WO1996015260A2 - Nucleotide sequences specifically hybridising with a genomic nucleic sequence of campylobacter fetus - Google Patents

Abstract

A nucleotide sequence which specifically hybridises with a genomic nucleic acid sequence of Campylobacter fetus. In particular, the sequence is selected from nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2, the respective complementary sequences thereof, and sequences distinguished therefrom by the mutation, insertion, deletion or substitution of one or more bases. Fragments of said sequence may be used as specific primers for amplifying sequences specific for Campylobacter fetus, and as nucleic probes specific for nucleic sequences of Campylobacter fetus. A method for detecting the presence of Campylobacter fetus strains in a biological sample, and a kit for carrying out the method, are also disclosed.

Description

 - I - Nucleotide sequences specifically hybridizing with a qenoic nucleic sequence of Campylobacter fetus

The present invention relates to a nucleic sequence specific for Campylobacter fetus, as well as to the applications of this sequence as a specific nucleotide probe for the detection of sequences of Campylobacter fetus or of fragments of this sequence as nucleotide primers for amplification. tion of CampylOabacter .fetus DNA or RNA in a biological sample.

Campylobacter infections are widespread worldwide, affecting humans as well as wild and domestic animals.

Although discovered at the beginning of the twentieth century, the bacteria currently called CampylOabacter were unknown for a long time, because their characteristics made their identification and their culture difficult. First isolated in ovids and bovids and called Vlbrlo fetus then, later, Campyloiacter fétus, it was not until 1946 that the first cases of human campylobacteriosis were described, but it was only from 1972, when selective media for Campylobacter began to be developed, the importance of Campylobacter infections could be proven and recognized.

Since the designation of the type species Campyloiacter fetus, a dozen other species and subspecies have been discovered, the exact number varying according to the authors and the taxonomic methods, which often propose new classification criteria. Among these species, the most encountered in human and / or animal pathology are CampylOabacter jejunl, Campylobacter coll and Campylobacter fetus.

Since the beginning of the century, the species C.. Fetus has been known as the bacterial agent responsible for many cases of abortion in ovids and bovids. This bacteria is also responsible for serious infections in humans. The first case of infection in men was described in 1947: a 6-month pregnant woman aborted following a septicemia due to C. fetus. This bacterial species has a tropism for the placental tissue, the consequences of the infection for the mother are limited but the fetal and neonatal mortality is close to 80%. Patients with compromised immune systems are also the target of this bacteria. The typical manifestation is then sepsis sometimes accompanied by an infection localized in the meninges, the peritoneum or an endovascular site. In the United States, septicemia with C. fetus reported to the Centers for Disease Control is almost as numerous as septicemia with C. jejunl. Statistics published by the CDC show that septicemia with C. f tus is three times more numerous in men than in women. On the other hand, it is now recognized that AIDS is a risk factor for extra-intestinal Campylobacter infections, and in particular for C. fetus septicemia. It is important to note that bacteriologists believe that the number of C. fetus infections is largely underestimated because of the difficulty in identifying the different species of CampylOabacter in general and C. fetus in particular. For example, many laboratories maintain blood cultures only for one week, use selective media containing cephalothin and incubate cultures at 42 ° C. However, most strains of C. fetus are sensitive to Cephalothin and do not grow to 42 ^β C, on the other hand 3 to 25 days of culture may be necessary for primary isolation in the blood. In the two foodborne C. fetus epidemics reported in the United States United, the strains were fortuitously isolated because they were thermotolerant and were able to grow at 42 ^β C, which is exceptional for C. fetus.

Like C. jejuni, C. fetus can be responsible for intestinal infections which, in the most severe cases can lead to high fluid losses, which can be particularly dangerous for children and infants, very sensitive to dehydration . However, C. fetus enteritis often remains uncomplicated and diarrhea may even spontaneously give way after a week. However, co-cultures can remain positive for a few weeks or even a few months, and in 5 to 10% of cases relapses can occur. Rigorous treatment and monitoring is therefore essential, especially for subjects who are immunocompromised or who have serious illnesses (AIDS, cirrhosis of the liver, cancer, leukemia, etc.), in which Campylobacter can behave like opportunistic bacteria. Used in time, intravenous antibiotic therapy can effectively cure C. fetus infections.

In animals, Campylobacter usually live in commensals in the digestive tract of many species: cattle, sheep, pigs, poultry, wild birds, dogs and cats. These animals, sick or healthy carriers, constitute a large reservoir of germs, and therefore a significant risk of contamination. Obvious cases of infection in cattle and sheep can result, in addition to the effect on livestock, transmission to humans through the spread of germs in the environment of animals (soil, water ). Even for asymptomatic animals, "healthy carriers", transmission to humans can occur: either by direct contact with these animals or their droppings, or by consumption of food or contaminated water (meats contaminated during preparation and undercooked, unpasteurized milk, polluted water, etc.).

