FR2721617A1 - Fragments of nucleic acids derived from the Mycobacterium xenopi genome and their applications. - Google Patents

Abstract

Nucleic acid fragments, derivatives of the Mycobacterium xenopi genome, their applications to the typing and specific identification of the Mycobacterium xenopi, and to the screening for Mycobacterium xenopi infections, as well as to plasmids containing said fragments. Such a nucleotide sequence is comprised of a nucleotide sequence, specific to M. xenopi which does not hybridize in stringent hybridation conditions, with any other nucleotide sequence of mycobacteria; it is particularly contained in a sequence PstI-PstI of 1200 pairs of bases (pb) or a fragment of such sequence, such as the fragment of 288 pb, which has formula (SEQ n DEG 1).

Description

 The present invention relates to nucleic acid fragments, derived from the genome of Mycobacterium xenopi, to their typing and specific identification Mycobacterium xenopi applications and to screening for Mycobacterium xenopi infections as well as plasmids containing said Mycobacterium xenopi. fragments.

 In Europe, M. xenopi is one of the most frequently isolated species of pathogenic mycobacteria and is, along with M. kansasii and M. avium, the main agent of opportunistic mycobacterial infections in patients who are seronegative to vis-à-vis the human immunodeficiency virus. However, pulmonary diseases due to these different mycobacteria can not be distinguished clinically, radiologically or histologically.

 In patients with AIDS, disseminated infections caused by M. xenopi have been reported in both Europe and the United States (see Ausina et al., 1988, Ann Intern Med, 109, 927-928). RW SHAFER et al., 1992, AIDS Clin, Infect Dis, 15, 161-162) and can be difficult to diagnose.

 Nosocomial lung diseases have also been demonstrated and are due to the presence of M. xenopi in the environment and in particular in tap water.

 The existence of pulmonary mycobacterioses due to several types of mycobacteria and the difficulty of distinguishing them by clinical signs, necessitates the development of rapid methods of detection and specific identification of each of these mycobacteria, especially insofar as the applicable treatments are different.

 In addition, in the various mycobacterial infections, the latter are often present in small quantities, and when they are in detectable quantities by the methods conventionally used, the disease is already evolving and the patients are contagious to those around them.

Several techniques are currently used clinically to identify the species involved in a mycobacterial infection; we can mention in particular
- the direct detection of microorganisms under the microscope; this technique is fast but does not allow identification of the observed mycobacterial species and lacks sensitivity, since a large number of microorganisms must be present in the sample (greater than 104 / ml) to allow reliable detection .

 the cultures, when positive, have a specificity approaching 100% and allow the identification of the isolated mycobacterial species (biochemical tests); however, it is a long-term process that requires at least 4 weeks for colony formation and when few mycobacteria are present at the site of infection, repeated cultures are required to ensure positive result.

 Serological techniques may prove useful under certain conditions, but their use is limited by their sensitivity and / or their low specificity.

 the presence or absence of mycobacteria can also be determined by hybridization with probes, preferably non-radioactive, which are either probes specific for the DNA sequences of the different species of mycobacteria; or probes specific for a conserved portion of the 16S RNA, which made it possible to simplify and reduce the identification duration of the mycobacteria.

These probes have shown their interest in identifying the bacillus of tuberculosis,
M. avium, M. intracellulare and M. gordonae (M. GOTO et al., J. Clin microbiol., 1991, 29, 2473-2476
L. Lebrun et al., 1992, J. Clin. Microbiol., 30, 247624778), but are not usable for M. xenopi.

 - Several methods have recently been developed, using PCR, for the amplification of a fragment selected for its hypervariable sequence in a conserved gene among mycobacteria; these are, in particular, fragments corresponding to the 16S RNA or the gene encoding the 65 kDa protein (PLIKAYTIS BB et al., J. Clin Microbiol., 1992, 30, 7, 1815-1822) .

 Direct sequencing of the amplified product provides a certain means of identification, but requires, for a routine application, an automatic sequencer, which is an investment far too important for most laboratories.

 in order to avoid sequencing of the amplified product, some authors have developed an analysis of the digestion fragments obtained from the PCR products (A. TELENTI et al., 1993, J. Clin Microbiol., 31, 175- 178, PLIKAYTIS BB et al., Supra).

