EP1472370A1

EP1472370A1 - Methods for specific rapid detection of pathogenic food-relevant bacteria

Info

Publication number: EP1472370A1
Application number: EP03704530A
Authority: EP
Inventors: Jiri Snaidr; Claudia Beimfohr
Original assignee: Vermicon AG
Current assignee: Vermicon AG
Priority date: 2002-02-04
Filing date: 2003-02-04
Publication date: 2004-11-03
Also published as: AU2003206830A1; WO2003066893A1; CA2474957A1; WO2003066893A8; JP2005516627A

Abstract

The invention relates to a method for the detection of pathogenic food-relevant bacteria, particularly to a method for simultaneous specific detection of bacteria of the genus Listeria and the species Listeria monocytogenes by in situ-hybridisation and to a method for specific detection of bacteria of the species Staphylococcus aureus by in situ hybridisation in addition to a method for simultaneous specific detection of bacteria of the genus Campylobacter and the species C. coli and/or C. jejuni by in situ- hybridisation. The invention also relates to corresponding oligonucleotide probes and kits with which the inventive methods can be carried out.

Description

Procedure for the specific rapid detection of pathogenic food-related

bacteria

The invention relates to a method for the detection of pathogenic food-relevant bacteria, in particular a method for the simultaneous specific detection of bacteria of the genus Listeria and the species Listeria monocytogenes by in situ hybridization and a method for the specific detection of bacteria of the species Staphylococcus aureus by in situ hybridization and 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 corresponding oligonucleotide probes and kits with which the methods according to the invention can be carried out.

Listeria are gram-positive, short, movable sticks. The genus Listeria (L.) includes six species: L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri. The worldwide distribution of these ubiquitous bacteria extends to aquatic areas as well as to the soil and vegetation.

Listeria are of particular medical importance due to the fectious disease caused in humans, domestic animals and wild animals, which is known as listeriosis. In humans, listeriosis, which has a very variable incubation period of a few days to two months, is caused by the species L. monocytogenes, although L. ivanovii, L. seeligeri and L. welshimeri have also been detected in some diseases. Listeria infection can manifest itself in serious clinical pictures such as sepsis, meningitis or encephalitis. Especially in newborns who can be infected via the placenta or during birth, and in the elderly, there is a high health risk in the case of listeriosis. The mortality rate in the case of newborn listeriosis is up to 50%. If the child is infected before birth, the fetus may also abort. The occurrence of listeriosis in elderly or otherwise defenseless people can be fatal in up to 30% of those affected. The transmission usually takes place through the consumption of contaminated food. Dairy products in particular are a common source of infection. But almost all other foods are potential sources of Listeria infections. So in the past, in addition to milk and various

Dairy products such as cheese, butter or ice cream also identify other foods as the source of listeriosis. These include various products such as coleslaw, mussels, pork, chicken, fish, corn flour or rice salad. In many cases, outbreaks of listeriosis caused by consumption of the foods mentioned have been accompanied by deaths.

It is particularly important in this context that Listeria are still capable of reproduction at 4 ° C (in milk even at -0.3 ° C). This means that despite refrigerated storage of food, listeria can multiply and accumulate in it. Even after cooking, roasting or smoking, listeria can accumulate in the food in question as a result of inadequate treatment or secondary contamination.

Therefore, a constant control of food for the occurrence of Listeria is an important part of the quality assurance in the manufacturing companies, as well as the daily routine in the hygiene institutes.

The classic method for the detection of L. monocytogenes is very time-consuming. This is initially carried out in a selective liquid medium, the so-called ^1A fraser broth, at 30 ° C. for 24 hours. A second enrichment step follows, now in Fraser broth, at 37 ° C for 48 h. Then both enrichments are plated on selective agar media (Oxford agar and PALCAM agar) and these are incubated at 30 ° C. or 37 ° C. for 24 h to 48 h. To confirm that the colonies grown in this way are Listeria or L. monocytogenes, further subcultures are carried out (on trypton Soy-yeast agar or on sheep's blood agar) for a period of at least 24 h, at most five days. The total duration of the classic verification procedure is five to ten days.

Staphylococcal intoxications are among the most common bacterial and foodborne diseases worldwide. These are triggered in particular by strains of Staphylococcus (S.) aureus. S. aureus is a gram-positive, immobile coagulase-positive bacterium and occurs on the skin, the mucous membrane of the nasopharynx, in stool, feces, abscesses and pustules. S. aureus is also widespread in the healthy population: Half of all healthy people have S. aureus in the nasopharynx.

Food poisoning as a result of infection with S. aureus is caused by the enterotoxins produced by these bacteria in the food and is characterized by vomiting and diarrhea. Enterotoxin A is most effective with an emetic dose below 1 μg. Already 0.1 - 0.2 μg enterotoxin lead to food poisoning. Also to be emphasized is the toxin F, which leads to a shock syndrome and is therefore also referred to as "Toxic Shock Syndrome Toxin" (TSST-1). Toxin F-induced shock syndrome is characterized by pulmonary edema, endothelial cell degeneration, kidney failure and shock.

S. aureus is usually also transmitted through the consumption of contaminated food, although the spectrum of possible sources of infection is quite broad. The following foods were involved in diseases: ready-made meat dishes, pies, cooked ham, raw ham, milk and milk products, egg-containing preparations, salads, creams, cake fillings, ice cream, pasta. Routine detection nowadays mostly takes place via the culture and confirmation tests of suspect colonies, since the detection of enterotoxins is quite difficult to carry out. If culture is detected, the sample to be examined is first incubated for 48 h on a suitable selective medium (eg Baird) at 37 ° C. If this first cultivation step was carried out in liquid medium, a second one closes

(again for 48 h) on solid medium (e.g. Baird-Parker). In the next step, suspect colonies are now tested for the presence of coagulase. There are two different methods available for this. As a rule, the so-called “tube test for the presence of the clumping factor” is first carried out, which takes about six to eight hours. If this test is negative, the result has to be passed through the so-called “tube test using rabbits -Plasma "can be confirmed. This test takes up to 24 hours. The total duration of the classic verification procedure is between 54 hours and five days.

