JP2001103981A - Kit for diagnosing mycobacterium tuberculosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a kit which allows rapid determination of the drug resistance of Mycobacterium tuberculosis and the infection by the bacterium. SOLUTION: This is a kit for diagnosing Mycobacterium tuberculosis containing a basal plate onto which an olignucleotide which is derived from a gene on the genome of the bacterium involved in the resistance against a drug used for the treatment of tuberculosis, and was covalently immobilized which was synthesized based on base sequences obtained from a wild-type strain of M. tuberculosis sensitive to a drug used for the treatment of tuberculosis and mutants of M. tuberculosis having drug resistance against the agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a kit for diagnosing Mycobacterium tuberculosis, and more particularly to a clinical test for a patient with Mycobacterium tuberculosis, particularly for obtaining information on drug resistance in order to select an appropriate therapeutic drug. The present invention relates to a kit that can rapidly perform drug resistance discrimination and infection determination of a kit. Furthermore, the present invention relates to a kit that can simultaneously determine and classify infection of atypical mycobacteria belonging to the genus Mycobacterium, like M. tuberculosis. Furthermore, the present invention relates to an oligonucleotide and a nucleic acid probe used in the kit and a method for producing the same.

[0002]

2. Description of the Related Art The Mechanism of Drug Resistance Acquisition in Mycobacterium tuberculosis Infection and the Importance of Prior Information on Drug Resistance The drug resistance of Mycobacterium tuberculosis is
It is known to be caused by spontaneous and random mutations. Its frequency varies with an agent, e.g., when viewed in the main drug, rifampicin (RFP): 10 ^-7, streptomycin (SM): 10 ^-6, ethambutol (EB): 10 ^-4, isoniazid (INH): 10 ^{- 6} , Kanamycin (KM): on the order of 10 ^-6 (1,15). No data are available on the frequency of pyrazinamide approved for use in 1996, but point mutations that induce resistant strains have already been reported (1,15).

[0003] It is generally assumed that the number of Mycobacterium tuberculosis on the order of 10 ⁸ is present in the tuberculosis cavity. If INH is administered as a single agent to a susceptible patient having 10 ⁹ bacteria, on average,
10 ³ or more bacteria survive as INH resistant bacteria. Although the patient appears to have recovered from this treatment, the disease reappears due to the growth of surviving INH-resistant bacteria. If RFP is used to treat this patient, the bacterium will have a similar mechanism.
The tuberculosis bacterium acquires resistance to RFP, and eventually acquires the resistance to the two drugs INH and RFP. This repetition results in intractable multidrug-resistant tuberculosis bacteria.

To address this problem, tuberculosis treatment has been
The mainstream is two-drug therapy of H and RFP, or triple therapy with one more drug. The use of other drugs, such as RFP, rather than INH alone in the first-line treatment of susceptible patients reduces the frequency of resistant bacteria to almost negligible levels. That is, theoretically, the frequency of emergence of resistant bacteria is 10 ⁻⁶ × 10 ⁻⁷ = 10 ⁻¹³ , and it can be considered that almost no resistant bacteria appear in normal tuberculosis patients. However, if the M. tuberculosis in a patient is resistant to one of the two drugs, it will in principle produce the same result as the single drug, and the probability of the other drug is high. You can think of it as acquiring resistance.

[0005] For this reason, if information on drug resistance is obtained before the start of treatment, it is possible to select the drug to be actually administered after excluding the drug having resistance, and the probability of treatment failure is low. Extremely low. Also, the emergence rate of new resistant bacteria is reduced to almost negligible levels.

[0006] 2. Genes involved in Mycobacterium tuberculosis drug resistance Research on the mechanism of Mycobacterium tuberculosis drug resistance acquisition has been carried out for a long time,
It has been clarified that it is not transmitted by R-plasmids, transposons and the like. Therefore, resistance is not acquired for many drugs at a time, and it is caused by individual mutation at the site where each drug acts on M. tuberculosis genomic DNA.

[0007] Rifampicin (RFP) resistance includes a gene encoding the RNA polymerase β-subunit (rpo
B) It has been shown that point mutations not found in M. tuberculosis (M. tuberculosis without resistance to RFP) exist in the narrow region above (1, 8, 9).

[0008] 16S Ri for streptomycin (SM) resistance
Gene (rrs) encoding bosome RNA and S12 Ribosom
A mutation in the gene encoding protein L (rpsL) is involved, and kanamycin (KM) is involved in a different region of the rrs gene from streptomycin.
(1,10,11,16,22).

It has been found that ethambutol (EB) resistance is related to mutations in the emb operon, and isoniazid (INH) resistance is in addition to the Catalase-peroxidase Gene (KatG), inhA and aphC / oxyR genes. The above mutations are involved (1,8,12,13,14).