 However, such methods require several digestions and several enzymes to establish a certain identification and are therefore also difficult to implement routinely.

 The different detection methods of the prior art therefore do not allow to identify and / or quickly detect a M. xenopi infection; however, such species identification is crucial to give patients the most appropriate treatment as soon as possible.

 It has therefore been the object of the present invention to provide a method for the identification and / or specific screening of M. xenopi, allowing rapid species identification and / or detection of small amounts of DNA. extracts of the germs, themselves in limited numbers, and to reveal the presence of said mycobacteria, directly in the pathological samples.

 It is also an object of the invention to provide specific identification and screening reagents for M. xenopi.

The subject of the present invention is a nucleotide sequence, characterized in that it consists of a nucleotide sequence specific for
M. xenopi and in that it does not hybridize, under stringent conditions of hybridization, with any other nucleotide sequence of mycobacterium.

 In the present invention, nucleotide sequence is understood to mean both a double-stranded DNA sequence, a single-stranded DNA sequence and the transcripts of said sequences.

 Such hybridization conditions may be defined in particular as follows: 6xSSC, 5x Denhardt's solution, 0.5% SDS, 100 g of denatured salmon sperm DNA / ml, 32 P labeled DNA probe (106 cpm ml), for 16 hours at 68 ° C.

 According to a preferred embodiment of said nucleotide sequence, it is contained in a 1200 base pair (bp) PstI-PstI sequence of M. xenopi genomic DNA (SEQ ID No. 1) or a fragment thereof.

 This 1200 bp sequence is unexpectedly common to all M. xenopi strains.

The present invention also encompasses fragments of this 1200 bp sequence, useful for the identification of M. xenopi species, and especially
a fragment of 288 bp, with the following formula (SEQ ID No. 2)

CACACCGATTGTG,
- GGAATAGTCAGAGCCCG (XEN1) (SEQ No. 4) and
GTGTTAGCCACACCGTC (XEN2) (SEQ ID NO: 5).

 The invention also relates to nucleotide fragments complementary to the preceding ones, as well as fragments modified, with respect to the preceding ones, by removal or addition of nucleotides in a proportion of approximately 15% with respect to the length of the fragments above, and or modified at the nature of the nucleotides, since the modified nucleotide fragments retain a hybridization capacity with the DNA sequence of M. xenopi, similar to that exhibited by the corresponding unmodified fragments.

 The subject of the present invention is also identification reagents for M. xenopi, characterized in that they comprise at least one nucleotide sequence or a fragment thereof, as defined above, optionally combined with a suitable marker.

 Such a reagent unexpectedly permits the specific identification of M. xenopi, to the exclusion of any other mycobacterium.

 According to an advantageous embodiment of said reagents, they consist of pairs of primers for the synthesis of a DNA or RNA fragment of M. xenopi, each primer comprising a sequence or a fragment of a nucleotide sequence such as defined above.

 According to an advantageous arrangement of this embodiment, a pair of primers according to the invention is in particular constituted by the oligonucleotide XEN1, paired with the oligonucleotide XEN2.

 These primers make it possible in particular to synthesize the n-3 sequence defined above and / or its complementary strand.

 According to another advantageous embodiment of said reagents, they consist of a probe for the detection and / or identification of a DNA or RNA fragment of M. xenopi.

 According to an advantageous arrangement of this embodiment, said detection probe is selected from the group constituted by the sequence n'1, the sequence n-2 and the sequence nQ3, as defined above.

 According to another advantageous embodiment, the marker is chosen from the group which notably comprises the radioactive isotopes, the appropriate enzymes, the fluorochromes, the appropriate chemical markers, the haptens and the antibodies or base analogs such as those described in FIG. French Patent 2,518,755 or European Patent Application 0 158 758.

 Said reagents can be used in a very large number of diagnostic techniques, based on the detection of nucleic acid by hybridization.

 The present invention also relates to a family of recombinant plasmids, characterized in that they contain at least one nucleotide sequence according to the invention.