With a view to prevention, it is therefore important, both in humans and in animals, to be able to identify the pathogen C. fetus as soon as possible, in order to prevent, by appropriate measures, any contamination. This is particularly the case in the food industry, where sterility conditions must be met. It is also important in human pathology, to carry out a correct follow-up of the patients treated following an infection with C. fetus, in order to avoid any new relapse.

Finally, in the case of declared infections, it is very important to properly identify the germ responsible, and this quickly after the onset of the disease, in order to be able to apply correct and effective treatment which would prevent the progression of infection or even the spread of epidemics. However, the identification of Campylobacter and the determination of the species accused is not easy. In fact, their isolation requires special media and their conventional detection is currently only done after enrichment by culture for at least 48 hours. This is very long when a rapid diagnosis is necessary. On the other hand, since microbiological diagnosis is currently made by bacteriological and / or biochemical techniques which exploit the phenotypic differences existing between the different species, diagnostic errors can occur, especially when mutants appear for a given trait. . In the specific case of CampylOabacter, hippurate hydrolysis is routinely used (C. jejuni can hydrolyze it while the others cannot).

However, the number of C. jejuni strains found to be negative hyppurate is increasing steadily; this observation makes this criterion less and less reliable for distinguishing the different species of the genus Campyloiacter.

On the other hand, since there are strains of C. coli resistant to nalidixic acid (5% in France), it is necessary to use other characteristics to differentiate C. coli and C. fetus.

Methods for identifying and classifying Campylobacter have been proposed, using either radioactive probes (Ng et al., Mol. Cell. Probes, 1987, _., 233-243), or non-radioactive probes (Chevrier et al., J. Clin. Microbiol., 1989, 27. 321-326), but these methods use total genomic probes and require enrichment by culture of the pathogen to be detected, because the detection threshold is quite high, around 10 ⁵ bacteria (Chevrier et al., above).

More sensitive approaches using molecular hybridization to identify C. jejuni and C. Coli species have been proposed, but no nucleic acid probe known to date can be used to carry out a rapid and specific test for detecting C. fetus in a biological sample.

The inventors have now isolated DNA sequences specific for Campylobacter fetus and have developed from these sequences an identification method based on the PCR technique and the molecular hybridization technique. The combination of these two techniques makes it possible to reach a very low detection threshold, which potentially makes it possible to detect and identify a single C. fetus bacterium in a biological sample.

The present invention thus relates to a nucleotide sequence which specifically hybridizes with a genomic nucleic acid from Campylobacter and which does not hybridize under usual conditions with a genomic nucleic acid from other Campylobacter nor with a genomic nucleic acid from close bacteria of the genus Ca pyloJbac er. The nucleotide sequence is in particular chosen from the nucleotide sequence SEQ ID no 1, l sequence SEQ ID No 2, the respective complementary sequences thereof, as well as the sequences which differ by mutation, insertion, deletion or substitution one or more bases.

By “sequences which differ therefrom by mutation, insertion, deletion or substitution of one or more bases” is meant the sequences which hybridize with the sequence SEQ ID No. 1, SEQ ID No. 2 or their complementary sequences under the conditions of usual stringency defined by SAMBROOK J., FRITSCH EF and MANIATIS T. (1989): Molecular Cloning: A Laboratory Manual, Ed. Cold. Spring Harbor Laboratory 9.47-9.62). These conditions are determined from the average stringency temperature T.

Preferably, the most advantageous sequences are those which hybridize in the temperature range (Tm -15 ° C) to (Tm -20 "C).

The invention also relates to a cloning vector containing a nucleotide sequence as defined above, in particular the plasmids pIGOl and pIG02, deposited at the National Collection of Culture of Microorganisms (CNCM), Institut Pasteur, Paris, October 4 1994 under the respective numbers 1-1479 and 1-1480. The deposits were made in accordance with the provisions of the Budapest Treaty.

The nucleotide sequences defined above can be DNA sequences or RNA sequences. The exact size of the sequence fragment IG01 SEQ ID No. 1 is 965 bp.

The fragment IG02 of sequence SEQ ID No. 2 has a length of 1581 bp.

These sequences are specific for the species C. fetus and do not hybridize with 8 other representative species of the genus Campylobacter.

A query of the "Genebank" and "EMBL" databases did not reveal any homology between on the one hand the sequences SEQ ID n ^* 1 and SEQ ID n "2 and on the other hand the known DNA sequences.

The sequences SEQ ID No. 1 and SEQ ID No. 2, parts or functionally equivalent variants thereof, can be used in molecular hybridization techniques for the detection and identification of Campylobacter fetus.

Functionally equivalent variants include sequences in which base pairs have been mutated, deleted, inserted or substituted, without affecting the properties essential to the specificity of these fragments.

The nucleotide sequences according to the invention have diagnostic and epidemiological applications in human or veterinary medicine, in particular as nucleic probes specific for Campylobacter fetus or as oligonucleotide primers for the amplification of a specific sequence for Campylobacter fetus.

