JP2002238563A - Identification of nucleotide sequence specific to mycobacterium and development of differential diagnosis strategy for mycobacterial species - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting non-tuberculous Mycobacterium strains(NTM) in a sample. SOLUTION: This method comprises the steps of (i) providing a non- tuberculous Mycobacterium species-specific upstream p34 gene region (us-p34) nucleotide probe, (ii) reacting the us-p34 nucleotide probe with the above sample under conditions that allow for the selective formation of nucleotide duplexes between the us-p34 nucleotide probe and the corresponding no-tuberculous Mycobacterium nucleic acid target present in the sample, and (iii) detecting any nucleotide duplexes containing the p34 nucleotide probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to novel gene sequences, diagnostic and / or quantification methods and to devices for the identification of various types of mycobacterial species using said sequences.

[0002] Mycobacterial diseases can be divided into three categories: M. tuberculosis or M. bo, all of which belong to the Mycobacterium tuberculosis complex (TUB).
tuberculosis caused by vis, leprosy caused by M. leprae, and diseases caused by nontuberculous mycoplasma (NTM) which cause all mycobacterial diseases except tuberculosis and leprosy. Historically, tuberculosis and leprosy are the two most prevalent mycobacterial diseases. However,
In recent years, NTM has become more frequent in developed countries, partly due to the development of acquired immunodeficiency syndrome (AIDS) (1). Tuberculosis only affects humans and animals, and no other carrier has been found to date. Conversely, N
Mycobacterial reservoirs responsible for TM disease are primarily in the natural environment, and eradication is not possible because the majority of these mycobacteria are naturally resistant to known anti-mycobacterial agents. The identification of mycobacterial species by rapid and specific methods is now essential. In particular, human and animal obligately pathogenic mycobacteria
(E.g., Mycobacterium tuberculosis, M. paratuberculosis, M. leprae
And other potentially pathogenic mycobacteria (e.g., M. avi
um-complex; M. chelonae, M. kansasii, M. xenop
i, M. simiae, M. malmoense, etc.) and normally non-pathogenic species (eg, M. gordonae; M. terrae; M. nonchrom)
genicum; M. flavescens; M. gastri; M. smegmati
s) must be quickly identified. Apart from obligate pathogens, most mycobacteria can actually be found everywhere in nature (soil, freshwater and seawater),
After ingestion or inhalation, they can temporarily or permanently colonize the human respiratory tract or digestive tract.

(Classification) a) Members of the Mycobacterium tuberculosis complex (TUB): This group comprises M. tuberculosis, M. bovis, M. bovis.
africanum, consisting of M. microti. b) Non-tuberculous mycobacteria (NTM):-A member of M. avium-complex (MAC group) is M. aviu
m and several environmental species with similar intermediate characteristics to M. intracellulare or common to these species (M. aviu
m; M. intracellulare; M. paratuberculosis, M. s
crofulaceum). -Mycobacterium other than TUB (also called MOTT) which does not belong to the MAC group (for example, M.
malmoense, M. sulzgai, M. kansasii, M. xenop
i, M. chelonae, M. simiae, M. marinum, M. gord
onae, M.fortuitum, etc.).

Preface Tuberculosis infects one-third of the world's population and kills more than three million people each year. Mycobacterium tuberculosis
Tuberculosis caused by (TUB) mycobacteria is still very prevalent in most parts of the world and may be introduced and transmitted in countries where it is rare (2). . In fact, tuberculosis has re-emerged in many industrialized countries in recent years as a result of AIDS and the changing demographics characterized by an increasing number of migrating workers, migrants, and homeless people (3). Of particular concern is the spread of drug-resistant species (4). Tuberculosis cases are becoming increasingly difficult to treat due to the increased morbidity of TUB species resistant to all first-line antituberculosis drugs. In HIV-1 infected patients, TUB is the most common opportunistic bacterial infection. At the stage when AIDS progressed, M. avium-intrace
Mycobacterial infection by members of the llulare complex (MAC) is the most common systemic bacterial opportunistic infection (1). In addition, the reduced and unprotected immune function found in newborns, infants, and immunosuppressed people has been identified by M.
avium-intracellulare, M. chelonae, M. fortuitu
m, M. kansaii, M. xenopi, M. marinum, M. ulcera
ns, M. tubec including M. scrofulaceum and M. szulgai
Allow opportunistic infections by mycobacteria (NTM) other than ulosis.

An increasing number of mycobacterial infections
The rapid identification of mycobacteria at the species level is of clinical importance. Diagnosing pathogenic species separately from non-pathogenic species is not only epidemiologically significant, but also meets the needs of patient management. People who are susceptible to contagious infections may want to be isolated to prevent the spread of the disease. In fact, antibiotic treatment varies depending on the species encountered. It is currently difficult to estimate the number of non-tuberculous infections, as there is no notification system as in the case of M. tuberculosis. In mild cases, there may be no further tests or misdiagnosis of M. tuberculosis
The currently reported incidence of these species appears to be underestimated. An increasing number of clinically isolated NTM bacteria are sent to clinical laboratories for identification, although the number of NTM diseases appears to be underestimated. This may reflect an increased prevalence of opportunistic mycobacterial diseases (especially in the context of AIDS) or an increase in the number and types of samples submitted for culture studies due to increased awareness of tuberculosis. It may also reflect.

[0006] If these mycobacterial diseases could be detected and identified at an early stage, it would have the potential to reduce morbidity and mortality and reduce socioeconomic consequences. The "golden rule" of definitive diagnosis remains a combination of mycobacterial culture and phenotypic testing (eg, analysis of fatty acid and mycolic acid content). However, conventional cultivation of human samples takes up to 8 months (depending on the type of mycobacteria). For example, primary culture of M. ulcerans under optimal conditions may take several months (5).