 According to an advantageous embodiment of said plasmid, it comprises the nucleotide sequence SEQ ID No. 1 or a fragment thereof.

 According to a preferred arrangement of this embodiment, said plasmid comprises said sequence associated with a vector pBS.

 According to another advantageous embodiment of said plasmid, it comprises the nucleotide sequence SEQ n-2.

 According to a preferred arrangement of this embodiment, said plasmid comprises said sequence associated with a pUC18 vector.

 The present invention also relates to a rapid and specific identification method of M.

xenopi, characterized in that it comprises
(1) a step in which the nucleic acid of the mycobacterium to be identified is contacted with a pair of primers as defined above for amplifying a nucleic acid fragment specific for M.

xenopi and
(2) a step in which the product obtained in step (1) is detected by any appropriate means.

 According to an advantageous embodiment of said identification method, prior to step (1), the mycobacteria are lysed and step (1) is carried out directly on said lysates, constituting the source of DNA, and this , without requiring further purification.

 According to another advantageous embodiment of said identification method, said pair of primers corresponds to the pair XEN1-XEN2, capable of hybridizing the 5 'and 3' ends of a specific fragment of M. xenopi.

 According to yet another advantageous embodiment of said identification method, step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a detection at using a suitable dye.

 According to another advantageous embodiment of said identification process, step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a membrane transfer. (Southern method) and hybridization with a non-radioactive probe as defined above.

The present invention also relates to a method for detecting a M. xenopi infection in a biological sample, characterized in that it comprises
(1) a step in which the biological sample is brought into contact with at least one diagnostic reagent according to the invention and
(2) a step in which the product obtained in step (1) is detected by any appropriate means.

 According to an advantageous embodiment of this method, the diagnostic reagent of step (1) is a pair of primers according to the invention and makes it possible to obtain amplification products of the nucleotide sequence to be detected. .

The amplification step is in particular one of the gene amplification techniques, such as the Qss-replicase method (Lizardi PM et al., Biotechnol., 1988, 6) or the PCR method (Polymerase Chain
Reaction) described in European Patent Applications 0 200 363, 0 201 184 and 0 229 701 filed by CETUS CO.

Alternatively, the amplification step of the DNA fragment containing the specific 288 bp sequence is performed according to the method described in US Pat.
French patent application 92 13562, in the name of
BioMérieux.

 According to the aforementioned embodiment, step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a detection with the aid of a dye appropriate.

 Alternatively, step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a membrane transfer (Southern method) and a hybridization with a non-radioactive probe as defined above.

 Such a method has the advantage of providing a sensitive and specific test, direct and rapid (less than 24 hours), of specific detection of a Mycobacterium xenopi infection, to the exclusion of any other mycobacterial infection (Bacillus Tuberculosis, MAIP group (M. avium, M. intracellulare, M. paratuberculosis) or M.

celatum).

 According to another advantageous embodiment of this method, the diagnostic reagent of step (1) is a probe as defined above.

The present invention further relates to a ready-to-use kit for carrying out the identification method and / or the screening method according to the invention, characterized in that it comprises: in addition to useful amounts of buffers and appropriate reagents:
appropriate doses of at least one pair of primers according to the invention,
and / or appropriate doses of at least one probe or nucleotide probe fragment according to the invention,
and / or reagents for visualizing the hybridization reaction of said probe and of the DNA of the sample to be tested.

According to an advantageous embodiment of the kit, it comprises
a pair of primers consisting of the sequences SEQ n-4 (XEN1) and SEQ ID No. 5 (XEN2) defined above,
the reagents necessary to carry out an amplification,
optionally a component making it possible to verify the sequence of amplified fragments, more particularly a nucleic probe having a length of at least 20 bases, capable of hybridizing with a part of the sequence n 2 or n 3, lying between the two fragments of the aforementioned primer pair.

 In addition to the foregoing, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method that is the subject of the present invention.

 It should be understood, however, that these examples are given solely by way of illustration of the object of the invention, of which they in no way constitute a limitation.