Only for over 20 years has a previously underestimated germ played a greater role than food poisoner: Campylobacter. Unlike salmonella, for example, it rarely reproduces in food, but a few hundred bacterial cells are sufficient for infection with this pathogen.

The Campylobacter (C.) genus comprises 20 species and subspecies. These bacteria, which have so far been difficult to cultivate, are gram-negative, slim, curved to spirally wound rods that require microaerophilic conditions for their growth.

The species C. jejuni, C. coli and C. laris are medically relevant. They colonize the small and large intestines and cause acute gastroenteritis accompanied by the following symptoms: diarrhea, abdominal pain, fever, nausea, vomiting. These symptoms are difficult to distinguish from those of a stomach ulcer. A careful differential diagnosis is therefore essential. Routine detection is currently carried out via multi-stage cultivation, starting with an 18-hour enrichment in selective liquid medium (Campylobacter-selective medium according to Preston), followed by twice 48 h on two different fixed media (Karmali agar followed by Columbia blood agar). These five-day cultivations are followed by biochemical or serological identification.

As a logical consequence of the difficulties, in particular the lengthiness, which the above-mentioned methods have for the detection of Listeria, S. aureus and Campylobacter, nucleic acid-based detection methods are therefore available.

In PCR, the polymerase chain reaction, a characteristic piece of the respective bacterial genome is amplified with specific primers. If the primer finds its destination, a piece of the genetic material multiplies millions of times. In the subsequent analysis, for example by means of an agarose gel separating DNA fragments, a qualitative assessment can take place. In the simplest case, this leads to the statement that the target sites for the primers used were present in the examined sample. No further statements are possible; these target sites can originate from a living bacterium as well as from a dead bacterium or from naked DNA. A differentiation is not possible here. Since the PCR reaction is positive even in the presence of a dead bacterium or naked DNA, false positive results often occur. A further development of this technique is quantitative PCR, in which an attempt is made to establish a correlation between the amount of bacteria present and the amount of amplified DNA. The advantages of PCR lie in its high specificity, ease of use and in a short amount of time. Significant disadvantages are their high susceptibility to contamination and thus false positive results as well as the already mentioned lack Possibility to differentiate between living and dead cells or naked DNA.

The fluorescence in situ hybridization method (FISH; RI Amann, W. Ludwig and.) Offers a unique approach to combine the specificity of molecular biological methods such as PCR with the possibility of bacterial visualization, as made possible by antibody methods K.-H. Schleifer, 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59, pp. 143-169). Bacteria species, genera or groups can be identified and visualized in a highly specific manner.

The FISH technique is based on the fact that there are certain molecules in bacterial cells that, due to their vital function, have undergone only little mutation in the course of evolution: the 16S and the 23S ribosomal ribonucleic acid (rRNA). Both are components of the ribosomes, the sites of protein biosynthesis, and due to their ubiquitous distribution, their size, and their structural and functional constancy, they can serve as specific markers (Woese, CR, 1987. Bacterial evolution. Microbiol. Rev. 51, p. 221 -271). Based on a comparative sequence analysis, phylogenetic relationships can be established based solely on this data. To do this, this sequence data must be aligned. In the alignment, which is based on knowledge of the secondary structure and tertiary structure of these macromolecules, the homologous positions of the ribosomal nucleic acids are reconciled.

Based on this data, phylogenetic calculations can be carried out. The use of the latest computer technology makes it possible to carry out large-scale calculations quickly and effectively, and to create large databases that contain the alignment sequences of the 16S rRNA and 23S rRNA. Thanks to the quick access to this data material newly obtained sequences can be analyzed phylogenetically in a short time. These rRNA databases can be used to construct species- and genus-specific gene probes. Here, all available rRNA sequences are compared with each other and probes designed for specific sequence sites that specifically detect a bacterial species, genus or group.

In the FISH (fluorescence-in-situ hybridization) technique, these gene probes, which are complementary to a specific region on the ribosomal target sequence, are introduced into the cell. The gene probes are usually small, 16-20 base long, single-stranded deoxyribonucleic acid pieces and are directed against a target region, which is typical for a type or group of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it and the cells can be detected in the fluorescence microscope due to their fluorescence.

The FISH analysis is fundamentally carried out on a slide, and the evaluation visualizes the bacteria by irradiating them with high-energy light, that is, makes them visible. However, this is one of the disadvantages of the classic FISH analysis: since only relatively small volumes can naturally be analyzed on a slide, the sensitivity of the method can be unsatisfactory and not sufficient for a reliable analysis. The present invention therefore combines the advantages of classic FISH analysis with those of cultivation. A comparatively short cultivation step ensures that the bacteria to be detected are present in sufficient numbers before the bacteria are detected using specific FISH.

The implementation of the methods described in the present application for the simultaneous and 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 simultaneous and specific detection of bacteria of the genus Campylobacter as well as the species C. coli and C. jejuni thus comprises the following steps:

- Cultivation of the bacteria contained in the sample examined - Fixation of the bacteria contained in the sample

Incubation of the fixed bacteria with nucleic acid probe molecules in order to bring about hybridization,

- Removing or washing off the non-hybridized nucleic acid probe molecules and - Detecting the bacteria hybridized with the nucleic acid probe molecules.