[0007] Other methods based on molecular biological techniques have allowed the development of genetic tests for the identification of some mycobacterial species. In the last decade, restriction fragment length polymorphism (RFRP) analysis, oligotyping and the use of nucleic acid probes have been proposed as a means of detecting and confirming infectious agents (6). These test methods are highly specific and rapid compared to conventional culture methods and have been applied to a wide variety of pathogenic bacteria, especially preferential microorganisms such as mycobacteria. To date, molecular test designs have speeded up the diagnosis of this preferred genus,
It still has some disadvantages. For species identification, the highly polymorphic region of the 16S rRNA gene has been shown to contain species-specific polymorphisms. This region is currently used in several commercially available assays, either directly on the sample (Accu-Probe; Gen-Probe) or after enzymatic amplification of the target to increase sensitivity (AMTD; Gen-Probe; Amplicor).
MTB, Roche) apply. However, most of these commercial kits are designed for diagnosis of the most common pathogenic mycobacterial species, namely M. tuberculosis and MAC strains, and each kit has only one type per test. Because they are designed to identify only mycobacteria, they are very expensive and are only a limited method of identifying mycobacterial species.

The newly identified and characterized sequences described in the present invention enable the design of differential diagnostics. These diagnostic methods can identify a wide range of mycobacterial species, including TUBs and NTMs, in a single assay. In addition, discrimination between members of the MAC group within the NTM group is also possible. In addition, these molecular targets open up new perspectives for the quantification of mycobacterial strains in clinical samples.

(Object of the invention)
The (us-p34) determinants provide new gene sequences, methods and devices for improved identification and / or quantification of various mycobacterial species, and these provide rapid epidemiological screening for their epidemiology. And rapid characterization in human clinical, animal and / or environmental samples.

[0010] Another object of the present invention is to identify similar gene sequences present in known and yet unknown mycobacterial species.

(Detailed Description of the Invention) The present invention more specifically relates to a method for detecting a non-tuberculous mycobacterial species (NTM), and (i) a non-tuberculous mycobacterial species-specific upstream p34. Providing a gene region (us-p34) nucleotide probe, and (ii) selectively generating a nucleotide duplex between the p-34 nucleotide probe and a corresponding non-M. Tuberculosis mycobacterial nucleic acid target present in the sample. Reacting the us-p34 with the sample under conditions and (iii) detecting all nucleotide duplexes containing the p-34 nucleotide probe. The probe may consist of all or part of the sequence of the species-specific us-p34 gene region aligned in FIG. 8 by those skilled in the art, as disclosed in FIG. Based on such an alignment (FIG. 8), it is within the knowledge of a person skilled in the art to actually design the appropriate probe to be used in the method.

More specifically, the present invention relates to a method wherein the non-M. Tuberculosis mycobacterium species-specific us-p34 nucleotide probe is a sequence selected from FIG.
Alternatively, the present invention relates to the aforementioned method, wherein T specifically hybridizes with at least a part of the corresponding sequence substituted with U.

More specifically, the present invention relates to a method wherein the non-tuberculosis mycobacterial species-specific us-p34 nucleotide probe is the sequence shown in FIG. A method as described above, wherein the method is selected from a group of sequences.

The invention further provides (i) at least two different non-M. Tuberculosis mycobacterium species-specific us-p34
Providing a nucleotide probe, and (ii) subjecting the us-p34 to the p-34 nucleotide probe under conditions that selectively generate a nucleotide duplex between the non-M. Tuberculosis Mycobacterium nucleic acid present in the sample. Reacting with the sample, (iii) detecting all nucleotide duplexes containing the us-p34 nucleotide probe, and (iv) the presence of a specific non-M. Tuberculosis Mycobacterium species from the generated nucleotide duplex. And a method for differentially detecting mycobacteria in a sample, comprising inferring the identification.

The probe according to the invention is preferably selected from Table 3.

The invention further provides (i) at least two different non-M. Tuberculosis mycobacterium species-specific us-p34
Providing a primer, (ii) reacting the us-p34 primer with the sample under conditions that permit selective amplification of the us-p34 sequence in the non-M. Tuberculosis Mycobacterium nucleic acid present in the sample; and , (Iii) step
(ii) a method for detecting a non-M. tuberculosis Mycobacterium strain in a sample, comprising detecting the amplification product of (ii).

The NTM-specific us-p34 primer is present in a particular species but not elsewhere in the us-p34 sequence.
(Part of the sequence shown in FIG. 3). Based on the sequence alignments given in FIG. 3 (see FIG. 8), it is within the knowledge of one of ordinary skill in the art to design the actual primer sequences that selectively amplify a particular type of Mycobacterium. is there.

The present invention further provides (i) at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2, (ii)
Reacting the us-p34 primer with the sample under conditions permitting selective amplification of the us-p34 sequence of the non-M. Tuberculosis Mycobacterium nucleic acid present in the sample,
ii) detecting the amplification product of step (ii), and
(iv) a method for differentially diagnosing mycobacteria in a sample, comprising inferring the presence and identity of a specific NTM from the generated amplification product. FIG. 2 shows the different amplification products that can be produced in step (ii).

The present invention further provides (i) at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (ii)
Reacting the us-p34 primer with the sample under conditions that allow for the selective generation of nucleotide duplexes between the us-p34 primer and the nucleic acid target of the MAC complex Mycobacterium strain in the sample; (iii) C) determining the us-p34 sequence of the Mycobacterium strain nucleic acid belonging to the MAC complex in the sample, the method comprising detecting the Mycobacterium strain belonging to the MAC complex in the sample.

The expression "determining the us-p34 sequence" means, for example, confirming the presence of the us-p34 sequence of the specific MAC complex by direct sequencing. As other methods, mass spectrometry, capillary electrophoresis, or HPLC can be used.

The present invention also provides (i) at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (ii)
The us-p34 nucleotide primer and the ust under conditions that allow for the selective generation of nucleotide duplexes between the MOTT Mycobacterium nucleic acid targets in the sample.
Reacting the -p34 primer with the sample; (iii) the us-p of the MOTT mycobacterium nucleic acid in the sample;
34. A method for detecting a MOTT Mycobacterium strain in a sample, comprising determining the sequence.

The expression "determining the us-p34 sequence" means, for example, confirming the presence of the us-p34 sequence of the specific MAC complex by direct sequencing. As other methods, mass spectrometry, capillary electrophoresis, or HPLC can be used.