[0010] In M. tuberculosis resistant to drugs, point mutations not found in wild-type M. tuberculosis have been detected in specific regions of the genes involved in these resistances. Conversely, point mutations on these genes have been detected. It has been clarified that the detection of a drug leads to the differentiation of drug resistance.

3. Current status of infection determination and drug susceptibility testing of tuberculosis and mycobacteria Mycobacterium (M. tuberculosis, leprosy, plaque, etc.)
Significant progress has been made in recent years on bacteriological testing of. In general, Mycobacterium bacteria are classified into three groups: Mycobacterium tuberculosis, atypical mycobacteria, and other special mycobacteria. Identification of atypical mycobacteria includes biochemical characterization or nucleic acid amplification. The method for detecting Mycobacterium tuberculosis is roughly classified into a culture method and a nucleic acid amplification method. The culturing method and the biochemical property test require 3 to 8 weeks to obtain the results, and the nucleic acid amplification method currently used requires much time and labor since tests are performed individually. Usually, it is a culture method that can perform a drug sensitivity test.

3-1. Culture method The culture method has been improved in various ways to improve detection sensitivity and shorten the number of culture days, and immunochromatography using an antibody against a tuberculosis-specific antigen has rapidly become a simple identification method. It is becoming popular. The procedure is to inoculate the sample on the slide into the medium without isolation and incubate it, or to grow the bacterial solution obtained by the isolation and culture on a solid medium or liquid medium to check for the presence of colonies and the growth of the bacteria. Thereafter, the resistance is discriminated by inoculating the medium into a sensitive medium for each drug. Since the detection of M. tuberculosis and the susceptibility test for drugs based on such a culture method usually take too much time, such as 6 to 8 weeks, to obtain results, various improved methods have been developed, particularly for M. tuberculosis culture. . For example, BACTEC system, MGIT method,
Septi-Check AFB method (Becton Dickinson), Bac
t / ALERT method (OrganonTechnica) and others (5,6,7).
However, even if these improved methods are used, it takes at least 2-3 weeks to identify drug resistance, and it cannot be said that the method can respond to the needs of medical sites in view of the mechanism of resistant bacterial development.

3-2. Nucleic acid amplification method The nucleic acid amplification method is a PCR method (Polymerase Chain Reaction method, Ro
che) or a method similar to MTD (using reverse transcriptase and RNA polymerase, Chugai Pharmaceutical) to amplify the gene (DNA or RNA) using a probe and to examine the presence of the M. tuberculosis gene. About 7 hours including sample preparation
(The processing time of only PCR and MTD is about 2 hours), which enables detection of Mycobacterium tuberculosis, which has many advantages as a rapid test method. Although these methods cannot be said to have been established as methods for examining drug resistance, a certain degree of drug resistance test is also possible by technical application.

For example, Telenti et al. Sequenced the rpoB gene with respect to rifampicin resistance using an automatic sequencer and identified the mutation site (8). However, this method requires the determination of the sequence of many genes and is expensive. Therefore it is not practical. For this reason, PCR-SSCP (PCR-Single Stran
d Conformational Polymorphism method, hetero-duplex method, PCR-RFLP (PCR-Restriction Fragment Len)
gth Polymorphism) method and molecular beacon method were developed.

The PCR-SSCP method utilizes the fact that DNA or RNA changes the electrophoretic mobility by a higher-order structure depending on its base sequence. In other words, the rpoB gene is amplified using nucleic acid amplification (PCR) using oligo primers, and then thermally denatured.Then, electrophoresis is performed to determine whether or not a mutation has occurred in the base based on the difference in the mobility of the obtained DNA. There is (17).

[0016] PCR-RFLP is a method of amplifying the rpoB gene site by PCR, separating fragments cut with restriction enzymes by electrophoresis, and classifying the obtained electrophoresis patterns to determine the presence or absence of resistance. There is (18).

The hetero-duplex method is similar to the PCR-SSCP method.
This is a method in which annealing is performed with the A fragment, and then the difference in the electrophoretic mobility is determined on gel electrophoresis.

The molecular beacon method uses an oligonucleotide having a stem-loop structure in which a fluorescent substance and a quencher are bonded at the 5 'end and the 3' end, respectively. It is an application of the fact that the physical distance between the fluorescent substance and the quencher increases when a double strand is formed (19).

These methods are based on the DNA which can be donated to the test.
Difficulties include limited fragment length, restriction sites required for mutations associated with drug resistance, and the need to perform several experiments in parallel to detect mutation patterns . Various efforts have been made to detect mutations, and research and development of rapid test methods have been attempted, but no effective and rapid drug resistance test methods have been reported so far.

3-3. DNA microarray technology A DNA microarray technology in which oligonucleotide probes are arranged in an array on silicon at a high degree of integration, and the technology of AFFYMAX is generally introduced as a representative (27, 28). This technique has one or two interrogations (INTERROGATION) at appropriate positions with respect to a DNA sequence as a reference, and a corresponding sequence (sense or antisense) probe in which different types of bases are assigned to the interrogations. Are arranged to form an array. Since the probes are shifted while overlapping on the reference sequence, the number thereof depends on the length of the sequence to be examined. In principle, the DNA sequence of the test sample is determined by applying the test sample DNA to a probe on the chip and determining the fusion thereof, in comparison with the reference base sequence.