In the context of the present invention, “cultivation” is understood to mean the multiplication of the bacteria contained in the sample in a suitable cultivation medium. For the detection of listeria, the cultivation can be carried out, for example, in V_ Fraser broth for 24 h at 30 ° C. For the detection of S Aureus can be cultivated, for example, as a blood culture (for example BACTEC 9240, Becton Dickinson Instruments) for 8 h to 48 h at 35 ° C. To detect Campylobacter, cultivation can be carried out, for example, in Preston selective medium for 24 h at 42 ° C. The person skilled in the art can in any case find the suitable cultivation methods from the prior art.

In the context of the present invention, “fixing” the bacteria is understood to mean a treatment with which the bacterial envelope is made permeable to nucleic acid probes. Ethanol is usually used for fixing. If the cell wall cannot be penetrated by the nucleic acid probes with these measures, the person skilled in the art will know Sufficient other measures are known which lead to the same result, for example methanol, mixtures of alcohols, a low-percentage paraformaldehyde solution or a dilute formaldehyde solution, enzymatic treatments or the like. In the context of the present invention, the fixed bacteria are incubated for the “hybridization” with fluorescence-labeled nucleic acid probes. These nucleic acid probes, which consist of an oligonucleotide and a marker attached to it, can then penetrate the cell envelope and adhere to the target sequence corresponding to the nucleic acid probe inside the cell The binding is to be understood as the formation of hydrogen bonds between complementary pieces of nucleic acid.

The nucleic acid probe 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 choose a nucleic acid probe that is complementary to an area that is present in the number of copies of more than 1 in the microorganism to be detected. The sequence to be detected is preferably 500-100,000 times per cell, particularly preferably 1,000-50,000 times. For this reason, the rRNA is preferably used as the target site, since the ribosomes in the cell as sites of protein biosynthesis are present thousands of times in each active cell.

The nucleic acid probe in the sense of the invention is a DNA or RNA probe, which will generally comprise between 12 and 1000 nucleotides, preferably between 12 and 500, more preferably between 12 and 200, particularly preferably between 12 and 50 and between 15 and 40, and most preferably between 17 and 25 nucleotides. The nucleic acid probes are selected on the basis 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 can be recorded. Complementarity should exist for a probe of 15 nucleotides over 100% of the sequence. With oligonucleotides with more than 15 nucleotides, one or more mismatching sites 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 according to the invention have the following lengths and sequences:

SEQ ID No. 1: 5 ^λ - ggc ttg cac egg cag tea et

SEQ ID No. 2: 5 "- egg ctt aca ccg gca gtc act

SEQ ID No. 3: 5 - cec ttt gta eta tec att gta

SEQ ID No. 4: 5 "- cec ttt gta cca tec att gta

SEQ ID No. 5: 5 "- cec ttt gta tta tec att gta g

SEQ ID No. 6: 5 ^Λ - cec ttt gta ctg tec att gta

The detection method for Listeria and L. monocytogenes is carried out, for example, as follows: The oligonucleotide SEQ ID No. 1 is specifically labeled, for example with a green fluorescent dye, and is used for the specific detection of all bacteria of the genus Listeria. The oligonucleotide SEQ ID No. 2 remains unlabeled and, as a competitor, prevents the labeled oligonucleotide SEQ ID No. 1 from binding to bacteria that do not belong to the Listeria genus. The oligonucleotide of SEQ ID No. 3 is also specific, but unlike the oligonucleotide SEQ ID No. 1, for example marked with a red fluorescent dye and is used for the specific detection of all bacteria of the species Listeria monocytogenes. The ohgonucleotides SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 again remain unlabeled and, as competitors, prevent the labeled oligonucleotide SEQ ID No. 3 from binding to bacteria that do not belong to the species L. monocytogenes. In this way, the simultaneous and highly specific detection of bacteria possible belonging to the genus Listeria or to the species L. monocytogenes. The different markers, for example a green fluorescent dye on the one hand and a red fluorescent dye on the other hand, are easily distinguishable from one another, for example by using different filters in fluorescence microscopy.

In the context of the method according to the invention for the specific detection of bacteria of the species S. aureus, the nucleic acid probe molecules according to the invention have the following lengths and sequences:

SEQID No.7: 5'- GAAGCAAGC TTC TCGTCC G

SEQID No.8: 5'- GGAGCAAGC TCC TCGTCC G

SEQID No.9: 5'- GAAGCAAGC TTC TCGTCATT

SEQ ID No.10: 5'- CTAATG CAG CGC GGATCC

SEQ ID NO.11: 5'- CTAATG CAC CGC GGATCC

SEQ ID No.12: 5 * - CTAATG CGG CGC GGATCC

SEQID No.13: 5'- CTAATG CAGCGC GGGTCC

The detection method for S. aureus is carried out as follows, for example: The ohgonucleotides SEQ ID No. 7 and SEQ ID No. 10 are specifically labeled, for example with a red fluorescent dye, and are used for the specific detection of all bacteria of the species Staphylococcus aureus. The ohgonucleotides SEQ ID No. 8 and 9 and SEQ ID No. 11, 12 and 13, on the other hand, remain unmarked and prevent competitors from binding the labeled ohgonucleotides to bacteria that 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 can be increased by using so-called "helper probes". These helper probes are unlabeled ohgonucleotides that have the following sequence:

SEQ ID No. 14: TCG CTC GAC TTG CAT GTA TTA GGC A

SEQ JD No. 15: ACC CGT CCG CCG CTA AC A TC A G

The use of the helper probes is not mandatory, but optional. The helper probes facilitate the binding of the marked probes to their target sites and thus improve the signal intensity. The detection method works very well even without these helper probes.