The present invention also provides (i) at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (ii)
Us-p in the mycobacterium nucleic acid target in the sample
Reacting the us-p34 primer with the sample under conditions permitting amplification of the 34 sequences; (iii) determining the sequence of the amplification product obtained in (ii).
It relates to a method for determining the sp-p34 sequence.

The expression "new us-p34 sequence" means the sequence of a new (unidentified) Mycobacterium species. Some of these new sequences are disclosed in FIG.

The present invention also provides (i) at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (ii)
Reacting the us-p34 primer with the sample under conditions that permit selective amplification of the us-p34 sequence in the non-M. Tuberculosis Mycobacterium nucleic acid target in the sample; (ii)
i) Selectively hybridizing the amplification product obtained in (ii) with at least one non-M. tuberculosis mycobacterium species-specific us-p34 nucleotide probe selected from the group of sequences shown in FIG. (Iv) the us
The mycobacteria in the sample, comprising detecting all nucleotide duplexes containing the p34 nucleotide sequence and (v) inferring the presence of a particular non-M. Tuberculosis Mycobacterium strain from the generated nucleotide duplexes. And a differential detection method. Preferably, the probes to be used in the method are disclosed in Table 3.

The present invention also relates to a non-tuberculosis, consisting of at least 8 contiguous nucleotides from one of the nucleic acid sequences represented in FIG. Fungal mycobacterium species-specific u
pertains to sp34 nucleotide probes or primers.

The present invention also relates to the above-mentioned non-tuberculosis mycobacterium species-specific us-p selected from Table 1 or 2.
For a 34 nucleotide primer.

The present invention also relates to the aforementioned non-M. Tuberculosis mycobacterium species-specific us-p34 nucleotide probe selected from Table 3.

The present invention also relates to a nucleic acid consisting of the sequence selected from FIG. Preferably, the sequence is any contiguous sequence of the sequence identification number having a length of from eight to the full length of the sequence identification number.

The present invention also relates to compositions comprising at least one nucleotide probe, primer or sequence as described above. The composition preferably contains two, three or more such components.

The present invention also relates to a diagnostic kit comprising a probe, primer or sequence as described above or a composition as described above.

The present invention also relates to a solid support for detecting mycobacteria, comprising fixing at least two capture probes selected from Table 3 to the support.

The present invention also relates to a solid support as described above for use in the above method.

Various techniques can be applied to implement the method of the present invention. These techniques may consist of amplifying the target nucleic acid and then immobilizing it on a solid support for hybridization with a labeled oligonucleotide probe. Alternatively, the probe of the invention may be immobilized (covalently or non-covalently) on a solid support and hybridization, optionally after amplification, is performed with a labeled target polynucleic acid. This technique is called reverse hybridization. Such techniques are well-known to those skilled in the art.

It should be understood that any of the other techniques for detecting an amplified target sequence described above are included in the present invention. Such techniques can include sequencing or other microarray methods known to those of skill in the art.

The following definitions and explanations will allow a better understanding of the present invention.

The target substance in the analysis sample is DNA or RNA, for example, genomic DNA, messenger RN.
A or an amplified product thereof. These molecules are also referred to in the present application as "polynucleic acids" or "nucleic acids".

For the isolation of RNA or DNA from a sample, well-known extraction and purification methods can be used (eg, S
ambrook et al., 1989).

The phrase “probe” according to the present invention is:
A single-stranded oligonucleotide designed to specifically hybridize to a target polynucleic acid is meant. Preferably, the probes of the present invention are about 5 to 50 nucleotides in length, more preferably about 10 to 25 nucleotides in length. Particularly preferred probe lengths are 10, 1
1, 12, 13, 14, 15, 16, 17, 18, 1
9, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides used in the present invention may be modified nucleotides, such as ribonucleotides, deoxyribonucleotides, and nucleotides containing inosine or a modifying group that does not substantially alter its hybridization properties.

The term "primer" according to the present invention means a single-stranded oligonucleotide sequence capable of acting as a starting point for the synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and sequence of the primers must be such that they initiate the synthesis of extension products. Preferably, the primer is about 5 to 50 nucleotides in length. The specific length and sequence is the required DNA or RN
A Depends on the complexity of the target and the circumstances in which the primers are used, such as temperature and ionic strength. It should be understood that the primers of the invention can be used as probes and vice versa if the experimental conditions are adapted.

The expression “suitable primer pair” in the present invention refers to a primer pair which enables the specific amplification of the specific us-p34 target polynucleic acid fragment defined above.

The phrase “target region” of a probe or primer according to the present invention refers to a probe or primer that is completely complementary or partially complementary.
Refers to the sequence within the polynucleic acid to be detected (ie, including some mismatches). It should be understood that the complement of the target sequence is also optionally a suitable target sequence.

"Specific hybridization" of a probe to a target region of a polynucleic acid is defined as that the probe forms a double helix with part or all of the present region under the experimental conditions used, and under those conditions. Means that the probe does not form a double helix with other regions of the polynucleic acid present in the analysis sample.

"Specific hybridization" of a primer to a target region of a polynucleic acid is that the primer forms a double helix with a portion or the entire region of the present region during the amplification step under the experimental conditions used; Under these conditions, it means that the primer does not form a double helix with other regions of the polynucleic acid present in the assay sample.
As used herein, "double helix" should be understood to mean a double helix leading to specific amplification.

The fact that amplification primers need not perfectly match the corresponding target sequence in the template to ensure proper amplification is well documented in the literature. However, if the primer is not completely complementary to its target sequence, it must be taken into account that the amplified fragment will have the sequence of the primer and will not have the target sequence. The primer may be labeled with a label of choice (eg, biotin). The amplification method used is
Polymerase chain reaction (PCR), ligase chain reaction (L
CR), nucleic acid sequence based amplification (NASBA), transcription based amplification system (TAS), strand displacement amplification (SDA) or Qβ
Amplification using replicase or any other suitable nucleic acid molecule amplification method known to those skilled in the art may be used.