EXAMPLE 1 Nucleotide Sequences in Accordance with the Invention

1) PstI-PstI Fragment of 1200 Db (SEQ nl)
at. DNA extraction
Mycobacteria are cultured on a medium
Middlebrook 7H9 containing glycine at 1.4% (w / v), lysozyme (400 Fg / ml) and D-cycloserine (200 Rg / ml) for 24 h at 37C.

The cultures are then centrifuged at 3000 rpm for 20 min. The pellets are resuspended in TEN buffer (pH 8), including 50 mM Tris, 50 mM EDTA and 10 mM NaCl, treated with 2.25 mg / ml Proteinase K and SDS ( Sodium Dodecyl
Sulfate) at 0.6% (w / v) and incubated overnight at 37 ° C.

 SDS is then added and the tubes are again incubated for 30 min at 60 ° C. The DNA is purified with phenol-chloroform, precipitated with ethanol and dissolved in TE buffer (10 mM Tris, 1 mM EDTA).

b. Digestion with PstI enzyme
About 1 μg of the genomic mycobacterial DNA, obtained in a., Is digested with the restriction enzyme
PstI, for 16 h at 37 C.

 The products of the digestion are separated for 1 night by electrophoresis on a 0.7% agarose gel at 1.5 volts / cm.

In particular, a PstI fragment is obtained.
PstI of 1200 bp, present in all M. strains

xenopi.

2) Frabing of 288 Db (SEQ No. 2)
This fragment is obtained by PCR, carried out either from the 1200 bp fragment or directly from a sample of M. xenopi, under the following conditions:
at. Lysis of mycobacteria
Mycobacteria of the M. xenopi species are suspended in distilled water and subjected to 3 boiling-freezing cycles, each comprising 5 min at 100 ° C and 5 min at -20 ° C; a cell lysate is thus obtained, which is used as a direct source of DNA, without the need for further purification.

b. Amplification of DNA
The amplification is performed as follows
100 μl of a reaction mixture containing
1 × of a Taq polymerase buffer (50 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl 2, 0.01% (w / v) gelatin),
200 RM of each deoxynucleoside triphosphate,
0.3 CLAM of a single primer, designated DKU49, of sequence 5'-CCGCCGACCGAG-3 '(PS PALITTAPONGARNPIM et al., 1993, J. Infect.Dis., 167,975-978), and
2 units of Taq polymerase, covered with mineral oil and subjected to 35 cycles each comprising 1 min at 95 ° C., 1 min at 60 ° C., 1 min at 72 ° C. and then at an extension of 10 min at 72 ° C. using a Techné thermal cycler.

 Unexpectedly, the use of this DKU49 primer (PALITTAPONGARNPIM P. et al., J. Infect.

Dis., 1993, 167, 975-978), previously used for the amplification of M. tuberculosis, effectively makes it possible to amplify under more stringent conditions, that is to say by carrying out the hybridization at 60 ° C. and not at 40 ° C, a 288 bp fragment specific for M. xenopi.

c. Analysis of the amplified sequence
Figure 1 illustrates the specificity of the 288 bp fragment against the M. xenopi species.

 Indeed, all strains of M. xenopi produce a 288 bp fragment, when their DNA is subjected to a PCR which uses primer DKU49, both after agarose gel electrophoresis of the amplified products (FIG. 1A), after Southern blot analysis of said amplified products (FIG. 1B), whereas the strains of other species do not produce such a fragment under the same conditions: lanes 1 to 3 and 14 correspond to isolates of M. xenopi; tracks 4, 8, 11, 12 and 13 respectively correspond to M.

celatum, M. chelonae, M. simiae and M. scrofulaceum; lanes 5 and 6 correspond to M. avium strains; lanes 7 to 9 correspond to strains of M. tuberculosis. Lane 10 corresponds to DNA k digested with the restriction enzyme PstI, used as a control product.

 Figure 1A corresponds to 1.5% agarose gel electrophoresis of the 288 bp sequence, which is then detected by ethidium bromide staining.

FIG. 1B corresponds to the analysis of the amplification product (fragment of 288 bp) by a method of
Southern performed under the following conditions
* Depurination
The gel obtained is stirred for 20 min in a 0.25 M HCl solution (13 ml HCl + 500 ml distilled water), then rinsed 3 times with distilled water.