The probes according to the invention advantageously comprise at least 18 consecutive nucleotides among the sequences or the fragments of sequences mentioned above.

The probes are DNA probes or RNA probes.

The nucleotide sequences described in this invention can thus be used as probes to specifically and directly detect strains of Campylobacter fetus in a biological sample.

The IGOl and IG02 fragments were labeled with ³² P and used as probes in a hybridization test, according to the Southern technique, on 9 species of the genus CampyloJbacter (C. jejuni, C. Coli, C. lari, C. fetus subsp. fetus, C. fetus siώsp. venerealis, C. sputorum siώsp. sputorum and C. feacalis) digested with the enzyme Hind III. As expected, hybridization was observed for the two probes with C. fetus. On the other hand, no hybridization is observed with the DNA of the other species. The IGOl probe hybridizes on several fragments: the number (between 5 and 10) and the size of these fragments depend on the strain studied. The IG02 hybrid probe with a single fragment whose size is between 1 and 2 kb. These observations make it possible to conclude that the IGOl and IG02 probes are specific for the species C. fetus and can be used in an identification test comprising molecular hybridization. In addition, the IGOl probe can be used for molecular typing experiments of strains belonging to the species C. fetus.

The unlabeled sequences can be used directly as probes, however the sequences are generally labeled with a radioactive element ( ³² p, ³⁵ S, ³ H, ¹²⁵ I) or with a non-radioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine) to obtain probes which can be used for numerous applications. In the latter case, one of the labeling methods described in FR 2 422 956 and FR 2 518 755 can be used. The hybridization technique can be carried out in various ways (Matthews, JA and Kricka, LJ, Anal. Biochem , 1988, 169, 1-25). The most general method is to immobilize the extracted nucleic acid Campylobacter fetus cells on a support (nitrocellulose, Nylon, polystyrene, ...) and to incubate, under well-defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe, etc.).

In another application, the nucleic acid probe (or derivatives) described herein can be used as capture probes. In this method, the probe is immobilized on a support and is used to capture by specific hybridization the target nucleic acid extracted from C. fetus. If necessary, the solid support is separated from the sample and the duplex formed between the capture probe and the target nucleic acid is then detected by means of a second detection probe marked with an easily detectable element.

When a sufficient quantity of Campylobacter fetus nucleic acid can be extracted from the samples to be analyzed, the sequences described in the patent can be used to detect and identify the strains belonging to Campylobacter fetus directly in these samples. Otherwise, a rapid culture in a liquid medium can be carried out before the extraction of the nucleic acid from CampyloJbacter fetus, or else the small quantity of nucleic acid extracted from the sample can be subjected to an amplification technique, such as for example the PCR technique, or any other in vitro enzymatic amplification system.

The sequences SEQ ID No. 1 and SEQ ID No. 2 or the fragments obtained from these sequences can also be used to select oligonucleotide primers, in particular for the PCR technique. This technique requires the choice of pairs of oligonucleotides surrounding the fragment which must be amplified (US Patent No. 4,683,202). These oligodexyxyonucleotide or oligoribonucleotide primers advantageously have; a length between 18 and 30 and preferably from 18 to 22 nucleotides. One of the two primers is complementary to the strand (+) of the matrix and the other primer is complementary to the strand (-). It is important that these primers do not have a secondary structure or a sequence complementary to one another. On the other hand, the length and the sequence of each primer must be chosen so that the primers do not hybridize with other nucleic acids originating from prokaryotic or eukaryotic cells, in particular with the nucleic acids of the CampyloJbacter not belonging to the fetus species and with DNA or human RNA which could possibly contaminate the sample.

The amplimers selected as specific primers for the amplification of nucleic sequences of strains belonging to CampyloJbacter fetus are chosen for example by following the method described by Griffais et al. (Nucleic Acids Res. 1991, 19, 3887-3891).

From the sequences SEQ ID No. 1 (IGO1), SEQ ID No. 2 (IG02), the inventors chose oligonucleotides to carry out a PCR test. Thanks to these oligonucleotides, they obtained a specific amplification of CampyloJbacter fetus, no amplification was visible with a nucleic acid of other Ca pylabacter, mentioned above, nor with the DNA extracted from the following cells: Escherichia coli, Helicobacter pylori,

Helicobacter mustelae, Salmonella typhi urium. Wollinella curva, Enterococcus faecalis, Streptococcus agalaetiae,

Mycobacterium avium, Edwardsiella tarda, Citrobacter amalonaticuε, Klebsiella pneumoniae, Enterobacter asburiae, Acinetobacter lwoffii, Pasteurella mul tocida, Vibrio harveyi, Serratia marcescens, Salmonella enterica subsp. salamae, Salmonella enterica subsp. diarizonae, Bacillus anthracis, human cells. A very particularly preferred pair of primers is represented by the oligonucleotides IG100 and IG101 derived from the sequence SEQ ID No. 1, of sequences:

IG100: ⁵ GCT AAG GGT GAG GTT GAT GGG ³ IG101: ⁵ AAG TTA TTC TTC AAA TCT ACC ^{3 '}

A second pair of primers which is very particularly preferred is represented by the oligonucleotides IG200 and IG201, derived from the sequence SEQ ID No. 2 of sequences.