Within the scope of the inventive method for simultaneous specific

Detection of bacteria of the genus Campylobacter and the species C. coli and C. jejuni, the nucleic acid probe molecules according to the invention have the following lengths and sequences:

SEQ ID No.16 5 'CTG CCT CTC CCT CAC TCT AG

SEQ ID No.17 5 'CTG CCT CTC CCTTAC TCT AG

SEQIDNr.18 5 'CTGCCT CTC CCC TAC TCTAG SEQ ID No. 19 5 'CTG CCT CTC CCC C AC TCT AG

SEQ ID No. 20 5 'CCT ACC TCT CCC ATA CTC TAG A

SEQ ID No. 21 5 '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 5 'AGT AGA TCG CCT TCG CAA T

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 ID 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 5 '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

SEQ ID 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 is carried out as follows: The ohgonucleotides SEQ ID No. 16 to SEQ ID No. 19 and the ohgonucleotides SEQ ID No. 24 to SEQ ID No. 28 and the oligonucleotide SEQ ID No. 30 and the ohgonucleotides SEQ ID No. 32 to 34 are specifically labeled, for example with a green fluorescent dye, and are used for the specific detection of all bacteria of the genus Campylobacter. The ohgonucleotides SEQ ID No. 20 to 23 and the oligonucleotide SEQ ID No. 29 and the oligonucleotide SEQ ID No. 31 remain unlabeled and prevent as

Competitors bind the abovementioned labeled ohgonucleotides specific for the genus Campylobacter to bacteria which do not belong to the genus Campylobacter.

The ohgonucleotides SEQ ID Nos. 35 and 36 and the oligonucleotide SEQ ID No. 39 are also specific, but different from the ohgonucleotides SEQ ID Nos. 16 to 19, 24 to 28, 30 and 32 to 34, i.e. distinguishable from them, e.g. marked with a blue fluorescent dye and are used for the specific detection of all bacteria of the species Campylobacter coli. The ohgonucleotides SEQ ID No. 37, 38 and 40 to 42 again remain unlabeled and, as competitors, prevent the labeled, for C. coli specific, ohgonucleotides from binding to bacteria which do not belong to the C. coli species.

The ohgonucleotides SEQ ID Nos. 43 and 48 are also specific, but again different from the aforementioned ohgonucleotides, that is to say distinguishable from them again, for example with a red fluorescent dye, and are used for the specific detection of all bacteria of the species Campylobacter jejuni. The ohgonucleotides SEQ ID Nos. 44 to 47 and 49 in turn remain unlabeled and, as competitors, prevent the labeled ohgonucleotides specific for C. jejuni from binding to bacteria which do not belong to the C. jejuni species. 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 is possible.

The intensity of the signals received can optionally be increased by using so-called helper probes. The helper probes are also unlabeled, but make it easier to bind the labeled probes to their target locations and thus improve the signal intensity. This is only an amplification of the signal intensity, the detection procedure naturally also works without these helper probes.

For example, the intensity of the signals obtained with the oligonucleotide SEQ ID No. 24 can be increased by using the following unlabelled ohgonucleotides as helper probes:

SEQ TO No.50: 5 'CAC GCG GCGTTG CTGCTG / 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

Competitors in the context of the present invention are understood to mean, in particular, ohgonucleotides which have a higher specificity for genus or species not to be detected than the marked ohgonucleotides which are specific for the genus or species to be detected.

The invention also relates to modifications of the above oligonucleotide sequences which, despite the deviations in the sequence and / or length, result in a specific hybridization with target nucleic acid sequences of the particular Show bacterium and are therefore suitable for use in a method according to the invention. This includes in particular a) Nuclemic acid molecules which (i) with one of the above ogonucleotide sequences (SEQ ID No. 1 to SEQ ID No. 51) in at least 60%, 65%, preferably in at least 70%, 75%, more preferably in at least 80 %, 84%, 87% and particularly preferably in at least 90%, 94%, 96%, of the bases agree (whereby the sequence region of the nucleic acid molecule is to be considered which corresponds to the sequence region of one of the above-mentioned ohgonucleotides (SEQ ID No. 1 to SEQ ID No. 51) corresponds to, and not approximately the entire sequence of a nucleic acid molecule which may be in the

Comparison to the above-mentioned oligonucleotides (SEQ ID No. 1 to SEQ ID NO. 51) is extended by one to numerous bases) or (ii) the above ohgonucleotide sequences (SEQ ID No. 1 to SEQ ID No. 51) by one distinguish one or more deletions and / or additions and which have a specific hybridization with nucleic acid sequences of

Allow 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 of the species C. coli and C. jejuni. Here, "specific hybridization" means that among those described here or those of the average expert in connection with in situ

Hybridization techniques known hybridization conditions bind only the ribosomal RNA of the target organisms, but not the rRNA of non-target organisms to the oligonucleotide. b) Nuclemic acid molecules which have a sequence which leads to one of the nuclemic acid molecules mentioned under a) or to one of the probes SEQ ID No. 1 to

SEQ ID NO. 51 is complementary, hybridize under stringent conditions. c) Nuclemic acid molecules that have an ohgonucleotide 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 in addition to the sequences mentioned or their Modifications according to a) or b) have at least one further nucleotide and enable specific hybridization with nucleic acid sequences of target organisms.

The degree of sequence identity of a nucleic acid molecule with the probes SEQ ID No. 1 to SEQ ID No. 51 can be determined using conventional algorithms. For example, the program for determining the sequence identity is suitable here, which is available at http://www.ncbi.nlm.nih.gov/BLAST (on this page e.g. the link "Standard nucleotide-nucleotide BLAST [blastn]").

In the case of detection of bacteria of the genus Listeria or of the species L. monocytogenes, the specific oligonucleotide probes preferably correspond to the oligonucleotides SEQ ID No. 1 or SEQ ID No. 3. However, modifications are also possible, as long as a specific hybridization between the probes is nevertheless possible and target sequence takes place. It may be sufficient that the oligonucleotide probe used in 15, preferably 16 and 17 and particularly preferably 18 and 19 successive nucleotides matches SEQ ID No. 1 or SEQ ID No. 3. The same applies to the ogonucleotides serving as competitors with regard to the sequences SEQ ID No. 2, 4, 5 and 6.