* Denaturation
The gel is then stirred for 20 minutes with buffer 1 (1.5M NaCl, 0.5M NaOH).

* Neutralization
The gel is then rinsed rapidly with buffer 2 (1.5M NaCl, 1M Tris-HCl pH 8), and then incubated with this same buffer 2 for 30 min.

* Transfer
Transfer to a Hybond nylon membrane, wetted in 1x SSC (NaCl, sodium citrate) solution, is performed overnight.

* Hybridization
The sequences obtained above are subjected to the following hybridization conditions
- the protocol
the membrane is pre-hybridized for 1 hour at 42 ° C. and at 6 rpm with 30 ml, for a blot of 320 cm 2, of a hybridization buffer provided in the Amersham ECL kit.
RPN 3001, and preheated to 42 C, for 30 min to 1 h)
a probe as prepared as specified in d is added. below, at a concentration of 10 ng / ml, then
the membrane is hybridized at 42 C for 1 night
. 4 washes are then carried out as follows
1st washing the membrane is washed for 10 min, at 55 ° C., in buffer A (1.6 ml of 10% SDS, 1 ml of 20 × SSC, 37.4 ml of distilled water per 320 cm 2, preheated to 55 ° C.)
2nd wash: same as first wash
3rd washing, carried out with stirring: the membrane is washed for 5 min at room temperature in a buffer B (2x SSC)
4th wash: identical to the third wash.

- 2nd protocol:
The membrane is hybridized with a buffer comprising 6xSSC (1xSSC: 0.15 M NaCl, 0.015 M sodium citrate buffer) 5x Denhardt's reagent (a 50x stock solution of Denhardt's reagent comprises: 5 g of
Ficoll, 5 g of polyvinylpyrrolidone, 5 g of bovine serum albumin and 500 ml of H2O), 0.5% SDS, 100 g of denatured salmon sperm DNA / ml, in the presence of a DNA probe labeled at 32P (106 cpm / ml) for 16 hours at 68 ° C.

4 washes are then carried out as follows
After washing, the membrane is washed for 10 min at 65 ° C. in a 2xSSC buffer.

 2nd wash: identical to the first.

 3rd wash: the membrane is washed for 30 min, at 65 ° C., in a buffer comprising 2xSSC and 0.1% SDS.

 4th wash the membrane is washed for 10 min, at 65 C, in a 0.1xSSC buffer.

* Detection
The membranes are then quickly dried.

The hybridized probe (1st protocol) is detected as follows
the membrane is incubated for 1 min in Amersham detection solution (ECL detection reagent, reference RPN 2105), the excess of detection solution is rapidly dried and revealed by autoradiography.

d. Demonstration of the hybridization of the 288 bp fragment with the 1200 bp sequence
- Preparation of the probe
The amplified DNA (288 bp sequence) is separated by electrophoresis on 1.5% agarose gel; the fragment thus separated is then recovered by electroelution through a dialysis membrane, immersed in sterile water. The DNA thus obtained is then purified by precipitation with chloroform and ethanol.

 The probe is then labeled with horseradish peroxidase using the Amersham ECL kit (Direct nucleic acid labeling and detection systems (RPN 3001)).

- DNA samples
A 1200 bp fragment as prepared in 1) above, or genomic mycobacterial DNA extracted from M. xenopi strains and digested with the PstI restriction enzyme, is used under the same conditions as above. above.

 The gels obtained are transferred to nylon membranes by the Southern method and hybridized with the labeled probe according to the invention, as specified above in FIG.

 A comparative hybridization test was carried out, under the same conditions as above, with 21 different species of mycobacteria listed below, in Table I.