IG200: ⁵ CGC ACA AGG CGG AGT ACG TC ³ IG201: ⁵ AGC AGG AAT GCC GCA TGC TG ³

The amplified fragments can be identified after an electrophoresis in agarose or polyacrylamide gel, or after capillary electrophoresis, or even after a chromatographic technique (gel filtration, hydrophobic chromatography or on ion exchanger). The specificity of the amplification can be checked by molecular hybridization using as probes the nucleotide sequences SEQ ID No. 1 or SEQ ID No. 2, fragments thereof, plasmids containing these sequences or fragments thereof. , oligonucleotides complementary to these sequences or fragments of sequences or amplification products. These probes may or may not be labeled with radioactive elements or with non-radioactive molecules.

The present invention also relates to a method for detecting the presence of strains of CampyloJbacter fetus in a biological sample, characterized by the following stages: i) bringing the biological sample into contact with a pair of so-called primers oligonucleotide fragments, as defined above, the nucleic acid contained in the sample having, where appropriate, been previously made accessible to hybridization and under conditions allowing hybridization of the primers to the nucleic acid of the strains belonging to Campyloacter fetus; ii) amplification of the nucleic acid of the CampyloJbacter fetus strains; iii) demonstration of the amplification of nucleic acid fragments corresponding to the fragment framed by the primers; iv) optional verification of the sequence of the amplified fragment, for example by hybridization of a specific probe, by sequencing or by analysis of restriction sites. The present invention further relates to a kit or kit for detecting the presence of strains belonging to Campylobacter fetus in a biological sample, characterized in that it comprises the following elements: - a pair of oligonucleotide fragments as defined above , ^*

- the reagents necessary to carry out an amplification of nucleic acid from strains belonging to Campylobacter fetus; - optionally, a component making it possible to verify the sequence of the amplified fragment, more particularly a nucleic probe as defined previously.

This kit more advantageously contains the probe or probes marked or not. These can be in solution or immobilized on a support. The kit can also contain the reagents necessary for the lysis of bacteria and the extraction of target nucleic acids, as well as the hybridization and washing solutions corresponding to the chosen method.

The invention also relates to the use of the sequence SEQ ID No. 1 or the derived sequences as defined above as an epidemiological tool, in molecular epidemiology. In fact, the specific fragment being present several times on the genome of C. fetus, its repeatability can be used as a tool to identify and classify identical strains and, obviously, to establish links as to the source and spread of the infection.

The invention is illustrated in more detail in the examples which follow, for which reference will be made to the appended figures in which:

- fig. 1 represents the results obtained after amplification with the pair of primers IG100 and

IG101;

- fig. 2 represents the results obtained after amplification with the pair of primers IG200 and IG201.

EXAMPLE 1:

Construction of the genomic library of C. fetus. Screening of the library and determination of the sequence of the specific fragment: The genomic DNA (200 μg) of the type strain

Campylobacter fetus fetus subspecies CIP 53.96T is partially digested with the Hind III restriction endonuclease (500 U of enzyme) in the buffer recommended by the supplier for 1 hour at 37 ° C. The genomic DNA thus digested is separated by gel electrophoresis 0.5% agarose. The fragments whose length is between 30 and 40 kb are electroeluted and precipitated in ethanol after extractions with phenol / chloroform

(1/1). The vector is the cosmid pHC79. It is digested in the same way and dephosphorylated to avoid any autoligation.

Ligation is carried out by mixing 900 ng of vector and 670 ng of DNA fragments 30/40 kb, the mixture is left at 14 ° C. for 18 hours after adding 2.5 units of T4-DNA ligase to the buffer appropriated.

The recombinant cosmids are packaged in vitro and used to transfect bacteria (E. coli HB 101). The transformed bacteria are incubated for 1 hour at 37 ° C. in LB medium, then spread on selective Agar medium containing 25 μg / ml 'ampillicin. The colonies resistant to ampicillin are all tested for their sensitivity to tetracycline, in fact the DNA fragment of 30/40 kb is inserted into the vector so as to inactivate the gene for resistance to tetracycline (Tet) and to keep the ampicillin (Am) resistance gene.

A mini DNA preparation of the first 96 colonies resistant to ampicillin (Amp ^r ) and sensitive to tetracycline (Tet ^s ) is carried out according to the technique of alkaline lysis. The DNA of these preparations is then digested with the Hind III restriction endonuclease, analyzed by electrophoresis on 0.8% agarose gel, then transferred to nylon filters. DNA is fixed irreversibly by 3 minutes of UV exposure at 254 nm.