The same applies to the detection of S. aureus. Here, the specific oligonucleotide probes preferably have a sequence which in 13 and 14 and preferably 15, 16 or 17 consecutive nucleotides corresponds to those of SEQ ID No. 7 or 10. The same applies to the ohgonucleotides serving as competitors with regard to the sequences SEQ ID No. 8, 9 and 11-13.

The same applies to the detection of bacteria of the genus Campylobacter and the species Campylobacter coli and Campylobacter jejuni. Here too, the specific oligonucleotide probes preferably have a sequence which is in 13 or 14, preferably 15 or 16 and particularly preferably 17 or 18 consecutive Nucleotides with SEQ ID Nos. 16-19, 24-28, 30, 32-36, 39, 43 and 48. The same applies to the ogonucleotides serving as competitors with regard to the sequences SEQ ID No. 20-23, 29, 31, 37, 38, 40-42, 44-47 and 49.

The nucleic acid probe molecules according to the invention can be used with various hybridization solutions as part of the detection method. Various organic solvents can be used in concentrations of 0 - 80%. Compliance with stringent hybridization conditions ensures that the nucleic acid probe molecule actually hybridizes with the target sequence. Moderate conditions in the sense of the invention are e.g. 0% formamide in a hybridization buffer as described below. Stringent conditions in the sense of the invention are, for example, 20-80% formamide in the hybridization buffer.

In the context of the method according to the invention for the 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, particularly preferably 40% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.5 mol / 1 - 1.0 mol / 1, more preferably 0.7 mol / 1 - 0.9 mol / 1 , particularly preferably of 0.9 mol / 1, the salt preferably being sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as 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%, particularly preferably in a concentration of 0.01%. Various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05 mol / 1, in a pH range of 6.0-9.0, preferably 7.0 to 8.0. The particularly preferred invention the execution of the hybridization solution contains 0.02 mol / 1 Tris-HCl, pH 8.0.

In the context 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 preferably 20% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.7 mol / 1 - 0.9 mol / 1, particularly preferably 0.9 mol / 1, where it is the salt is preferably sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as e.g. 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%, particularly preferably in a concentration of 0.01%. Various compounds such as Tris-HCl, sodium citrate, PIPES or HEPES can be used to buffer the hybridization solution, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05 mol / 1, in a pH range of 6.0-9.0, preferably 7.0 to 8.0. The particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.

Within the scope of the method according to the 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, particularly preferably 20% formamide. It also has a salt concentration of 0.1 mol / 1 - 1.5 mol / 1, preferably 0.7 mol / 1 - 0.9 mol / 1, particularly preferably 0.9 mol / 1, where it is the salt preferably around

Sodium chloride. Furthermore, the hybridization solution usually comprises a detergent, such as 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%, particularly preferably in a concentration of 0.01%. Various compounds, such as tris HC1, sodium citrate, PIPES or HEPES are used, which are usually used in concentrations of 0.01-0.1 mol / 1, preferably from 0.01 to 0.05, in a pH range of 6, 0 - 9.0, preferably 7.0 to 8.0. The particularly preferred embodiment of the hybridization solution according to the invention contains 0.02 mol / 1 Tris-HCl, pH 8.0.

It is understood that the person skilled in the art can select the stated concentrations of the constituents of the hybridization buffer in such a way that the desired stringency of the hybridization reaction is achieved. Particularly preferred embodiments reflect stringent to particularly stringent hybridization conditions. Using these stringent conditions, the person skilled in the art can determine whether a specific nucleic acid molecule enables specific detection of nucleic acid sequences of target organisms and thus was used reliably in the context of the invention. The person skilled in the art is able, by changing the parameters of the hybridization buffer, to increase or decrease the stringency if necessary or depending on the probe or target organism.

The concentration of the nucleic acid probe in the hybridization buffer depends on the type of its labeling and the number of target structures. In order to enable rapid and efficient hybridization, the number of nucleic acid probe molecules should exceed the number of target structures by several orders of magnitude. However, with fluorescence in situ hybridization (FISH) care must be taken to ensure that an excessively large amount of fluorescence-labeled nucleic acid probe molecules leads to increased background fluorescence. The concentration of the nucleic acid probe molecules should therefore be in a range between 0.5-500 ng / μl, preferably between 1.0-100 ng / μl and particularly preferably between 1.0-50 ng / μl. The preferred concentration in the context of the method according to the invention is 1-10 ng of each nucleic acid probe molecule used per μl of hybridization solution. The volume of the hybridization solution used should be between 8 μl and 100 ml, in a particularly preferred embodiment of the method according to the invention it is 30 μl.

The duration of the hybridization is usually between 10 minutes and 12 hours; hybridization is preferably carried out for about 1.5 hours. The hybridization temperature is preferably between 44 ° C. and 48 ° C., particularly preferably 46 ° C., the parameter of the hybridization temperature, as well as the concentration of salts and detergents in the hybridization solution, depending on the nucleic acid probes, in particular their lengths and the degree of complementarity can be optimized for the target sequence in the cell to be detected. The person skilled in the art is familiar with the relevant calculations here.

After the hybridization has taken place, the non-hybridized and excess nuclear acid probe molecules should be removed or washed off, which is usually done using a conventional washing solution. If desired, this washing solution can contain 0.001-0.1% of a detergent such as SDS, preferably 0.005-0.05%, particularly preferably 0.01%, as well as Tris-HCl in a concentration of 0.001-0.1 mol / 1. preferably 0.01 - 0.05 mol / 1, particularly preferably 0.02 mol / 1, the pH of Tris-HCl in the range from 6.0 to 9.0, preferably at 7.0 - 8, 0, particularly preferably 8.0. A detergent may be included, but is not essential. The washing solution usually also contains NaCl, the concentration depending on the stringency required being from 0.003 mol / 1 to 0.9 mol / 1, preferably from 0.01 mol / 1 to 0.9 mol / 1. A NaCl concentration of 0.07 mol / l (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) is particularly preferred. aureus) or from 0.215 mol / 1 (process for simultaneous specific Detection of bacteria of the genus Campylobacter and the species C. coli and C. jejuni). Furthermore, the washing solution can contain EDTA, the concentration preferably being 0.005 mol / 1. Furthermore, the washing solution can also contain preservatives known to the person skilled in the art in suitable amounts.

