution D, diluted 1:10 with distilled water) and the slide is incubated in
the chamber (46°C,
1 S min).
Then the chamber is filled with distilled water, the slide is briefly immersed
and then air-dried
in lateral position (46°C, 30 min or until completely dry).
Then the slide is embedded in a suitable medium (finisher).
Finally, the sample is analyzed with the help of a fluorescence microscope.
CA 02474957 2004-07-30
SEQUENCE LISTING
<110> Vermicon AG
<120> Method for specific rapid detection of pathogenic food-relevant bacteria
<130> 67761/00002
<140>
<141> 2003-02-04
<150> DE 102 04 447.3
<151> 2002-02-04
<160> 51
<170> PatentIn Ver. 2.1
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 1
ggcttgcacc ggcagtcact 20
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 2
cggcttacac cggcagtcac t 21
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 3
ccctttgtac tatccattgt a 21
21305004.1
CA 02474957 2004-07-30
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 4
ccctttgtac catccattgt a 21
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 5
ccctttgtat tatccattgt ag 22
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 6
ccctttgtac tgtccattgt a 21
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 7
gaagcaagct tctcgtccg lg
<210> 8
<211> 19
<212> DNA
<213> Artificial sequence
21305004.1
CA 02474957 2004-07-30
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 8
ggagcaagct cctcgtccg lg
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 9
gaagcaagct tctcgtcatt 20
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 10
ctaatgcagc gcggatcc lg
<210> 11
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 11
ctaatgcacc gcggatcc lg
<210> 12
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 12
21305004.1
CA 02474957 2004-07-30
ctaatgcggc gcggatcc lg
<210> 13
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 13
ctaatgcagc gcgggtcc lg
<210> 14
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 14
tcgctcgact tgcatgtatt aggca 25
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 15
acccgtccgc cgctaacatc ag 22
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 16
ctgcctctcc ctcactctag 20
<210> 17
<211> 20
21305004.1
CA 02474957 2004-07-30
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 17
ctgcctctcc cttactctag 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 18
ctgcctctcc cctactctag 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 19
ctgcctctcc cccactctag 20
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 20
cctacctctc ccatactcta ga 22
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
21305004.1
CA 02474957 2004-07-30
Oligonucleotide probe
<400> 21
ccatcctctc ccatactcta gc 22
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 22
cctacctctc cagtactcta gt 22
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 23
cctgcctctc ccacactcta ga 22
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 24
cgctccgaaa agtgtcatcc tc 22
<210> 25
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 25
ctaaatacgt gggttgcg lg
21305004.1
CA 02474957 2004-07-30
<210> 26
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 26
ctaaacacgt gggttgcg lg
<210> 27
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 27
agcagatcgc cttcgcaat lg
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 28
agcagatcgc tttcgcaat lg
<210> 29
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 29
agtagatcgc cttcgcaat 19
<210> 30
<211> 19
<212> DNA
<213> Artificial sequence
21305004.1
CA 02474957 2004-07-30
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 30
tcgagtgaaa tcaactccc lg
<210> 31
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 31
tcgggtgaaa tcaactccc lg
<210> 32
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 32
cgtagcatgg ctgatctac 19
<210> 33
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 33
cgtagcatag ctgatctac 19
<210> 34
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
21305004.1
CA 02474957 2004-07-30
<400> 34
cgtagcattg ctgatctac lg
<210> 35
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 35
gccctgacta gcagagcaa lg
<210> 36
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 36
ttcttggtga tctctacgg 19
<210> 37
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 37
ttcctggtga tctctacgg 19
<210> 38
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 38
ttcttggtga tatctacgg 19
<210> 39
21305004.1
CA 02474957 2004-07-30
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 39
ttgagttcta gcagatcgc lg
<210> 40
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 40
ttgagttcca gcagatcgc lg
<210> 41
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 41
ttgagttcta gcagatagc
19
<210> 42'
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 42
ttgagttcca gcagatagc
19
<210> 43
<211> 21
<212> DNA
<213> Artificial sequence
<220>
21305004.1
CA 02474957 2004-07-30
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 43
cgcgccttag cgtcagttga g 21
<210> 44
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 44
cacgccttag cgtcagttga g 21
<210> 45
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 45
cgcgccttag cgtcagttaa g 21
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 46
cacgcattag cgtcagttga g 21
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 47
cgagcattag cgtcagttga g 21
21305004.1
CA 02474957 2004-07-30
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 48
tacactagtt gttggggtgg 20
<210> 49
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 49
ttcgcgcctc agcgtcagtt acag 24
<210> 50
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 50
cacgcggcgt tgctgctkc 19
<210> 51
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence:
Oligonucleotide probe
<400> 51
tcttttycch rahaaaagga gttacg 26
21305004.1