Although it is suggested in the patent application of AFFYMETRIX that this method is applicable to the drug resistance test of M. tuberculosis, the examples are not described in a clear form. D
Although the NA microarray technology is an excellent method for decoding a large number of unknown sequences, its suitability in cases where an immediate diagnostic result is required, such as drug resistance differentiation, is questionable.
Chip fabrication costs, precise array construction on silicon chips, and high-resolution devices (scanners) for reading results remain issues from the technical and economical aspects.

3-4. Line Hybridization Assay Applying PCR Method Recently, Line Hyb for the purpose of detecting rifampicin resistance
ridization Assay (Inno-Lipa Rif.TB kit, Innogeneti
cs, Belgium) (26, not introduced to Japan). This is obtained by adsorbing four fragments of the rpoB gene sequence containing a frequently occurring point mutation and five fragments obtained from the rpoB gene sequence site of a rifampicin-sensitive strain on a filter as a detection probe. It is. Mycobacterium tuberculosis DNA amplified by PCR is applied to this probe, and their fusion is detected by gel electrophoresis. When a band appears in any of the probes containing the four mutations and when no band can be detected in the probe derived from M. tuberculosis, it is regarded as rifampicin resistance. The test takes less than 96 hours after receiving the sample. Although this method is a significant improvement over conventional methods, there are still some challenges in meeting the needs of diagnostic accuracy and speed in medical practice.

Since the method is such that oligonucleotides are adsorbed on a filter, the number of lines that can be arranged per unit area is limited. For example, in the case of a mutant sequence related to rifampicin resistance, if the coverage of the resistance sequence is to be increased, the number of positive sequences alone will increase to nearly 20 types, and many types of filters must be prepared.
In the Inno-Lipa Rif.TB kit, in order to avoid such a problem, five sequences derived from wild-type M. tuberculosis having different positions on the genome are placed. The method of judging is adopted. However, it is unclear whether the mutation is necessarily associated with resistance and may produce spurious resistance information. In order to further expand this kit for multidrug resistance, there is a size problem, and in principle, a large amount of sample DNA is required. Therefore, it may take time to obtain DNA from cells.

4. Identification of drug-resistant M. tuberculosis and countermeasures for treatment In order to counteract drug-resistant bacteria, a rapid drug resistance identification method (ideally, information on drug resistance is obtained before treatment) is desirable and inevitable The development of new test methods based on genetic (DNA or RNA) diagnosis is expected.

As mentioned above, many of the genes involved in drug resistance of major antituberculosis agents have been elucidated by many researchers including the research group of the present inventors. For example,
Mutations associated with rifampicin resistance are concentrated in a core region of only 81 bases on the rpoB gene, and mutations in this region account for 95% of the total (8,9). It has been found that isoniazid resistance involves katG, inhA, and aphC / oxyR genes in addition to katG (13, 21). In addition, streptomycin resistance involves multiple distant regions on the rrs gene and the rpsL gene (10,11), while kanamycin resistance involves a different region of the rrs gene from streptomycin. Ethambutol has been found to be involved in mutations on the emb operon (12,23). Analyzing data on mutations in specific regions of these genes can explain about 70-95% of the development of resistance to each drug, and using this information effectively, a rapid differentiation method for drug resistance can be used. Being developed will have crucial implications for TB control.

If it becomes possible to obtain information on drug resistance before the start of treatment, it is possible to not only give an effective prescription for treatment but also prevent the emergence of resistant bacteria, which is a recent social problem. It can lead to a dramatic reduction in nosocomial and outbreak infections.

[0027]

The most important consideration in addressing the problem of tuberculosis is to be able to provide advance information on the main drugs for treating patients.

The current general drug resistance test method is a culture-based method, which requires a period of at least two to three weeks before a colony of M. tuberculosis is formed, and further inoculates a drug-sensitive medium. It is necessary to check for tolerance. Although various approaches have been made for the nucleic acid amplification method and its improved method, there are drawbacks in its simplicity and accuracy, and it is not in a state where it can sufficiently respond to the needs of medical sites.

Line Hybridization Ass, a new technology
Although ay incorporates various improvements to these disadvantages, the problem is that it is difficult to adsorb too many probes in one test kit, and the concentration of the sample to be applied tends to be small like a smear. That is, in order to enable a multidrug resistance test and to increase the mutation detection rate, an improvement is required that allows the number of nucleotide sequences having a mutation to be increased. Also,
There is also a need for an improved system that can be tested with a small amount of sample when the fusion state is good. It is desirable that the detection time be further reduced.