These different filters are incubated for 16-18 hours at 65 ° C in a 6X SSC buffer (IX SSC corresponds to 0.15M NaCl and 0.015M Na citrate) containing 10% dextran sulfate, a concentrated Denhardt 5X solution (a Denhardt IX solution corresponds to 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin), lOmM EDTA, 0.5 % of SDS, 100 μg / ml of denatured salmon sperm DNA and genomic DNA, radiolabeled with ³² P by multipriming, of one of the following two species: C. hyointestinalis CIP 103746T and C. fetus subsp. fetus CIP 53.96T.

After hybridization, the filters are washed once 5-15 minutes in 2X SSC at 65 ° C, twice 15 minutes in 2X SSC + 0.1% SDS at 65 ° C and finally 1 time 15-30 minutes in 0.1X SSC at 65 ° C. The filters that are still wet are put into autoradiography at -80 ° C with an intensifying screen from 15 minutes to 3 days. The results of these hybridizations made it possible to isolate two cosmid clones: one (C35) contains a fragment of approximately 0.9 kb (fragment called IGOl) and the other (C3) a fragment of approximately 1.7 kb (fragment named IG02). These fragments were cloned into a pUC18 vector and prepared in large quantities. The resulting plasmids were designated pIGOl and pIG02.

The specificity of the fragments was checked as described in Example No. 2.

The IGO1 and IG02 fragments were cloned and sequenced according to the Sanger method using the sequencing kit "T7 sequencing kit" (Pharmacia).

All sequencing reactions were performed with ³⁵ S-labeled dATP.

The complete sequences of the two fragments are shown in the listing attached to the description. The comparison between the "Genebank" and "EMBL" databases and the 965 nucleotides of the IGOl fragment (sequence SEQ ID no. 1) and the 1581 nucleotides of the IG02 fragment (sequence SEQ ID no. 2) thus determined, does not reveal no significant homology with sequences known today.

EXAMPLE 2:

Molecular hybridization experiments (Southern technique) between the DNAs of different bacteria belonging to the genus Campylobacter and the nucleic acid sequences of the invention:

After culturing in an icroaerophilic atmosphere on an appropriate medium (5% sheep blood agar, Biomérieux), the Campylobacter are treated as follows: the bacteria from each Petri dish are harvested with 3 ml of TE-100 buffer (Tris- 100 mM HCl pH 8, 100 mM EDTA), centrifuged for 5 minutes at 5000 g, the pellet is dispersed in 8 ml of TE-100. To this suspension, 1 ml of TE-100 containing 9 g of lysozyme is added. The mixture is incubated for 30 minutes at 37 ° C. A ml of 10% SDS is added, the mixture is then incubated at 56 ° C for 30 minutes. Then 55 μl of a proteinase K solution are added, the mixture is incubated at 56 ° C for 5 to 10 minutes and then left at 37 ^β C overnight. The RNAs are eliminated by adding 10 μl of RNAse A (10 g / ml) and by incubating the mixture for 30 minutes at 37 ° C. Finally, the DNAs are extracted using a phenol / chloroform / isoamyl alcohol mixture (25: 24: 1) and precipitated with ethanol. The DNAs thus extracted undergo total digestion with the enzyme Hind III. The fragments obtained are then separated by electrophoresis on an agarose gel at 0.8% in TAE before being transferred to a nylon membrane according to the Southern technique.

The transferred fragments are analyzed by molecular hybridization. In this example, we used as probes, separately, the IGOl fragment and the IG02 fragment obtained after hydrolysis of the plasmids pIGOl and pIG02. pIGOl was cut by the enzymes Hind III and Hae III, pIG02 was digested with the enzyme Hind III. Both probes were labeled with ³² P.

Autoradiographs show that the IGOl probe detects 5 to 10 fragments in the DNA of the species C. fetus. With the same probe, no hybridization is visible with the DNA of different strains belonging to other species of Campylobacter even after 72 hours of exposure.

The IG02 probe specifically detects the DNA of the species C. fetus (C. fetus subsp. Fetus and C. fetus subsp. Venerealis) and does not hybridize to the genomic DNA of other Campylobacter. A single fragment, the size of which is close to 1.7 kb, hybridizes with the IG02 probe. All of these results lead us to conclude that the IGOl and IG02 fragments can be used for typing and for the specific detection of bacteria belonging to the species C. fetus.

EXAMPLE 3:

In vitro enzymatic amplification of C. fetus DNA with the primers defined from the nucleic acid sequences which are the subject of the invention. Choice of primers:

It has been demonstrated that it is essentially the 3 ′ end of the oligonucleotide primers which determines the specificity of the PCR (PCR Protocols, M. Innis et al. Académie Press Inc.). It is therefore important that this 3 ′ region is perfectly specific for the target to be amplified.

Since the Campylobacter genome has a very low percentage of guanine and cytosine (between 28 and 38% G + C), the inventors have thought that primers whose 3 'end was rich in G + C could have a high degree of specificity.