In general, buffer solutions are used in the washing step, which in principle may look very similar to hybridization buffers (buffered sodium chloride solution), except that the washing step is usually carried out in a buffer with a lower salt concentration or at a higher temperature. The following formula can be used to theoretically estimate the hybridization conditions:

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 sodium ions

% GC = proportion of guanine and cytosine nucleotides in the number of bases n = length of the hybrid

% FA = formamide content

With the help of this formula e.g. the proportion of formamide (due to the toxicity of the

Formamids should be as low as possible) of the wash buffer be replaced by a correspondingly lower sodium chloride content. However, it is known to the person skilled in the art from the extensive literature on in situ hybridization methods that the components mentioned can be varied and in what manner. With regard to the stringency of the hybridization conditions, what has been said above in connection with the hybridization buffer applies.

The "washing off" of the unbound nuclear acid probe molecules usually takes place at a temperature in the range from 44 ° C. to 52 ° C., preferably from 44 ° C to 50 ° C and particularly preferably at 46 ° C for a period of 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 specified target organisms. The Fast FISH method is known to the person skilled in the art and e.g. described in patent applications DE 199 36 875 and WO 99/18234. Reference is hereby expressly made to these documents with regard to their disclosure in order to carry out the detection methods described there.

The specifically hybridized nucleic acid probe molecules can then be detected in the respective cells. The prerequisite for this is that the nucleic acid probe molecule is detectable, e.g. in that the nucleic acid probe molecule is linked to a marker by covalent binding. As detectable markers e.g. fluorescent groups such as CY2 (available from Amersham Life Sciences, Inc., Arlington Heights, USA), CY3 (also available from Amersham Life Sciences), CY5 (also available from Amersham Life Sciences), FITC (Molecular Probes Inc., Eugene, USA), FLUOS (available from Röche Diagnostics GmbH, Mannheim, Germany), TRITC (available from

Molecular Probes ie. Eugene, USA), 6-FAM or FLUOS-PRTME, all of which are well known to those skilled in the art. Chemical markers, radioactive markers or enzymatic markers such as horseradish peroxidase, acid phosphatase, alkaline phosphatase, peroxidase can also be used. A number of chromogens are known for each of these enzymes, which can be converted instead of the natural substrate and can be converted into either colored or fluorescent products. Examples of such chromogens are given in the table below: table

Enzymes Cl romogen

1. Alkaline phosphatase and 4-methylumbelliferyl phosphate (*), acidic phosphatase bis (4-methylbelliferyl phosphate), (*) 3-O-methylfluorescein, flavone-3-diphosphate triammonium salt (*), p-nitrophenylphosphate disodium salt

2. peroxidase tyramine hydrochloride (*), 3- (p-hydroxyphenyl) propionic acid (*), p-hydroxyphenethyl alcohol (*),

2,2'-azino-di-3-ethylbenzthiazolinesulfonic acid (ABTS), ortho-phenylenediamine dihydrochloride, o-dianisidine, 5-aminosalicylic acid, p-Ucresol (*),

3,3'-dimethyloxybenzidine, 3-methyl-2-benzothiazoline hydrazone, tetramethylbenzidine 3. Horseradish peroxidase H ₂ O + diammonium benzidine H ₂ O + tetramethylbenzidine

4. β-D-galactosidase o-nitrophenyl-β-D-galactopyranoside, 4-methylumbelliferyl-β-D-galactoside

5. Glucose oxidase ABTS, glucose and thiazolyl blue

*Fluorescence

Finally, it is possible to design the nucleic acid probe molecules so that at their 5 'or 3' end there is another nucleic acid suitable for hybridization. sequence is present. This nucleic acid sequence in turn comprises approximately 15 to 1,000, preferably 15-50 nucleotides. This second nucleic acid region can in turn be recognized by a nucleic acid probe molecule which can be detected by one of the means mentioned above.

Another possibility is to couple the detectable nucleic acid probe molecules with a hapten, which can then be brought into contact with an antibody that recognizes the hapten. Digoxigenin can be cited as an example of such a hapten. The skilled worker is also well known about the examples given.

The final evaluation is possible depending on the type of marking of the probe used with a light microscope, epifluorescence microscope, chemiluminometer, 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 extraordinary speed. In comparison to conventional cultivation methods, which take up to ten days, the result is available within 24-48 hours when using the methods according to the invention.

Another advantage is the simultaneous detection of bacteria of the genus Listeria and the species L. monocytogenes. So far, only bacteria of the species L. monocytogenes have been detected with more or less reliability. However, epidemiological studies have shown that in addition to L. monocytogenes, other species of the genus Listeria also can trigger dangerous listeriosis. The sole evidence of

L. monocytogenes is therefore not sufficient according to the current state of knowledge.

Another advantage is the ability to distinguish between bacteria of the genus Listeria and those of the species L. monocytogenes. This is easily and reliably possible through the use of different labels for the respective genus or species-specific nucleic acid probe molecules.

Another advantage is the specificity of these processes. The nucleic acid probe molecules used can be used to specifically detect and visualize all species of the genus Listeria, but also highly specifically only the species L. monocytogenes. The species S. aureus is just as reliable and all species of the genus Campylobacter, but also highly specifically only the species C. coli and C. jejuni are detected.