It is an object of the present invention to provide a new method for rapidly drug-diagnosing drug resistance of M. tuberculosis, which solves most of the problems of currently available test methods, and a kit for use in the method. Another object of the present invention is to provide a kit and a method designed to simultaneously determine tuberculosis and atypical mycobacterial infection.

[0031]

DISCLOSURE OF THE INVENTION The present invention provides a wild-type tuberculosis bacterium which is susceptible to a drug used in the treatment of tuberculosis, the gene being derived from a gene on the genome of the tuberculosis bacterium involved in resistance to the drug. A tuberculosis diagnostic kit comprising a substrate on which an oligonucleotide synthesized based on a base sequence obtained from each of the mutant tubercle bacilli having resistance to is immobilized by a covalent bond, which is derived from a test microorganism. This is a diagnostic kit for performing drug resistance discrimination or infection determination of Mycobacterium tuberculosis by hybridization with a nucleic acid to be used. Furthermore, a nucleic acid derived from a test bacterium including a substrate on which an oligonucleotide synthesized based on a base sequence obtained from a gene on the genome of a bacterium belonging to the genus Mycobacterium other than Mycobacterium tuberculosis is immobilized by covalent bonds It is also a kit for identifying Mycobacterium infection by hybridization with E. coli.

The present invention also relates to a mutant gene having a point mutation relating to the resistance, which is provided by a mutant Mycobacterium tuberculosis having resistance to any of rifampicin, streptomycin, kanamycin, isoniazid and ethambutol, and And a synthesized oligonucleotide designed to have a length of 12 to 24 bases including the mutation site. The present invention further provides a gene of a wild-type Mycobacterium tuberculosis in which the position on the genome of Mycobacterium tuberculosis corresponds to the base sequence of each of the nucleotide sequences on the 5 'side and / or 3' side of the nucleotide sequence of the gene from which the oligonucleotide is derived. An oligonucleotide having a nucleotide sequence obtained by extending or shortening the sequence, wherein the mutation site contained in each original nucleotide sequence is not substituted is provided.

[0033] The present invention also relates to an oligosaccharide derived from a wild-type gene of a wild-type Mycobacterium tuberculosis susceptible to rifampicin, streptomycin, kanamycin, isoniazid and ethambutol, wherein the gene is derived from a mutant gene involved in the resistance of the drug. Provided is an oligonucleotide designed and synthesized to have a length of 12 to 24 bases obtained from a gene sequence whose position on the genome of Mycobacterium tuberculosis corresponds to the base sequence of the nucleotide. Oligonucleotides include DNA, RNA or peptide nucleic acids (PN
A) is included.

The diagnostic kit of the present invention is characterized by containing the following combinations of (a) and (b). That is, (a) the number of bases derived from a mutant gene having a point mutation relating to drug resistance, which is present in a mutant tubercle bacillus having resistance to any of rifampicin, streptomycin, kanamycin, isoniazid and ethambutol, is 12 to The base sequence shown in SEQ ID NOS: 1 to 66 as a representative example of the sequence designed in No. 24. (B) a sequence designed to have 12 to 24 nucleotides derived from a gene of wild-type Mycobacterium tuberculosis having susceptibility to rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol; The base sequence shown in 101. The present invention can further include, in the diagnostic kit, the following oligonucleotides (c) and (d) in addition to the synthetic oligonucleotides, or independently provide the oligonucleotides in the diagnostic kit. (C) a positive control derived from a gene region having a species-specific nucleotide sequence that is highly conserved in each of Mycobacterium tuberculosis and atypical mycobacteria and is not homologous between species of Mycobacterium; (D) in the base sequence of the oligonucleotide of (c), at least one base and at least 2 bases of the length of the base sequence;
An oligonucleotide as a negative control having a base sequence containing a base substitution within a range of less than 0%.

The present invention also provides a method for obtaining a labeled nucleic acid probe derived from the nucleic acid of a test microorganism for determining drug resistance and determining infection with atypical mycobacteria and Mycobacterium tuberculosis. That is, DNA is extracted from the cells of a test microorganism isolated from sputum or a test microorganism obtained from a test microorganism obtained by culture, and using this DNA as a template, either the 5 ′ side or the 3 ′ side A nucleic acid probe labeled with a fluorescent substance is provided by performing nucleic acid amplification using a labeled primer labeled at the end with a fluorescent substance or a hapten. Further, the present invention provides a two-stage amplification method for effective gene amplification, and a primary primer and a secondary primer related thereto.

The present invention also relates to a pair of oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 134 and 135 for amplifying the rpoB gene, and SEQ ID NO: 136 for amplifying the rrs gene, which are used for preparing the nucleic acid probe.
An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOs: 138 and 139 for amplifying the rpsL gene;
SEQ ID NOS: 140 and 14 for amplifying the inhA gene
An oligonucleotide pair having the base sequence shown in 1,
an oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOs: 142 and 143 for amplifying the tG gene, and e
SEQ ID NOs: 144 and 145 for amplifying the mbB gene
The present invention provides a primary primer, which is any one or any combination selected from oligonucleotides having the nucleotide sequence shown in (1).