Probe and primer sequences are referred to throughout the specification as single stranded DNA oligonucleotides, 5 'to 3'. It will be obvious to those skilled in the art that any of the probes specified below can be used as such, or in a form complementary thereto, or in their RNA form (where T is replaced by U).

Probes, primers and other nucleic acid sequences according to the invention can be prepared by cloning a recombinant plasmid containing an insert containing the corresponding nucleotide sequence, if necessary, The plasmid can be excised from the cloned plasmid using a suitable nuclease, and recovered and prepared, for example, by fractionation based on molecular weight. Probes according to the invention can also be chemically synthesized, for example, by the conventional phospho-triester method.

[0048] Oligonucleotides used as primers or probes may also consist of nucleotide analogs such as phosphorothioates, alkyl phosphorothioates or peptide nucleic acids, or may contain intercalating agents. Like most other changes or modifications introduced into the original DNA sequence of the invention, these changes are tailored to the conditions under which the oligonucleotide is used to obtain the required specificity and sensitivity. Would need to. However, the end result of the hybridization will be essentially the same as that obtained with the unmodified oligonucleotide. The introduction of these modifications would be advantageous because it would positively affect properties such as hybridization kinetics, reversibility of hybridization, biological stability of the oligonucleotide molecule, and the like.

The phrase "solid support" refers to any substrate to which an oligonucleotide probe can be coupled if it retains hybridization properties and the background level of hybridization is kept low. Can be. Usually the solid phase substrate is
It could be a microtiter plate, a membrane (eg, nylon or nitrocellulose) or a microsphere (bead) or chip (biochip). Prior to addition to the membrane or prior to immobilization, it may be advantageous to modify the nucleic acid probe to facilitate immobilization or improve hybridization efficiency. Such modifications include addition of homopolymer tails, coupling with different reactive groups such as fatty groups, NH ₂ groups, SH groups, carboxylic acid groups, or coupling with biotin, haptens or proteins, and the like. Will do.

The phrase "labeled" refers to the use of labeled nucleic acids. Labeling may be performed by using labeled nucleotides or labeled primers that are incorporated during the polymerase step of the amplification, or by any other method known to those skilled in the art. The body of the label may be based on an isotope ( ³² P, ³⁵ S, etc.) or non-isotope (biotin, digoxigenin, etc.).

The phrase "biological sample or sample" refers to, for example, nasopharyngeal aspirate, throat or nasopharyngeal swab, nasopharyngeal lavage or tracheal aspirate, or other airway sample consisting of DNA or RNA.

The following useful guidelines known to those skilled in the art can be applied to design probes with desired properties.

Since the extent and specificity of a hybridization reaction as described herein is affected by a number of factors, will one or more manipulations of these factors be completely complementary to the target? Whether or not
The exact sensitivity and specificity of a particular probe will be determined. The importance and effect of the various assay conditions will now be described in more detail.

The stability of the [probe: target] nucleic acid hybrid should be chosen to suit the assay conditions. This can be achieved by avoiding long AT-rich sequences, terminating the hybrid at G: C base pairs, and designing probes with appropriate Tm values. The starting and ending points of the probe should be chosen so that their length and GC% will cause the Tm to be about 2-10 ° C. above the temperature at which the final assay will be performed. The base composition of the probe is important because GC base pairs are more temperature stable than AT base pairs due to additional hydrogen bonding. That is, high G-
Hybridizations containing C-complementary nucleic acids will be more stable at elevated temperatures.

Probe usage conditions, such as ionic strength and incubation temperature, should also be considered when designing probes. It is known that the degree of hybridization increases as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrid increases with increasing ionic strength. On the other hand,
Chemical reagents that break hydrogen bonds, such as formamide, urea, DMSO and alcohol increase the stringency of hybridization. Destabilization of hydrogen bonds by such reagents can significantly lower Tm. Generally, optimal hybridization of a synthetic oligonucleotide probe of about 10 to 50 bases in length will occur at a temperature approximately 5 ° C. below the melting temperature of a given duplex. Incubation at suboptimal temperatures will allow hybridization of the mismatched base sequence, and may thus result in reduced specificity.

It is desirable to have a probe that hybridizes only under high stringency conditions. Under conditions of high stringency, only highly complementary nucleic acid hybrids form,
Hybrids without a sufficient degree of complementarity will not be produced. Thus, the stringency of the assay conditions determines the degree of complementarity required between the two nucleic acid strands forming a hybrid. The degree of stringency is chosen to maximize the difference in stability between hybrids generated with target and non-target nucleic acids.

Target DNA or RN known to produce a strong internal structure that inhibits hybridization
The region in A is less preferred. Similarly, probes with significant self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acid to form a hydrogen-bonded strand. It will be understood without further remarks that if one of the two strands is involved in whole or in part in the hybrid, it will be less likely to participate in the formation of a new hybrid. If there is sufficient self-complementarity, within one type of molecule of the probe,
Intramolecular and intermolecular hybrids can form. Such structures can be avoided by careful probe design. By designing probes so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization can be greatly increased. Computer programs are available that search for this type of interaction. However, in certain cases, it may not be possible to avoid this type of interaction.

Standard hybridization and washing conditions are disclosed in the Experimental Materials and Methods section of the Examples. Other conditions are, for example, using 3 × SSC (aqueous sodium citrate solution), 20% deionized FA (formamide) at 50 ° C. If the specificity and sensitivity of the probe are maintained,
(SSPE (sodium phosphate EDTA saline solution), T
MAC (such as tetramethylammonium chloride) and temperature can also be used. If necessary, probe length or sequence modifications must be made to maintain the required specificity and sensitivity in a given environment.

The phrase "hybridization buffer" refers to a buffer that allows the hybridization reaction to take place under appropriate stringent conditions between the probe and the polynucleic acid or amplification product present in the sample.

The phrase "wash solution" means a solution that allows washing of the hybrids produced under appropriate stringency conditions.