TABLE I

<tb><SEP> Number <SEP> of <SEP> strains <SEP> tested
<tb><SEP> Species <SEP> Identification <SEP> Analysis <SEP> en
<tb><SEP> PCR <SEP> Southern <SEP> blot
<tb> M. <SEP> xenopi <SEP> 38 <SEP> 28
<tb> M. <SEP> avium <SEP> 8 <SEP> 3
<tb> M. <SEP> celatum <SEP> 5 <SEP> 2
<tb> M. <SEP> chelonae <SEP> 5 <SEP> NF <SEP> * <SEP>
<tb> M. <SEP> flavescens <SEP> 5 <SEP> 2
<tb> M. <SEP> fortuitum <SEP> 5 <SEP> 2
<tb> M. <SEP> gastric <SEP> 2 <SEP> 1
<tb> M. <SEP> gordonae <SEP> 6 <SEP> 2
<tb> M. <SEP> kansasii <SEP> 6 <SEP> 1
<tb> M. <SEP> malmoense <SEP> 5 <SEP> 1
<tb> M. <SEP> marinum <SEP> 5 <SEP> NF
<tb> M. <SEP> no <SEP> chromogenicum <SEP> 3 <SEP> 1
<tb> M. <SEP> scrofulaceum <SEP> 3 <SEP> 1
<tb> M. <SEP> shimoïdei <SEP> 1 <SEP> 1
<tb> M. <SEP> simiae <SEP> 5 <SEP> 1
<tb> M. <SEP> smegmatis <SEP> 1 <SEP> 1
<tb> M. <SEP> szulgai <SEP> 1 <SEP> 1
<tb> M. <SEP> terrae <SEP> 5 <SEP> 1
<tb> M. <SEP> ulcerans <SEP> 1 <SEP> 1
<tb> M. <SEP> vaccae <SEP> 1 <SEP> 1
<tb> M. <SEP> tuberculosis <SEP> 6 <SEP> 2
<tb> M. <SEP> bovis <SEP> 1 <SEP> 1
<tb> M. <SEP> bovis <SEP> BCG <SEP> 1 <SEP> 1
<tb> M. <SEP> africanum <SEP> 1 <SEP> 1
<tb> NF: not done
FIG. 3 illustrates the results obtained and shows that under the hybridization conditions specified above, the 288 bp fragment hybridizes exclusively with strains of the M. xenopi species (28 strains tested) and more particularly with the PstI digest fragment of about 1200 bp found in all M. xenopi strains, whereas this probe does not recognize any fragments of other mycobacterial DNAs.

 The comparison of FIGS. 1A and 3 shows a perfect correlation between the results obtained by the two methods: PCR and Southern blot analysis.

 In this FIG. 3 (Southern blot analysis of the DNA digested with the PstI restriction enzyme of different mycobacterial strains with the M. xenopi specific probe of 288 bp), lanes 1 to 4 and 11 to 16 correspond to M. xenopi strains; lanes 5 and 8 correspond to M. tuberculosis strains; tracks 6 and 7 correspond to strains of M.

avium, tracks 9 and 10 correspond to strains of
M. bovis and M. celatum respectively.

e. sequencing
This 288 bp fragment, produced by PCR using primer DKU49 as specified above in 2) b, was cloned into a vector pUC18, previously digested with the restriction enzyme SmaI.

 The clones obtained were transformed into E. coli strains. coli TG1.

 The recombinant colonies were selected on LB solid medium supplemented with isopropyl-D-thiogalactoside, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) and ampicillin 100 Rg / ml.

 The pUC18 plasmids which contain the inserts are purified using Qiagene mini-columns (Qiagen3 "Midi" kit, Qiagen Inc, USA).

The sequences of the mycobacterial inserts of three different strains of M. xenopi were thus carried out by the SANGER method (F. SANGER et al., 1977,
Proc. Natl. Acad. Sci. USA, 74, 5463-5467) using sequenase.

 In the 3 strains studied, the two strands were sequenced. The nucleotide sequence of the amplified fragment is identical in the 3 strains and the complete sequence of the 288 bp fragment is represented in the n-2 sequence.

 This sequence does not show significant homology with sequences previously described.

3) Fragment of 263 Db (SEQ No. 3)
Under the same conditions as those of 2), when the PCR is carried out with the primers according to the invention, XEN1 (SEQ No. 4) and XEN2 (SEQ ID No. 5), the fragment of 263 bp is obtained, which is a highly specific probe for the detection of M. xenopi.