[0037] The present invention further provides a method for hybridizing these nucleic acid probes to oligonucleotides immobilized on a substrate and obtaining oligonucleotides derived from a gene associated with drug resistance of a mutant tuberculosis bacterium having a completely identical nucleotide sequence. A method for distinguishing drug resistance by identifying nucleotides, and a method for judging tuberculosis and atypical mycobacterial infection by comparing binding to a positive control and a negative control are provided. By using the present invention for a method of diagnosing tuberculosis infection, it is possible to simultaneously perform both drug resistance discrimination and infection determination about 6 hours after receiving a test sample from a patient.

[0038]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, sensitivity and resistance to a drug are relative concepts, and the degree of sensitivity or resistance is not limited. If a mutant M. tuberculosis grows better than a wild-type M. tuberculosis in the presence of a certain concentration of the drug, the mutant M. tuberculosis is resistant to a drug to which the wild-type M. tuberculosis is susceptible.

In the present invention, the drug is not particularly limited as long as a mutant tubercle bacillus whose sensitivity is lower than that of a wild-type tubercle bacillus can be used. Examples of the drug include rifampicin, streptomycin, kanamycin, isoniazid, ethambutol and the like. Is a major drug. Usually, in Mycobacterium tuberculosis, 50 μg / μL rifampicin, 20 μg / μL streptomycin, 100 μg / μL
Of kanamycin, 1 μg / μL of isoniazid, or
If it can grow in the presence of 5 μg / μL ethambutol, it can be said that it is resistant to these drugs.

The mutant M. tuberculosis having resistance to a drug may have resistance to only a single drug or may have resistance to two or more drugs. Good.

The construction of the kit of the present invention and a method for producing the kit, a method for identifying drug resistance and a method for identifying tuberculosis and atypical mycobacterial infection will be described below.

Hereinafter, the structure of the kit of the present invention and a method for producing the kit will be described.

<1> Synthetic Oligonucleotide Used in the Present Invention The synthetic oligonucleotide used in the present invention is derived from a gene on the genome of Mycobacterium tuberculosis involved in resistance to a tuberculosis therapeutic agent, and is sensitive to wild-type tuberculosis tuberculosis. Oligonucleotides synthesized based on base sequences obtained from bacteria and mutant tubercle bacilli having resistance to any drug. Hereinafter, these oligonucleotides are also referred to as “capture oligos (capture oligos)”, capture oligos derived from wild-type Mycobacterium tuberculosis are referred to as “wild-type capture oligos”, and capture oligos derived from mutant Mycobacterium tuberculosis are also referred to as “mutant capture oligos”.

Other synthetic oligonucleotides of the present invention include:
Synthetic oligos derived from the region of the gene having a species-specific nucleotide sequence that is consistent between drug-sensitive wild-type M. tuberculosis and drug-resistant M. tuberculosis, but is not homologous between Mycobacterium species Nucleotides (hereinafter, also referred to as “positive control oligos”) and, in the base sequence of the positive control oligo, at least one base and an artificial base within a range of less than 20% of the number of bases of the sequence. (Hereinafter, also referred to as “negative control oligo”). These oligonucleotides are used to determine the infection of M. tuberculosis bacteria and atypical mycobacteria, respectively.

Hereinafter, the oligonucleotide used in the present invention will be described. (1) Capture Oligo Design Mycobacterium tuberculosis acquires drug resistance by a point mutation of a specific gene. As described above, several genes involved in such drug resistance have been identified, and the mutation sites in each gene sequence have been identified (1,8,9,10 to 15). ). Some of these genes, such as the rrs gene, are involved in resistance to different types of drugs (streptomycin and kanamycin). The gist of the present invention is to determine whether or not a tubercle bacillus to be tested has drug resistance based on the presence or absence of a mutation in a gene related to drug resistance as described above.

In the present invention, the gene relating to drug resistance includes not only a gene of a mutant tubercle bacillus having drug resistance but also a gene of a wild type tuberculosis bacterium having a nucleotide sequence identical to that of the gene except for a mutation site. Point. The relationship when the positions of the mutant gene and the wild-type gene related to drug resistance on the M. tuberculosis genome are identical is represented by the term "corresponding".

The presence or absence of a mutation in a gene involved in drug resistance can be determined by detecting a nucleic acid fragment probe having a part of the nucleotide sequence of a gene involved in drug resistance in the chromosomal DNA of Mycobacterium tuberculosis (hereinafter referred to as "nucleic acid probe") and a wild-type capture oligo. The mutant oligo can be hybridized with the mutant capture oligo, and the determination can be made based on which of the capture oligos is hybridized. In other words, it is possible to know exactly which drug has resistance based on the position of hybridization on the substrate.