The present invention, which has been described so far in general, may be more readily understood by reference to the following examples and figures, which merely describe certain aspects of the present invention. The embodiments are included for illustrative purposes only and do not limit the invention in any way. All references cited herein are incorporated by reference.

(Brief Description of the Drawings) FIG. 1: M. bovis (MB), M. tuberculosis (MT), M. av
ium subsp.paratuberculosis (MPT) and M. avium ss
Multiple nucleotide sequence alignment of the us-p34 gene of (MA). Gaps between sequences are indicated by dots (.). Vertical bar
(|) Indicates identity between sequences. The start codon (ATG) of the p34 ORF is bold. The sequences of the primers are shown in shaded boxes. Arrows indicate M. tuberculosis and
1 shows point mutations between M. bovis and M. avium subsp. Paratuberculosis and M. avium ss. Figure 2: Primers U1, U2, U in the us-p34 region
3, Amplification by U4 and U9. Figure 3: New us-p34 sequence (5 'to 3' orientation). The primers used to obtain the sequence (U2-U1; U3-
U1; U4-U1; U2-U9; U3-U9 or U
4-U9) and the size of the amplicon. Additional internal primers (U1, U2, U3) are indicated in the sequence. Sequence changes (point mutations) found in the same species (eg, M. ulcerans) are also shown when known. FIG. 4: U1-U4 consensus amplification of the us-p34 region of different mycobacterial species. Figure 5: Specific and non-specific hybridization. FIG. 6 discloses a species-specific mycobacterial probe,
Differential reverse hybridization of mycobacterial target amplicon on nylon membrane. a) Unlabeled amplification D specific for various mycobacterial species
The NA segment was first transferred onto a nylon membrane (M. tuberc
ulosis (TB), M. avium (AV), M. szulgai (SZ), M.
kansasii (KA), M. xenopi (XE), M. simiae (SI) and M. malmoense (ML)). b) M. tuberculosis (TB ^* ), M. avium (AV ^* ), M. szulg
ai (SZ ^* ), M. kansasii (KA ^* ), M. xenopi (XE ^* ) and
A digoxigenin-labeled amplicon derived from M. simiae (SI ^* ) was hybridized on a nylon membrane. Specific differential hybridization is obtained. Figure 7: Example of a biochip that specifically detects M. gordonae. FIG. 8: Alignment of some mycobacterial us-p34 sequences.

[0063]

EXAMPLES Preparation of Mycobacterial DNA a) Short Protocol (Mycobacteria obtained from the Pasteur Institute): DNA was obtained by boiling a sample to elute mycobacterial DNA into solution. b) Long protocol (mycobacteria obtained from the Institute of Tropical Medicine): Mycobacteria (10 mg (wet weight) were suspended in 200 μl of lysis solution (0.1 M NaOH, 1 M NaCl, and 5% sodium dodecyl sulfate [SDS]). And heated for 20 minutes (100 ° C.) The suspension was then cooled and 3 volumes of 0.1 M Tris-HCl (pH
7.4) Neutralize with buffer and centrifuge (5,000 xg, 5
Minutes). The supernatant was extracted with phenol-chloroform, the DNA was precipitated with ethanol, collected by centrifugation, dissolved in 50 μl of H ₂ O and stored at 20 ° C.

(PCR Amplification) For amplification, an aliquot (10 μl) of the DNA sample was transferred to 10 mM Tris-HCl (p
_{H8.8), 1.5mM MgCl 2, 50mM} KC
1, 0.1% Triton X-100, 0.25m
M (each) deoxynucleoside triphosphate, each primer
(Tables 1 and 2) 10 pmol and DyNAzyme
DNA polymerase (Finnzymes Inc., Espoo, Finland) was added in 90 μl of a PCR mixture consisting of 0.625 U. After the first heat denaturation step (96 ° C for 3 minutes)
A 30 cycle amplification was performed as follows: 9
Denaturation at 6 ° C. for 30 seconds, annealing at 58 ° C. for 45 seconds, and DNA extension at 72 ° C. for 30 seconds, increasing the denaturation and extension steps by 1 second per cycle. Final extension was performed at 72 ° C. for 15 minutes. Amplification was performed using a DNA 2400 thermocycler (Perkin-Elmer Applied Biosystems, Fost).
er City, Calif.). The PCR product is 2% (w
(t / vol) Agarose gel was checked. Electrophoresis was performed using 0.1 M Tris HCl (pH 8.6) containing 0.5 μg ethidium bromide per ml, 80 mM
Performed in boric acid, 1 mM EDTA buffer. DNA fragments are visualized at 300 nm on a UV transilluminator. The PCR product was cloned using the TOPO XL PCR cloning kit (Invitrogen, Carlsbad, Calif.) According to the manufacturer's protocol. Clones were also Taq Dye Deoxy T
emitter Cycle Sequencing Kit and ABI377 DNA Sequencer (Perkin-Elmer Applie
d Biosystems).

(DNA Sequence Analysis) Nucleotide sequence analysis was performed using Genetics obtained from the University of Wisconsin.
Using Computer Group software, B
Elgin EMBnet Node facility was used. The sequences were aligned using the Pileup program and pairwise compared using the Bestfit program.

(Reverse hybridization analysis) The us-P34 cloned DNA obtained from the standard strain was
And U4 amplification to create a mycobacterial species-specific capture probe. The amplified DNA fragments were sequentially subjected to a nylon membrane (Hybon
d-N +; Amersham, Little Chalfont, UK). After pre-hybridization for 2 hours, standard or clinical mycobacterial genomic DNA was
Primers U1 and U4 in the presence of G-11-dUTP
The us-p34 digoxigenin-labeled target probe obtained by PCR amplification in Table 1 was hybridized with the membrane. Hybridization of the heat denatured target probe (95 ° C. for 5 minutes) was performed using 2 × SSC (1 × SSC was 0.15 M
NaCl and 0.015 M sodium citrate), 1
Performed for 4 hours at 50 ° C. in% blocking reagent, 0.1% SDS, 0.1% N-lauroyl sarcosine, 5 mg / ml salmon sperm DNA and 5% formamide. The filter was then passed through 2xSSC (1xSSC was 0.15
(MNaCl plus 0.015M sodium citrate)
Wash twice with 0.1% SDS at 37 ° C. for 5 minutes each.
The plate was washed twice with 2 × SSC-0.1% SDS at 50 ° C. for 5 minutes. Hybridized digoxigenin-labeled DN
A fragment was treated with alkaline phosphatase labeled anti-DIG Fab
Detected by fragment. Nitro BlueTetra
zodium Chlorure and 5,3-bromo-4-indolyl phosphate (BCIP) (Boerhinger
Colorimetric detection was carried out using Mannhaim) according to the manufacturer's instructions.