They searched, within the IGOl and IG02 sequences, for zones rich in G + C and which are present only once on these sequences. It is from these regions that the primer sequence was oriented and completed so as to obtain a length of approximately 20 nucleotides.

Synthesis of the oligonucleotide primers: The primers derived from the sequence IGO1 and

IG02 were synthesized on a "Cyclone Plus" (Millipore) automaton based on the chemistry of phosphoramidites. After synthesis, the oligonucleotide solution is transferred to a tube and incubated with concentrated ammonium hydroxide for 16 hours at 55 ° C. The oligonucleotide is precipitated with ethanol, then the pellet is washed with 70% ethanol and dried. Finally, the pellet is taken up in 1 ml of sterile distilled water. The concentration of each primer is determined using a spectrophotometer.

Amplification:

The in vitro enzymatic amplification (PCR) technique is carried out according to the protocol described by Saiki et al. (Science, 1988, 239, 487-491) using the oligonucleotide primers at a concentration of 10 μM, 30-100 ng of DNA from different Campylobacter strains and 0.5 units of Taq polymerase in a buffer containing 25 mM KCl, 20 mM Tris-HCl pH 8.5, 1.5 mM MgCl ₂ , 200 μM deoxyribonucleotide triphosphates and 100 μg / ml of bovine serum albumin, the final reaction volume being 50 μl. The parameters of the PCR steps were chosen as follows: 5 minutes at 94 "C, 1 minute at 50 ° C, 1 minute at 72 ° C (1 time), then 1 minute at 94 ° C, 1 minute at 50 ° C, 1 minute at 72 ° C (38 times) and 1 minute at 94 ° C, 1 minute at 50 ° C, 5 minutes at 72 ° C (1 time). Thirty cycles are thus carried out using an automatic device. After the last cycle, the samples are kept at 4 ° C until analysis. Electrophoretic analysis on an agar gel of the amplified samples:

Ten μl of the amplified samples are placed on a 2% agarose gel in a TBE buffer (0.04 M Tris-borate, 0.001M EDTA) containing 1 μg / ml of ethidium bromide. The amplified fragments are visualized under UV and the gels are photographed using a Polaroid 667 film.

FIGS. 1 and 2 show the results of the amplifications obtained with, on the one hand, the primers IG100 and IG101, and, on the other hand with the primers IG200 and IG201 on different DNAs of bacteria belonging to the genus Campylobacter or to others genres. Only the DNA of the strains belonging to the species C. fetus gives a fragment whose length corresponds to the theoretical length expected with these pairs of primers.

It is important to note that these pairs of primers make it possible to specifically detect the species C. fetus; isolates from patients and biological samples infected with C. fetus can therefore be identified, thus making the method usable in the field of clinical bacteriology and veterinary medicine.

LIST OF SEQUENCES

5 I - GENERAL INFORMATION

(1) APPLICANT: Institut Pasteur

(2) TITLE OF THE INVENTION: 10

Nucleotide sequences hybridizing specifically with a genomic nucleic sequence of Campylobacter fetus, application in the diagnosis of infection with Campylobacter 15 fetus and as an epidemiological tool.

(3) NUMBER OF SEQUENCES: 6

20

II - INFORMATION FOR SEQ. ID N ° 1:

CHARACTERISTICS OF THE SEQUENCE

TYPE: Nucleotide

LENGTH: 965 base pairs 25 NUMBER OF STRANDS: double

CONFIGURATION: linear

TYPE OF MOLECULE: DNA

ORGANISM: Campylobacter fetus

NAME: IGOl

30

DESCRIPTION OF THE SEQUENCE

35 GGCCTTTGAAGTTATTCTTCAAATCTACCTTAAGCACATCATTACCAGCACCACCATCTA TCTTATCCCCAGGATTTAAAGTACTCTCAGCAGCAGTACCTACCACCCCACTAATAAGAT CTCCACCCTCAGTACCAGTAATAGTATCATTCTCAGTAGTAAGAGCTATCTTATTTAAAC CAGCCTCATCTATACTCTCTTTTAACCCATCAACCTCACCCTTAGCACTCTCAACACTAC TACTATTACTAGTAACATTATTAATTATATCAACAGTCTTTTGTATTAAAGCATTTTGTT GATCAGGAGTAAGACCATCAAGTGGAAGTTTAGCTATAGTATCAGCAACATAATTACTAA CAATTACCTTATTATTAAATAGATCTTGATTAGCCCCAGCATTACTAGGATCTTTAGCAG CATTCACGAAGTGTCCTACTAAATCAGCCTTACTTACTGTACCATTATCTAAAAGATCAA CCCAATACTTAAGACCAGCTTTTGCTCATCACTAACAAACTCAGTTAAATTAACAGCATT CTTAAATAAATGCTTTACAAACGACTCATTATTATTTAAAGTCTCGCCAAGAAAACTCTT AGTAATATCTAAATTAAGTATAGCATTAGATGTCTCAATCATACCCCATCCATTAGCACT ACTCTCATTTACCCAATAAGTATTACCTTCACCCTCTGTAGGTCTTCCAAAAAGAACTAT AAATAGCTCTGAAACTTCTGATTTTGAAATCATACGAACTCCTTAATAAAATAAAATAAA ATCCAGACAAACAAACACTAACTAGTCTTATACTTTATTATTATACTTACAAGGTATCAA AGAGTATTTGGTTTGTTTATTAGGAGTATTAAGCTATTTGTAACTTTTATCCGATATAAT TATGTTTATTAACATAATGAAAGATAACATAATGTTTATTAACCGTATTAATGAATTACA AGCTT