The capture oligo can be prepared as a synthetic oligonucleotide having a nucleotide sequence of 12 to 24 nucleotides including a mutation point in the nucleotide sequence of a gene involved in drug resistance. When designing a capture oligo, the position of the mutation point in the capture oligo is
It is not particularly limited as long as it can hybridize with the nucleic acid probe, but usually it is preferably present in the central part of the capture oligo. If the capture oligo is too short, it will be difficult to detect hybridization,
If it is too long, hybridization will not be inhibited by the mutation point, so the range of 12 to 24 bases is preferable. However, the experiment of the present invention showed that the optimization of the oligo length mainly depends on the characteristics of the sequence (content of a specific base, repeat of the same base), and those having good binding properties may be short chains. Has been confirmed.

Examples of the base sequence of the capture oligo are shown in SEQ ID NOs: 1 to 101 and Table 1. Among the capture oligos, SEQ ID NOS: 1 to 66 are mutant capture oligos, and SEQ ID NOs: 67 to 101 are wild type capture oligos. These capture oligos were designed based on published literature and data analysis results on the frequency of drug resistance obtained through the inventors' own tests, and should cover a wide range of point mutations that occur for each drug. Can be raised to the level that can be done.

[0050] Differentiation of resistance to a particular drug can be carried out by selecting an oligonucleotide from these and subjecting it to a test. Capture oligo length is about 14 ~
The size was set to 22 bases, and 16 to 17 bases were set as the central size from the viewpoint of hybridization efficiency. In addition, in addition to the nucleotide sequences shown in SEQ ID NOs: 1 to 66, 5 ′ of each nucleotide sequence
An oligonucleotide having a base sequence obtained by extending the wild-type Mycobacterium tuberculosis gene sequence corresponding to each base sequence on the side or 3 ′ side or both, or the 5 ′ side of the base sequence shown in SEQ ID NOs: 1 to 66 Alternatively, an oligonucleotide having a shortened base sequence on the 3 ′ side or both may be used as the capture oligo. In addition, one of the 5 ′ side and the 3 ′ side of the base sequence shown in SEQ ID NOs: 1 to 66 may be extended and the other may be shortened, but in any case, the capture oligo is 12 to 2
The range is 4 bases. The nucleotide sequence of a gene related to drug resistance of wild-type Mycobacterium tuberculosis can be obtained from the GenBank / EMBL / DDBJ database or the like. The accession number of each gene is shown below. The sequence of the extended oligonucleotide can be designed according to these sequences.

RpoB: MTCI376 rrs: MTRRNOP rpsL: MTV040 inhA: MTCY277 katG: MTKATGCP embB: MSGY126

With regard to isoniazid resistance, aphC / oxyR
Although it is possible to design from the gene region, the contribution to the frequency of drug resistance is low, and by targeting inhA and katG, almost the same detection probability (coverage) is achieved. It is considered unnecessary to use a gene-derived capture oligo. However, aphC / oxyR
It is safe to use a gene-derived capture oligo, and if resistance in the aphC / oxyR gene that cannot be covered by mutations derived from other genes is found in the future, incorporate the mutant sequence and the corresponding wild-type sequence as a capture oligo. Is preferred.

In addition, since some genes have a plurality of mutation sites related to drug resistance, a capture oligo corresponding to each mutation site can be prepared. The inventors retrospectively (retrospe) the oligonucleotide sequences designed in the present invention against the drug resistance gene sequence database accumulated from their own findings and literature reports.
ctive), the expected detection rate is as shown in Table 2.

The capture oligo designed in the present invention well reflects the results of research and investigation up to the filing date. However, if additional information is obtained on these drug-resistant mutant sequences, New capture oligos will be designed based on the methods described in this application, which are included in the claims of this application.

[0055]

[Table 1]

[0056]

[Table 2]

Synthesis of oligonucleotide, preparation of chromosomal DNA described later, hybridization, PCR
Techniques are well known to those of ordinary skill in the art (Maniat
is, T. et al., "Molecular Cloning A Laboratory Manu
al, Second Edition ", ColdSpring Harbor Laboratory
Press (1989)).
Oligonucleotides can be synthesized using a commercially available DNA synthesizer. When the oligonucleotide has a hairpin structure, a loop structure or a steric structure that prevents hybridization with other test nucleic acids,
The three-dimensional structure can be released by substituting one or more nucleotides constituting the oligonucleotide with inosine or a nucleic acid not paired with any nucleotide.

(2) Design of Control Oligo The determination of whether the bacterial infection in the patient is a Mycobacterium tuberculosis infection or an atypical mycobacterium infection is based on whether the test bacterium is a Mycobacterium tuberculosis or an atypical mycobacterium. This can be done depending on whether or not each has a unique gene sequence. This determination is performed by hybridization with a positive control oligo. A negative control oligo is used for the purpose of confirming that the hybridization between the oligonucleotide and the nucleic acid probe is specific, that is, that no false-positive hybridization signal is generated.