(Analysis on "Mycobacterial Biochip") Design of Biochip. Mycobacterial microarrays are being developed by ATT (Namur, Belgium). The array is a glass slide with different mycobacterial species-specific capture nucleotide sequences on the surface (Table 3). Immobilized, positive and negative hybridization control capture probes were also included in the array, and the capture probes of the array consisted of four replicates each.

Preparation of biotinylated amplicon. Amplification of the mycobacterial us-p34 sequence was performed at 2.5 mM M
gCl ₂ , 75 mM Tris-HCl, pH 9.0,
50 mM KCl, 20 mM (NH ₄ ) ₂ SO ₄ , primers U4 and U5 0.5 μM, 200 μM dA each
TP, 200 μM dCTP, 200 μM dGTP,
150 μM dTTP, 50 μM biotin-16-dU
TP, uracil-DNA-glycosylase (Boehringer
Mannheim, Germany) 0.5 U, 1.25 U Taq DNA polymerase (Biotools, Madrid, Spain) and 50 μl final volume containing 10 μl DNA template.
1 is performed. The reagents were first incubated at 94 ° C. for 5 minutes, then the DNA9600 thermocycler (Per
(Kin Elmer, Foster City, CA, USA) Perform 40 cycle reactions using the following temperatures and cycle times: 94 ° C for 30 seconds, 49 ° C for 45 seconds, 72 ° C for 30 seconds.
Seconds. Perform a final extension step at 72 ° C. for 10 minutes. PCR products are used directly or stored at -20 ° C.

Hybridization and colorimetric detection.
The procedure for detecting the PCR product is carried out according to the manufacturer's instructions (ATT, Namur, Belgium) as follows:
5 μl of the CR product and 5 μl of the positive control provided in the kit are denatured with fresh 0.05 N NaOH for 5 minutes at room temperature. This solution is then used as hybridization solution 3
5 μl and mixed in a hybridization chamber (Bio
zym, Landgraaf, Netherlands). The chamber is closed with a plastic cover slip and hybridization is performed at 53 ° C. for 30 minutes. Wash slides 4 times for 1 minute each in wash buffer. The glass sample was then treated with blocking buffer for 100
Incubated with 800 μl of 0-fold diluted streptavidin conjugate for 45 minutes at room temperature. After washing five times, the slides are incubated three times for 10 minutes each with 800 μl of the color mixture, then washed with water, dried and imaged using a colorimetric microarray reader (ATT, Namur, Belgium). A visual glance already provides most of the information related to the image, but the resulting image is analyzed by algorithms that quantify the intensity of the spots and pattern recognition algorithms.

(Cloning and Sequencing of the P34 Region Upstream of Mycobacterium sp.) M. tuberculosis (Gen
Bank registration number Z79700) and M. avium su
bsp. paratuberculosis (GenBank registration number X
No. 68102) and its regulatory sequences (upstream p34 or us-p34) were compared to M. tuberc
This indicated that there was a deletion in the region upstream of the start codon of the p34 protein of ulosis (FIGS. 1 and 2). To confirm and extend these findings, M. bovis BCG and M. avium D
Four us-p34 sequences were amplified and sequenced. A multiple sequence alignment of this region is based on M. tuberculosis
And M. bovis Bacile de Calmette et Guerin [BCG])
And non-tuberculosis bacteria (M. avium sp and M. avium subsp.
ratuberculosis) revealed an interspecies polymorphism specific to both mycobacteria, ie, the us-34 fragment was 79 bases shorter in M. tuberculosis. Conversely, us-p34 was highly conserved within each group: M. tub
The difference between E. erculosis and M. bovis is dependent on a single T to C transposition at position 41 of us-p34, and is due to M. avium ss and M. avium subsp. paratubercu.
Losis relied on a single base conversion from C to G at position 264 of us-p34 (FIG. 1).

M. tuberculosis / M. Bovis and M. aviu
Based on sequence homology between m / M. avium subsp. paratuberculosis, several oligonucleotides U1, U2, U3, U4 that match the conserved sequence of polymorphic us-p34
And U9 (Tables 1 and 2) were designed to produce other standard bacteria of the genus Mycobacterium, M. szulgai, M. africanum.
gordonae, M. gastri, M. kansasii, M. marinum,
M. ulcerans, M. intracellulare, M. scrofulaceu
m, M. xenopi, M. malmoense and M. simiae (Table 3)
The corresponding region was amplified. These fragments were cloned and sequenced (FIG. 3).

(Consensus P of Mycobacterium sp.)
Development of CR Amplification) A consensus PCR assay was developed based on the sequence of mycobacterium us-p34. Using primers U1 and U4, M. szulgai
(163 bp), M. non chromogenicum (169 bp),
M. tuberculosis, M. bovis, M. africanum (178
bp), M. gordonae (182 bp), M. gastri (22
3 bp), M. kansasii (225 bp), M. marinum and M. ulcerans (236 bp), M. intracellulare,
M. avium, M. paratuberculosis (256 bp), M. s
crofulaceum (259 bp), M. xenopi (256 b
p), M. leprae (269 bp), M. malmoense (29
0 bp) and M. simiae (298 bp).
The corresponding fragment spanning 3 bp to 298 bp was amplified (FIG. 4; Table 4). All mycobacterial sequences and
As illustrated by the pairwise partial alignment with the us-p34 region of M. tuberculosis, the sequence alignment shows polymorphisms in both size and nucleotide sequence.