III - INFORMATION FOR SEQ. ID No. 2; CHARACTERISTICS OF THE SEQUENCE

TYPE: Nucleotide LENGTH: 1581 base pairs NUMBER OF STRANDS: double CONFIGURATION: linear TYPE OF MOLECULE: DNA

ORGANISM: Campylobacter fetus NAME: IG02

DESCRIPTION OF THE SEQUENCE AAGCTTATGGGAATGAGTGCGACTTTGTAAATTTGAGATAACTCCACCGCTTGCAATACC GTTTTGTGTTAAAGCGATCTTAAAAAAACTAAAATTTCCCAAACTCCAAGCCAAAAAAGG CTCTAAAAGTTCATCTGCTTTCTCTTTGCAAACTCCTATTTTGGTCTTTATACCGCTATT TTTTAAAATTTGCTCACCGCCTTTTGCATTTTCGCTACTATCAATTGCCCCTATAATAAC TTGCTTAAAACCAAGCTCTTTTAACAAATTCGCACAAGGCGGAGTACGTCCTTGATGTGA GCAAGGTTCAAGCGTCACATAAGCAGTTGATCCATTTAGTAAATTTGAATGATTTTGCAT TATGAAATTATAAAGTTCGTTTGTATCATTTATCTCGTCAAAATCTTGCTTAAAAGCCGG ATTTTTCATCTCTAAAGCATATTTTATAGCGTTTAATTCAGCATGCGGCATTCCTGCTTT TTCATGAGCATTTATCGATAAAATTTCACCATTTTTACCTGTTATAACACATCCCACGGC AGGATTAGGATACGTAAGAAACTGAAACTCCCATGCTTTATTTATAGCTATATTCATATA ATATTCATTACACATCTTAATCTTTCTTTTATAAAATCAATTCCTTATTATAGTCAATTT GCAATTAAAAGCTACCTTAAACGGTAGTTTTGATTCTCTCTAGCTAATTTTTGCATCTCT TCAAAATTCTGCTTACTTAACCATAAAAGTTCGTCGAATTTAAATCCAAGCATAACTATA CGATAGAGCTCTGGTTTGTTTTTCTTCCAATTATATAAAGTTTTTACATCAATTTTAAGG ATACCTGCCATATCTCTTTTGCTTAGCTTTCTTTCCATAATCGTTCCTTGAAATATAAAA ACAAAATTTATCAAAAATTACCATCTTTTGCAACAAAGAAAATATTACTATATATTATAA TAAATCCATAGAAATATTACTATG TTTTATTTTAATAGACAAATGTATATATTTATAAAA AATTGAGACTAGAAAAAAGGTGCAAAATTATGGAGTACATAATATATTGCGAAAGTATCT GTGGAGAAAAAAGATACTTTATGATTCAAGACAAATTTAGCACTTTACCATACATAGTAA GGAATAGCGGAATCATATATAAAGTAATAAAATACATGAAAATAAACGAAAAAAATATAG AAAATGAAATTATGGAAAAGGTTTACCACCCAAACTGCTTTTTTTACGATATAAACACTC TTTATGCAGAGAAAAATTCATGGCAAAAAGTTGAGTGGTAAACTACCACTCCATATAGGT TTTAGCCTTCTTTATATTTTCAAATTTGACTTCAATTGATTCATTATTTTTGATATTTAT ACATATATTATCATCTTTTACGGATACTATCTTACCTTTAATCTCGGTCTTATCATTTAG TATAATTTTAGCAAATTCGCCTATACTAAGCGTAAAATGTTCTATTTTACTAAGTTTTCT CTCAAGCCCAGGGCTTGAAACCTCTAAAATCCACTCTCCGCTTACTGGCGGCATTACATC GTAAATAGGAGAAAGAAGCTT

IV - INFORMATION FOR SEQ. ID N '3: CHARACTERISTICS OF THE TYPE SEQUENCE: Nucleotide LENGTH: 21 bases NUMBER OF STRANDS: simple CONFIGURATION: linear TYPE OF MOLECULE: oligonucleotide ORGANISM: Campylobacter fetus NAME: IG100

DESCRIPTION OF THE SEQUENCE:

IG100: ⁵ GCT AAG GGT GAG GTT GAT GGG ³

V - INFORMATION FOR SEQ. ID N ° 4: CHARACTERISTICS OF THE SEQUENCE:

TYPE: Nucleotide

LENGTH: 21 bases

NUMBER OF STRANDS: simple

CONFIGURATION: linear TYPE OF MOLECULE: oligonucleotide

ORGANISM: Campylobacter fetus

NAME: IG101

DESCRIPTION OF THE SEQUENCE:

IG101: ⁵ AAG TTA TTC TTC AAA TCT ACC ³

VI - INFORMATION FOR SEQ. ID N ° 5: CHARACTERISTICS OF THE SEQUENCE: TYPE: Nucleotide LENGTH: 20 bases

NUMBER OF STRANDS: simple CONFIGURATION: linear TYPE OF MOLECULE: oligonucleotide ORGANISM: Campylobacter fetus NAME: IG200

DESCRIPTION OF THE SEQUENCE:

IG200: ⁵ CGC ACA AGG CGG AGT ACG TC ³ VII - INFORMATION FOR SEQ. ID N ° 6: CHARACTERISTICS OF THE SEQUENCE: TYPE: Nucleotide 5 LENGTH: 20 bases

NUMBER OF STRANDS: simple CONFIGURATION: linear TYPE OF MOLECULE: oligonucleotide ORGANISM: Campylobacter fetus 10 NAME: IG201

DESCRIPTION OF THE SEQUENCE:

IG201: ⁵ AGC AGG AAT GCC GCA TGC TG

Claims

CLAIMS 1. Nucleotide sequence which specifically hybridizes with a genomic nucleic acid from Campylobacter fetus and which does not hybridize under usual conditions with a genomic nucleic acid from other Campylobacter nor with a genomic nucleic acid from bacteria close to the genus Campylobacter, characterized in that it is chosen from the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2, the respective complementary sequences thereof, as well as the sequences which differ therefrom by mutation, insertion , deletion or substitution of one or more bases. 2. Nucleic probe specific for the nucleic acids of Campylobacter fetus, characterized in that it comprises at least 18 consecutive nucleotides chosen from the nucleotide sequences according to claim 1. 3. Nucleic probe specific for the nucleic acids of Campylobacter fetus according to claim 2, characterized in that it is chosen from the nucleotide sequence SEQ IDn ′ 1, the nucleotide sequence SEQ ID n ° 2, the sequences complementary to these, as well as the sequences which differ therefrom by mutation, insertion, deletion or substitution of one or more bases. 4. Nucleic probe according to claim 2 or 3, characterized in that it is immobilized on a support and used as a capture probe. 5. Couples of specific oligonucleotide primers for the amplification of a nucleic sequence of Campylobacter fetus, characterized in that they comprise pairs of oligonucleotides having from 18 to 30 nucleotides, preferably 18 to 22 nucleotides chosen from the sequences according to claim 1. 6. Oligonucleotide primer pairs specific for the amplification of a nucleic sequence of Campylobacter fetus according to claim 5, characterized in that they consist of the oligonucleotide pairs of sequences: IG100: ⁵ GCT AAG GGT GAG GTT GAT GGG ^{3 '} IG101: ⁵ AAG TTA TTC TTC AAA TCT ACC ^3' IG200: ⁵ CGC ACA AGG CGG AGT ACG TC ^{3 '} IG201: ⁵ AGC AGG AAT GCC GCA TGC TG ^3' 7. Nucleic acid fragment obtained by amplifying a nucleic acid from C. fetus by a gene amplification technique, in particular by the PCR technique using a pair of oligonucleotide primers according to claim 5 or claim 6 . 8. Cloning vector, characterized in that it contains a nucleotide sequence according to claim 1. 9. Plasmids deposited in the National Collection of Cultures of Microorganisms under the numbers 1-1479 and I-1480. 10. Method for detecting the presence of Campylobacter fetus in a biological sample, characterized by the following steps: a) bringing the biological sample into contact with a pair of primers according to claim 5 or 6, the nucleic acid of Campylobacter fetus contained in the biological sample having, where appropriate, made previously accessible to hybridization, and under conditions allowing hybridization of the primers to the nucleic acid belonging to Campylobacter fetus; b) amplification of the nucleic acid belonging to Campylobacter fetus; c) highlighting the amplification of Campylobacter fetus nucleic acid fragments corresponding to the fragment framed by the primers; d) possible verification of the sequence of the amplified fragment. 11. Kit for detecting the presence of Campy¬ lobacter fetus in a biological sample, characterized in that it comprises the following elements: - a pair of primers according to claim 5 or 6; - the reagents necessary to carry out an amplification of a nucleic acid of Campylobacter fetus; - possibly a component making it possible to verify the sequence of the amplified fragment, more particularly a nucleic probe according to one of claims 2 to 4. 12. Use of the nucleotide sequence SEQ ID No. 1 as well as the sequence complementary thereto and of the sequences which differ therefrom by mutation, insertion, deletion or substitution of one or more bases as an epidemiological tool for identifying and classifying Campylobacter fetus strains.