As a typical model of the nucleotide sequence of the positive control oligo, the nucleotide sequence shown in SEQ ID NO: 102 or 160 for infection or identification of M. tuberculosis, the nucleotide sequence of the negative control oligo is 103 or 161,
Similarly, Mycobacterium avium complex has SEQ ID NOs: 162 and 165, M. kansasii has SEQ ID NOs: 164 and 163,
M. xenopi has SEQ ID NOs. 206 and 219, M. ulcerans has SEQ ID NOs. 207 and 220, and M. szukgai has SEQ ID NO.
8 and 221, and M. simiae in SEQ ID NOs: 209 and 222
However, in M. shimoidei, SEQ ID NOs: 210 and 223 correspond to M. scr.
ofulaceum has SEQ ID NOS: 211 and 224 as M. nonchromog
en is SEQ ID Nos. 212 and 225; M. marinum is SEQ ID Nos. 213 and 226; M. malmoense is SEQ ID Nos. 214 and 2
27, M. intracellular has SEQ ID NOs: 215 and 228
However, for M. gordonae, SEQ ID NOs: 216 and 229
uitum includes SEQ ID NOs: 217 and 230, and M. chelonae includes SEQ ID NOs: 218 and 231. On the other hand, the base sequence of the negative control oligo is one in which one or more bases have been substituted and which has 80% or more homology. The range of nucleic acid amplification is set so that the nucleic acid probe obtained from the test sample DNA also covers this region.

The results of hybridization of a nucleic acid probe to a capture oligo derived from wild-type Mycobacterium tuberculosis for a multidrug resistance test may already provide sufficient information to determine M. tuberculosis infection. Comparison of the positive and negative controls further confirms the determination of M. tuberculosis infection and is clearly distinguishable from infection with other Mycobacterium bacteria.

<2> Production of Substrate Used in the Present Invention The material of the substrate on which the oligonucleotide is immobilized is not particularly limited as long as the oligonucleotide can be immobilized stably. And synthetic resins such as plastic. The form of the substrate is not particularly limited, and examples thereof include a plate shape and a film shape. A substrate having a uniform and flat surface is suitable.

The oligonucleotide is immobilized on the substrate by
Although techniques used in ordinary hybridization methods such as physical adsorption, electrical bonding or molecular covalent bonding can be used, in the present embodiment, a substrate having a carbodiimide group or an isocyanate group on the surface is used. (JP-A-8-23975), a method of irradiating ultraviolet rays and immobilizing oligonucleotides by covalent bonds was employed. This is because the substrate having a carbodiimide group or an isocyanate group on the surface was irradiated with ultraviolet rays, thereby succeeding in significantly improving the immobilization efficiency.

When spotting oligonucleotides,
If the spot amount of the oligonucleotide is too small, sufficient reactivity between the oligonucleotide and the nucleic acid probe cannot be sufficiently ensured, and the determination may be difficult. In addition, spotting with a high degree of integration is costly at the same time as technical problems, and a more precise and expensive detection device (fluorescent labeling of nucleic acid probes and the determination of the presence or absence of hybridization using chemical coloring, etc.) (Typically a scanner). Therefore,
The oligonucleotide has a diameter of 10 to 1,000 on the surface of the substrate.
It is preferable to fix the size to 0 μm. The oligonucleotide can be immobilized by, for example, spotting an oligonucleotide solution on a substrate using a spotting machine. Oligonucleotide solutions are usually spotted approximately circularly.

Each oligonucleotide is spotted on a single substrate at a plurality of spots, and the spots are preferably arranged in a grid. The capture oligo containing the point mutation and the corresponding wild-type strain-derived oligonucleotide are preferably arranged for easy comparison. The number of spots is preferably 1600 or less in total per cm ² if the spot size is 1000 μm in diameter, and preferably 40 × 40 or less in the case of spotting in a square shape. If the spot size is 10 μm in diameter, cm ²
In the case of spotting in a square shape, the spots are preferably 20 × 20 or less. Also,
When the vertical and horizontal sizes are different, the number of vertical and horizontal directions may be adjusted according to the shape.

<3> Arrangement of Oligonucleotides on Substrate The arrangement of each oligonucleotide on the substrate was made by comparing the wild-type capture oligo with the mutant-type capture oligo in order to easily distinguish the resistance or sensitivity of M. tuberculosis to each drug. It is preferable to arrange the capture oligos side by side so that they can be compared with each other. The arrangement of the spots on the substrate is usually within 1 cm ² .