 These primers are significantly more specific than the primer DKU49. Indeed, the latter, although allowing the production of the 288 bp fragment described above and also highly specific for M. xenopi, when it is used to carry out amplifications, results in the production of an additional fragment of 800 bp ( Figure 1A), for the majority of strains.

 Both the 288bp product and the 263bp product are highly specific for M. xenopi and do not hybridize with any of the other mycobacterial species; even M. celatum, a new species of recently isolated mycobacteria with phenotypic similarities to M. xenopi does not cross-hybridize with these specific probes.

The XEN1 and XEN2 primers which allow the amplification of this 263 bp fragment in all M. xenopi strains, even in those which also accumulate an 800 bp fragment when the primer is used.
DKU49 (FIG. 4) are particularly interesting for obtaining a single PCR profile, shared by all M. xenopi strains and consisting of a single band.

EXAMPLE 2: Identification test of M. xenopi

1) This test is carried out according to the protocol described in Example 1, namely obtaining a fragment
PstI-PstI, from genomic DNA, amplification with primers XEN1 and XEN2 and detection of amplified fragments.

2) Specificity and sensitivity of the test according to the invention
The sensitivity of the PCR assays was determined by amplification performed with 10th serial dilutions, from the template DNA.

 The results are shown in Figure 2, in which lanes 1 to 10 correspond to 10th dilutions, starting at about 10 ng of DNA. Lane 11 corresponds to DNA X digested with restriction enzyme PstI (control); dilutions were made in sterile water.

 The 288 bp fragment can be detected by staining with ethanol bromide up to 100 μg of DNA, while hybridization with the probe increases the sensitivity of the detection by a factor of 1000 (FIGS. 2A and 2B).

 Since 108 cells correspond to approximately 1 mg, it can be considered that the limit of detection of the product of the agarose gel PCR corresponds roughly to 103 cells, which is therefore a threshold of sensitivity that is significantly lower than those required for PCR tests carried out for the purpose of identifying strains.

 To determine the specificity of this PCR test, the 288-bp fragment was used as a probe after its labeling with horseradish peroxidase under the conditions of Example 1, 2).

 The autoradiograms show the specific hybridization of this probe with the 288 bp PCR fragment and there is no other band, nor with the 800 bp product found for some M. strains.

xenopi, nor with strains of other species (Figure 1B and Table I above).

 The PCR test developed for the detection of this single target has sufficient sensitivity for an identification test (Figure 2).

 The detection threshold is as low as cells, which is well below the minimum required when considering that a colony contains 108 cells.

 EXKMPLE 3: N. xenopi Screening Test.

 This test can be carried out under the same conditions as those of Example 2.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (29)

 1) nucleotide sequence, characterized in that it consists of a nucleotide sequence specific for M. xenopi and in that it does not hybridize, under stringent conditions of hybridization, with any other nucleotide sequence of mycobacterium.  2) Nucleotide sequence according to claim 1, characterized in that it is contained in a PstI-PstI sequence of 1200 base pairs (bp) of the genomic DNA of M. xenopi (SEQ ID No. 1) or a fragment of thereof.

 3) Fragment of a sequence according to claim 1 or claim 2, characterized in that it comprises 288 bp and has the following formula (SEQ n-2)