Another advantage is the ability to differentiate between bacteria of the genus Campylobacter and those of the species C. coli or C. jejuni. This is due to the use of different markings for the respective genus or. Species-specific nucleic acid probe molecules easily and reliably possible.

By visualizing the bacteria, simultaneous visual control can take place. False positive results, which often occur in the polymerase chain reaction, are therefore excluded.

Another advantage of the method according to the invention is that it is easy to handle. The method can be used to easily test large quantities of samples for the presence of the bacteria mentioned.

The methods according to the invention can be used in a variety of ways. For example, food samples (e.g. poultry, fresh meat, milk, cheese, vegetables, fruits, fish, etc.) can be examined for the presence of the bacteria to be detected.

For example, environmental samples can also be examined for the presence of the bacteria to be detected. These samples can e.g. taken from the ground or also be parts of plants.

The method according to the invention can also be used for the investigation of wastewater samples or silage samples.

The method according to the invention can also be used to examine medical samples, e.g. of stool samples, blood cultures, sputum, tissue samples (including sections), wound material, urine, samples from the respiratory tract, implants and catheter surfaces are used.

Another area of application for the method according to the invention is the control of foods. In preferred embodiments, the food samples are taken from milk or milk products (yoghurt, cheese, curd cheese, butter, buttermilk), drinking water, beverages (lemonades, beer, juices), baked goods or meat products.

Another area of application for the method according to the invention is the examination of pharmaceutical and cosmetic products, e.g. Ointments, creams, tinctures, juices, solutions, drops etc.

According to the invention, kits for carrying out the corresponding methods are also made available. The hybridization arrangement contained in these kits is described, for example, in German patent application 100 61 655.0. On the disclosure in this document regarding the in situ hybridization arrangement is hereby expressly incorporated by reference.

In addition to the hybridization arrangement described (referred to as the VIT reactor), the kits comprise, as the most important constituent, the respective hybridization solution with the nucleic acid probe molecules (VIT solution) which are described above for the microorganisms to be detected. The corresponding hybridization buffer (Solution C) and a concentrate of the corresponding washing solution (Solution D) are also included. Fixation solutions (Solution A (50% ethanol) and Solution B are also included

(Absolute ethanol)) and, if necessary, a mounting solution (finisher). Finishers are commercially available, among other things they prevent the rapid fading of fluorescent probes under the fluorescence microscope. If necessary, solutions for the parallel execution of a positive control and a negative control are included.

The following example is intended to illustrate the invention without restricting it. The buffers and solutions used have the compositions given above.

example

Specific quick detection of pathogenic food-relevant bacteria in one sample

A sample is appropriately cultured for 20-44 hours. To detect listeria, cultivation can be carried out, for example, in V_ Fraser broth for 24 h at 30 ° C. To detect S. aureus, the cultivation can be carried out, for example, as a blood culture (for example BACTEC 9240, Becton Dickinson Instruments) for 8 h to 48 h at 35 ° C respectively. To detect Campylobacter, cultivation can be carried out, for example, in Preston selective medium for 24 h at 42 ° C.

The same volume of fixative solution (Solution B) is added to an aliquot of the culture. Alternatively, an aliquot of the culture can be centrifuged

(4,000 g, 5 min, room temperature) and, after discarding the supernatant, the pellet in 4 drops of fixative solution.

To carry out the hybridization, a suitable aliquot of the fixed cells (preferably 40 μl) is applied to a slide and dried (46 ° C., 30 min or until completely dry). Alternatively, the cells can also be applied to other carrier materials (e.g. a microtite latte or a filter). The dried cells are then completely dehydrated by adding the fixing solution again (Solution B, preferably 40 μl). The slide is dried again (room temperature, 3 min or until completely dry).

The hybridization solution (VIT solution) with the nucleic acid probe molecules described above for the microorganisms to be detected is then applied to the fixed, dehydrated cells. The preferred volume is 40 ul. The slide is then incubated in a chamber moistened with hybridization buffer (Solution C, corresponds to the hybridization solution without probe molecules), preferably the VIT reactor (46 ° C., 90 min).

The slide is then removed from the chamber, the chamber is filled with washing solution (Solution D, 1:10 diluted in distilled water) and the slide is incubated in it (46 ° C, 15 min). The chamber is then filled with distilled water, the slide is briefly immersed and then air-dried in the lateral position (46 ° C, 30 min or until completely dry).

The slide is then embedded in a suitable medium (finisher).

Finally, the sample is analyzed using a fluorescence microscope.

Claims

PATENT CLAIMS

1. Oligonucleotide for simultaneous specific detection of bacteria of the genus Listeria and of the species L. monocytogenes with a nucleic acid sequence, selected from the group consisting of (all sequences in 5 '-> 3' direction): i)

SEQ ID No. 1 5 "- ggc ttg cac egg cag tea et SEQ DO No. 2 5'- egg ctt aca ccg gca gtc act

SEQ ID No. 3 5 ^Λ - cec ttt gta eta tec att gta

SEQ ID No. 4 5 ^λ - cec ttt gta cca tec att gta

SEQ ID No. 5 5 '- cec ttt gta tta tec att gta g

SEQ ID No. 6 5'- cec ttt gta ctg tec att gta

ii) oligonucleotides which correspond to one of the ohgonucleotides under i) in at least 60%, preferably at least 80% and particularly preferably at least 90%, 92%, 94%, 96% of the bases and a specific hybridization with nucleic acid sequences of bacteria of the genus Listeria and / or of the species L. monocytogenes, iii) oligonucleotides which differ from one of the ohgonucleotides under i) and ii) in that they are extended by at least one nucleotide, iv) oligonucleotides which have a sequence which leads to a Oligonucleotide under i), ii) and iii) is complementary, hybridize under stringent conditions.