(Consensus my for clinical isolates)
Validation of c-U1-mycU4 PCR) The reliability of this consensus amplification method was examined for a series of clinical isolates of mycobacteria (Table 4). This method is characterized by being highly sensitive to most mycobacteria. The low sensitivity seen for some is
This is probably related to DNA samples rather than the insensitivity of this method. In fact, amplification of M. marinum-M. Ulcerans is more sensitive to the "long protocol" DNA sample (20/20) than to the "short protocol" DNA sample (4/10). The quality of these "short protocol" DNA preparations needs to be checked before any further conclusions can be made. This is actually the case with other mycobacterial species (for example, M. phlei,
M. flavescens, M. nonchromogenicum, M. chelonae
Etc.) may affect the further identification of potentially specific amplicons.

(Development of Species-Specific PCR Amplification Method) Between U1 and U4 (including most interspecies differences) or most widely U9
Based on all upstream p34 alignments between (and likely to include specific strain differences) between
Primers that amplify one strain (two new primers or one new primer and one shown in Tables 1 and 2)
Primer combinations) were designed. This method allows the differentiation of Mycobacterium species by PCR.

(Inverse Hybridization Method) An inverse hybridization assay was set up based on homologous and heterologous hybridization of amplicons from the same species and amplicons from two different species, respectively (FIG. 5). In the first step, a species-specific probe corresponding to each mycoplasma species was prepared by amplifying the cloned DNA of the standard strain us-p34 using two consensus primers U1 and U4, and was applied to a nylon strip. Fixed. The DNA of the species to be identified was amplified in the presence of the same primer, biotinylated at the 5 'end, hybridized with the immobilized probe, and subjected to colorimetric assay. M. tuberculosis, M. avium, M. szulga
The assay that correctly identified i, M. xenopi, M. simiae and M. malmoense is shown in FIG.

(Development of "Mycobacterial Biochip") Polymorphic us-p34 sequences can also be applied to low-density microarray technology to develop methods for simultaneous species-specific detection of mycobacteria. In preliminary studies, mycobacterial microarrays with attached various mycobacterial species-specific capture probes were identified as A
Developed by TT (Namur, Belgium). These probes can be used as us-derived from different mycobacterial species.
Hybridized with p34 biotinylated amplicon. M. gordonae, M. tuberculosis, M. gastri, M.
kansasii, M. intracellulare, M. leprae, M. aviu
m, M. marinum, M. simiae, M. xenopi, M. malmoe
FIG. 7 shows an example of specific detection for nse, M. szulgai and M. scrofulaceum-specific capture probes.

References 1. Portaels f. Epidemiology of mycobacterial diseases. Clin
ics in Dermatology 1995; 13: 207-222. 2. Raviglione MC, Snider DE, Kochi A. Global Epidemiology of Tuberculosis: Global Epidemic Morbidity and Mortality. JA
MA 1995; 273: 220-26. 3. Cobelens FG, van Deutekom H, Draayer-Jansen IW
Risk of Mycobacterium tuberculosis infection in travelers traveling to the area where the tuberculosis rises. Lancet 2000; 356: 461-465. 4. Pablos-Mendez A, raviglione MC, Laszlo A et al;
Global surveillance of antituberculous drug resistance from 1994 to 1997. New Engl J Med 1998; 3
38: 1641-49. 5. Tsang AY and Farber ER. Mycobacterium ulceran
Primary separation of s. Am. J. Clin. Pathol. 1973; 59: 688-69.
2. 6. Portaels F, Fonteyne PA, De Beenhouwer H, de R
ijk P, Guedenon A, Hayman J, Meyers WM.Mycobacter
Variability at the 3 'end of 16S rRNA of ium ulcerans is associated with the geographical origin of the isolate. J. Clin Microbi
ology 1996; 34: 962-965.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Brief description of the drawings]

[Fig. 1] M. bovis (MB), M. tuberculosis (MT),
M. avium subsp.paratuberculosis (MPT) and M. avi
Multiple nucleotide sequence alignment of the us-p34 gene of um ss (MA). Gaps between sequences are indicated by dots (.).
A vertical bar (|) indicates identity between sequences. The start codon (ATG) of the p34 ORF is bold. The sequences of the primers are shown in shaded boxes. Arrows indicate M. tuberculosis and M. bovis and M. avium subsp.paratuberculosis
And point mutations between M. avium ss.

FIG. 2: Primers U1, U2 of the us-p34 region,
Amplification by U3, U4 and U9.

FIG. 3. Novel us-p34 sequence (5 ′ to 3 ′ orientation).
The primers used to obtain the sequence (U2-U1; U
U1-U9; U2-U9; U3-U9 or U4-U9) and the size of the amplicon. Additional internal primers (U1, U2, U3) are indicated in the sequence. Sequence changes (point mutations) found in the same species (eg, M. ulcerans) are also shown when known.

FIG. 4. U1-U4 consensus amplification of the us-p34 region of different mycobacterial species.

FIG. 5. Specific and non-specific hybridization.

FIG. 6. Differential reverse hybridization of mycobacterial target amplicons on nylon membranes, disclosing species-specific mycobacterial probes. a) Unlabeled amplification D specific for various mycobacterial species
The NA segment was first transferred onto a nylon membrane (M. tuberc
ulosis (TB), M. avium (AV), M. szulgai (SZ), M.
kansasii (KA), M. xenopi (XE), M. simiae (SI) and M. malmoense (ML)). b) M. tuberculosis (TB ^* ), M. avium (AV ^* ), M. szulg
ai (SZ ^* ), M. kansasii (KA ^* ), M. xenopi (XE ^* ) and
A digoxigenin-labeled amplicon derived from M. simiae (SI ^* ) was hybridized on a nylon membrane. Specific differential hybridization is obtained.

FIG. 7 shows an example of a biochip for specifically detecting M. gordonae.

FIG. 8. Us-p34 of some mycobacteria.
Sequence alignment.