 4) Fragment of a sequence according to claim 1 or claim 2, characterized in that it comprises 263 bp and has the following formula (SEQ n-3) CACACCGATTGTG.  5) Fragment of a sequence according to claim 1 or claim 2, characterized in that it comprises 17 bp and has the following formula (SEQ No. 4): GGAATAGTCAGAGCCCGGG (XEN1).  6) Fragment of a sequence according to claim 1 or claim 2, characterized in that it comprises 17 bp and has the following formula (SEQ ID No. 5): GTGTTAGCCACACCGTC (XEN2).  7) Identification reagents of M. xenopi, characterized in that they comprise at least one nucleotide sequence or a fragment thereof according to any one of claims 1 to 6, optionally associated with a suitable marker.  8) Reagents according to claim 7, characterized in that they consist of primer pairs for the synthesis of a DNA or RNA fragment of M. xenopi, each primer comprising a sequence or a fragment of nucleotide sequence according to any one of claims 1 to 6.  9) Reagents according to claim 8, characterized in that said pair of primers is constituted by the oligonucleotide XEN1 according to claim 5, paired with the oligonucleotide XEN2 according to claim 6.  10 ') Reagents according to claim 7, characterized in that they consist of a probe for detecting and / or identifying a DNA or RNA fragment of M. xenopi.  11 ') Reagents according to claim 10, characterized in that said detection probe is selected from the group consisting of the sequence n'l, the sequence n-2 and the sequence n 3, according to any one of claims 2 to 4.  12-) Reagents according to any one of claims 7 to 11, characterized in that the marker is selected from the group which includes in particular the radioactive isotopes, the appropriate enzymes, fluorochromes, appropriate chemical markers, haptens and antibodies or base analogs.  13-) Family of recombinant plasmids, characterized in that they contain at least one nucleotide sequence according to any one of claims 1 to 6.  14-) Plasmid according to claim 13, characterized in that it comprises the nucleotide sequence SEQ n'l or a fragment thereof, according to claim 2.  15-) Plasmid according to claim 14, characterized in that it comprises the nucleotide sequence SEQ not associated with a vector pBS.  16-) Plasmid according to claim 13, characterized in that it comprises the nucleotide sequence SEQ n-2 according to claim 3.  17-) Plasmid according to claim 16, characterized in that it comprises the nucleotide sequence SEQ n-2 associated with a vector pUC18.  (2) a step in which the product obtained in step (1) is detected by any appropriate means.  (1) a step in which the nucleic acid of the mycobacterium to be identified is contacted with a pair of primers according to claim 8 or claim 9 for amplifying a nucleic acid fragment specific for M. xenopi and  18-) Method for rapid and specific identification of M. xenopi, characterized in that it comprises  19 ') Identification method according to claim 18, characterized in that, prior to step (1), the mycobacteria are lysed and step (1) is carried out directly on said lysates.  20 ') Identification method according to claim 18 or claim 19, characterized in that said pair of primers corresponds to the pair XEN1-XEN2, capable of hybridizing the 5' and 3 'ends of a specific fragment of M. xenopi.  21-) Identification method according to any one of claims 18 to 20, characterized in that step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by detection with a suitable dye.  22) Identification method according to any one of claims 18 to 20, characterized in that step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed membrane transfer and hybridization with a non-radioactive probe according to claim 10.  23) Method for screening for M. infection  (2) a step in which the product obtained in step (1) is detected by any appropriate means.  (1) a step in which the biological sample is contacted with at least one diagnostic reagent according to any one of claims 7 to 12 and xenopi, in a biological sample, characterized in that it comprises  24) A screening method according to claim 23, characterized in that the diagnostic reagent of step (1) is a pair of primers according to claim 8 or claim 9 and allows obtaining amplification products of the nucleotide sequence to be detected.  25) A screening method according to claim 23 or claim 24, characterized in that step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a detection with a suitable dye.  26) A screening method according to claim 23 or claim 24, characterized in that step (2) comprises an electrophoretic separation of the amplification products obtained at the end of step (1), followed by a membrane transfer and hybridization with a non-radioactive probe according to claim 10.  27) Screening method according to claim 23, characterized in that the reagent of step (1) is a probe according to claim 10.  and / or reagents for visualizing the hybridization reaction of said probe and the DNA of the sample to be tested.  and / or appropriate doses of at least one probe or a nucleotide probe fragment according to claim 10,  appropriate doses of at least one pair of primers according to claim 8 or claim 9,  28) Kit, ready to use, for carrying out the identification method and / or the screening method according to any one of claims 18 to 27, characterized in that it comprises, in addition to useful quantities appropriate buffers and reagents  optionally a component making it possible to verify the sequence of amplified fragments, more particularly a nucleic probe having a length of at least 20 bases, capable of hybridizing with part of the n-2 or n3 sequence, lying between the two fragments of the aforementioned primer pair.  the reagents necessary to carry out an amplification,  a pair of primers consisting of the sequences SEQ ID NO: 4 (XEN1) and SEQ ID NO: 5 (XEN2) according to claim 5 or claim 6,  29) Kit according to claim 28, characterized in that it comprises