2. Oligonucleotide for the specific detection of bacteria of the species S. aureus with a nucleic acid sequence, selected from the group consisting of (all sequences in 5 '->3' direction): i) SEQ ID No.7: 5'- GAA GCAAGC TTC TCGTCC G SEQ ID No. 8: 5>'' - GGA GCA AGC TCC TCGTCC G

SEQ ID No. 9: 5> '' - GAA GCAAGC TTC TCG TCA TT

SEQ ID No. 10: 5> '' - CTA ATG CAG CGC GGA TCC

SEQ TD No. 11: 5> '' - CTA ATG CAC CGC GGA TCC

SEQ ID No. 12: 5> '' - CTAATG CGG CGC GGATCC

SEQ ID No. 13: 5> '' - CTA ATG CAG CGC GGG TCC

ii) oligonucleotides which correspond to one of the ohgonucleotides under i) in at least 60%, preferably at least 80% and particularly preferably at least 90%, 92%, 94%, 96% of the bases and a specific hybridization with nucleic acid sequences of the species S. aureus enable iii) oligonucleotides which differ from one of the ohgonucleotides under i) and ii) in that they are extended by at least one nucleotide, iv) oligonucleotides which have a sequence which leads to an oligonucleotide under i), ii) and iii) is complementary, hybridize under stringent conditions.

3. Oligonucleotide for simultaneous specific detection of bacteria of the genus Campylobacter and of the species C. coli and / or C. jejuni with a nucleic acid sequence selected from the group consisting of (all sequences in 5 '-> 3' direction):

SEQ JD NO.16 5 'CTG CCT CTC CCT CAC TCT AG SEQ ID NO.17 5' CTG CCT CTC CCT TAC TCT AG SEQ JD 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 TAGA SEQ ID NO. 21 5' CCA TCC TCT CCC ATA CTC TAG C SEQ ID NO. 22 5 'CCT ACC TCT CCA GTA CTC TAGT SEQ ID NO.23 5 'CCT GCC TCT CCC ACA CTC TAGA SEQ ID NO.24 5' CGC TCC GAAAAG 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 5' AGT AGA TCG CCT TCG CAA T

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 ID 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 JX) NO.39 5' TTG AGT TCT AGC AGA TCG C

SEQ LD NO.40 5 'TTG AGT TCC AGC AGA TCG C

SEQ LD NO.41 5 'TTG AGT TCT AGC AGA TAG C

SEQ ID NO.42 5 'TTGAGT TCC AGC AGA TAG C

SEQ ID NO.43 5 'CGC GCC TTA GCG TCA GTT GAG SEQ ID NO.44 5' CAC GCC TTA GCG TCA GTT GAG

SEQ LD NO.45 5 'CGC GCC TTA GCG TCA GTT AAG

SEQ LD NO.46 5 'CAC GCA TTA GCG TCA GTT GAG

SEQ ID 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

ii) oligonucleotides which correspond to one of the ohgonucleotides under i) in at least 60%, preferably at least 80% and particularly preferably at least 90%, 92%, 94%, 96% of the bases and a specific hybridization with Enable nucleic acid sequences of bacteria of the genus Campylobacter and / or of the species C. coli and / or C. jejuni, iii) oligonucleotides which differ from one of the ohgonucleotides under i) and ii) in that they are extended by at least one nucleotide, iv) oligonucleotides which hybridize with a sequence which is complementary to an oligonucleotide under i), ii) and iii) under stringent conditions.

4. A method for the simultaneous specific detection of bacteria of the genus Listeria and the species L. monocytogenes in a sample, comprising the steps: a) cultivating the pathogenic food-relevant bacteria contained in the sample, b) fixing the pathogenic food-relevant bacteria contained in the sample, c) incubating the fixed bacteria with at least one oligonucleotide according to claim 1 in order to bring about hybridization, d) removing non-hybridized ohgonucleotides, e) detecting and visualizing and optionally quantifying the pathogenic food-relevant bacterial cells with the hybridized oligonucleotides.

5. A method for the specific detection of bacteria of the species S. aureus in a sample, comprising the steps: a) cultivating the pathogenic food-relevant bacteria contained in the sample, b) fixing the pathogenic food-relevant bacteria contained in the sample, c) incubating the fixed ones Bacteria with at least one oligonucleotide according to claim 2 in order to bring about hybridization, d) removing non-hybridized ohgonucleotides, e) Detecting and visualizing and, if necessary, quantifying the pathogenic food-relevant bacterial cells with the hybridized oligonucleotides.

6. A method for the simultaneous specific detection of bacteria of the genus Campylobacter and the species C. coli and / or C. jejuni in a sample, comprising the steps: a) cultivating the pathogenic food-relevant bacteria contained in the sample, b) fixing the bacteria in the Sample containing pathogenic food-relevant bacteria, c) incubating the fixed bacteria with at least one oligonucleotide according to claim 3 in order to bring about a hybridization, d) removing non-hybridized ohgonucleotides, e) detecting and visualizing and optionally quantifying the pathogenic food-relevant bacterial cells with the hybridized oligonucleotides.

7. The method according to any one of claims 4 to 6, wherein the sample is a food sample.

8. The method according to any one of claims 4 to 7, wherein the detection is carried out by means of light microscope, epifluorescence microscope, chemiluminometer, fluorometer, flow cytometer.

9. Kit for carrying out the method according to claim 4, comprising at least one oligonucleotide according to claim 1.

10. Kit for carrying out the method according to claim 5, comprising at least one oligonucleotide according to claim 2.

11. Kit for carrying out the method according to claim 6, comprising at least one oligonucleotide according to claim 3.

12. Kit according to one of claims 9 to 11, in which the at least one oligonucleotide is contained in a hybridization solution.

13. Kit according to one of claims 9 to 12, further comprising a washing solution and optionally one or more fixing solutions.