────────────────────────────────────────────────── ─── of the front page continued (51) Int.Cl. ⁷ identification mark FI theme Court Bu (reference) G01N 33/566 C12R 1:32) 33/569 ( C12Q 1/68 a // (C12N 15/09 ZNA C12R 1:32) C12R 1:32) C12N 15/00 ZNAA (C12Q 1/68 F C12R 1:32) C12R 1:32) (72) Inventor Pascal Vannuffel Belgium- 7133 Buvuline, Ludo La Passage Egypt No. 138 F term (reference) 4B024 AA13 CA01 CA09 HA14 4B029 AA21 AA23 BB20 CC03 4B063 QA01 QA18 QQ06 QQ42 QR08 QR32 QR39 QR56 QR62 QR84 QS14 QS25 QS34 QS39 QX01

Claims (18)

[Claims] 1. A method for detecting a non-tuberculous mycobacterial strain (NTM), comprising: (i) providing a nucleotide probe for a non-tuberculous mycobacterial species-specific upstream p34 gene region (us-p34); Reacting the us-p34 with the sample under conditions that selectively produce a nucleotide duplex between the -34 nucleotide probe and the corresponding non-M. Tuberculosis mycobacterial nucleic acid target present in the sample; and (iii) C.) Detecting all nucleotide duplexes containing said p-34 nucleotide probe. 2. The non-M. Tuberculosis mycobacterium species-specific us-p34 nucleotide probe is specific for at least a part of the sequence selected from FIG. 3, or its complement, or the corresponding sequence in which T is replaced by U. The method according to claim 1, which hybridizes specifically. 3. The non-M. Tuberculosis mycobacterium species-specific us-p34 nucleotide probe is selected from the sequence shown in FIG. 3, or a complement thereof, or a corresponding sequence wherein T is replaced by U. A method according to claim 1 or 2. 4. A method for differentially detecting mycobacteria in a sample, comprising: (i) providing at least two different non-tuberculosis mycobacterium species-specific us-p34 nucleotide probes; and (ii) the p-34 nucleotides. Under conditions that selectively generate nucleotide duplexes between the probe and the non-M. Tuberculosis mycobacterial nucleic acid present in the sample,
Reacting p34 with the sample, (iii) detecting all nucleotide duplexes containing the p-34 nucleotide probe, and (v) specific non-tuberculosis mycobacteria from the generated nucleotide duplexes. A method consisting of inferring the presence and identity of a species. 5. A method for detecting a non-M. Tuberculosis mycobacterial strain in a sample, comprising: (i) providing at least two different non-M. Tuberculosis mycobacterial species-specific us-p34 primers; Reacting the us-p34 primer with the sample under conditions that permit selective amplification of the us-p34 sequence in the non-M. Tuberculosis mycobacterial nucleic acid present in (a), and (iv) the amplification product of step (ii). A method comprising detecting 6. A method for differential diagnosis of mycobacteria in a sample, comprising: (i) providing at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. ii) reacting the us-p34 primer with the sample under conditions that permit selective amplification of the us-p34 sequence of the non-M. tuberculosis mycobacterial nucleic acid present in the sample; And (iv) inferring the presence and identity of specific NTMs from the resulting amplification. 7. A method for detecting a mycobacterial strain belonging to the MAC complex in a sample, comprising: (i) using at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. (Ii) the us-p34 primer and the MA in the sample;
Reacting the us-p34 primer with the sample under conditions that permit selective generation of nucleotide duplexes between the C complex mycobacterial nucleic acid targets; and (iv) the mycobacteria belonging to the MAC complex in the sample. A method comprising determining the us-p34 sequence of a nucleic acid. 8. A method for detecting a MOTT mycobacterial strain in a sample, comprising: (i) providing at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (ii) reacting the us-p34 primer with the sample under conditions permitting selective generation of nucleotide duplexes between the us-p34 nucleotide primer and the MOTT mycobacterial nucleic acid target in the sample; Determining the us-p34 sequence of the MOTT mycobacterial nucleic acid in said sample. 9. A novel us-p34 sequencing method in a sample, comprising: (i) providing at least one sense and antisense us-p34 primer selected from Table 1 or 2 as shown in FIG. 2; (iv) us in the mycobacterial nucleic acid target in the sample
The us-p3 under conditions permitting amplification of the p34 sequence.
4) reacting the primers with the sample; (v) determining the sequence of the amplification product obtained in (ii). 10. A method for differentially detecting mycobacteria in a sample, wherein (vi) at least one sense and antisense us-p34 selected from Table 1 or 2 as shown in FIG.
Providing primers; (vii) reacting the us-p34 primer with the sample under conditions that permit selective amplification of the us-p34 sequence in the non-M. Tuberculosis mycobacterial nucleic acid present in the sample; (viii) selectively hybridizing the amplification product obtained in (ii) with at least one non-M. tuberculosis mycobacterial species-specific us-p34 nucleotide probe selected from the group of sequences depicted in FIG. ix) detecting all nucleotide duplexes containing the us-p34 nucleotide sequence, and (x) inferring the presence of a particular non-M. tuberculosis mycobacterial strain from the generated nucleotide duplexes. 11. A non-M. Tuberculosis mycobacterium comprising at least 8 contiguous nucleotides from one of the nucleic acid sequences represented in FIG. 3, or the complement thereof, or the corresponding sequence in which T is replaced by U. Species-specific us-p34
Nucleotide probe or primer. 12. The non-tuberculous mycobacterium species-specific us-p34 of claim 11, selected from Table 1 or 2.
Nucleotide primer. 13. The non-M. Tuberculosis mycobacterium species-specific us-p34 nucleotide probe of claim 11, selected from Table 3. 14. A nucleic acid comprising the sequence selected from FIG. 15. A composition comprising at least one nucleotide probe, primer or sequence according to any of claims 11 to 14. 16. A probe, primer or sequence according to any of claims 11 to 14, or claim 1.
A diagnostic kit comprising a composition according to claim 5. 17. A solid support for detecting mycobacteria, comprising fixing at least two capture probes selected from Table 3 to the support. 18. A solid support according to claim 16 for use in a method according to any of claims 1 to 4 or 10.