For example, if the drug is rifampicin,
A mutant capture oligo derived from a rifampicin-resistant mutant tubercle bacillus and a wild-type capture oligo corresponding to the capture oligo are arranged at positions where they can be compared. When a plurality of types of mutant capture oligos are used, they are arranged side by side. When there are a plurality of medicines, the above arrangement may be arranged collectively for each medicine.
Drugs include rifampicin, streptomycin,
One or more selected from the group consisting of kanamycin, isoniazid, and ethambutol. Among these, preferred combinations include a combination of rifampicin and isoniazid, or a combination containing all of rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol.

Further, in order to facilitate determination of tuberculosis and infection with atypical mycobacteria, it is preferable to arrange positive controls and negative controls side by side so that they can be compared.
FIG. 1 shows an example of the arrangement of each oligonucleotide. This example is designed so that the resistance of all five drugs of rifampicin, streptomycin, kanamycin, isoniazid and ethambutol can be simultaneously discriminated, and the infection of M. tuberculosis can be determined. When only one agent, for example, for distinguishing resistance to rifampicin, the “R” in FIG.
What is necessary is just to manufacture the part of the substrate enclosed by the above. Also, 2
When discriminating the resistance to an agent such as rifampicin and isoniazid, a substrate having both a portion surrounded by "R" and a portion surrounded by "I" in FIG. 1 may be prepared.

<4> Preparation of Nucleic Acid from Test Sample and Preparation of Nucleic Acid Probe Nucleic acid can be prepared from the test sample in the same manner as in the usual method for preparing nucleic acid from bacteria. For example, DNA
Is the US Department of Healthand H
According to the method (25) introduced by Uman Services), bacteria can be separated from sputum by alkali treatment, and the genome of the bacterial cell can be disrupted and DNA extracted using the method of Jacobs et al. (2
Four). Extraction of DNA from cells obtained by culturing
This can be done using the method of acobs et al. While these are all standard test methods, there are many alternatives,
Either may be adopted.

Based on the obtained DNA, a nucleic acid probe to be used for discriminating drug resistance or determining infection is prepared. A nucleic acid probe can be prepared by amplifying a nucleic acid using a primer designed according to the base sequence of the capture oligo or control oligo. The nucleic acid probe is usually DNA, but may be RNA. As a method of nucleic acid amplification, for example, PCR
(Polymerase Chain Reaction) by DN
RN by the method of amplifying as A or the in vitro transcription method
A includes a method of amplifying.

When nucleic acid amplification is performed by PCR, in order to enhance the specificity of the amplification reaction, first amplification is performed using primary primers that amplify a wider region than the target nucleic acid probe, and the amplified DNA is subjected to amplification. It is preferable to perform nucleic acid amplification using a primer for obtaining a target nucleic acid probe as a template (two-step nucleic acid amplification). The primary nucleic acid amplification may be omitted depending on the amount and purity of the sample.

The primer used in the final PCR is designed so that the nucleic acid probe contains a base sequence complementary to the capture oligo or control oligo except for the mutation site. The nucleic acid probe may be longer or shorter than the capture oligo or control oligo as long as hybridization is possible.

Since some genes have a plurality of mutation sites related to drug resistance, a nucleic acid probe corresponding to each mutation site can be prepared. By labeling the primers used in the final PCR in advance, a labeled nucleic acid probe can be obtained. The nucleic acid probe may be labeled during or after the PCR reaction. As the labeling substance, a labeling substance similar to a probe used for ordinary hybridization, such as a fluorescent substance or a hapten, can be used. Specifically, for example, fluorescein (FITC), rhodamine (Rodamine), phycoerythrin (PE), Texas Red, cyanine-based fluorescent dyes and the like as fluorescent substances, and biotin (BIOTI
N), digoxigenin (Dig), dinitrophenyl (DNP), fluorescein and the like.

Table 3 shows examples of the first PCR primer and the second PCR primer used for the two-step amplification. Table 4 shows the nucleotide sequence of the nucleic acid probe amplified by each primer.

[0074]

[Table 3]

The nucleic acid probe corresponding to the positive control oligo having the nucleotide sequence shown in SEQ ID NO: 102 and the nucleic acid probe corresponding to the negative control oligo having the nucleotide sequence shown in SEQ ID NO: 103 have the nucleotide sequences shown in SEQ ID NOS: 104 and 105, respectively. Can be prepared by PCR using a genomic DNA of Mycobacterium tuberculosis as a template by using a primer having SEQ ID NO: 160 and SEQ ID NO: 161 correspond to Mycobacterium tuberculosis genomic DNA, and SEQ ID NO: 162 and SEQ ID NO: 165 correspond to Myco.
bacterium avium complex genomic DNA, SEQ ID NO: 16
No. 4 and SEQ ID NO: 163 are Mycobacterium kansasii genomic DNs
A, SEQ ID NO: 206 and SEQ ID NO: 219 are Mycobacterium x
enopi genomic DNA, SEQ ID NO: 207 and SEQ ID NO: 220 are My
cobacterium ulcerans genomic DNA, SEQ ID NO: 208 and SEQ ID NO: 221 are Mycobacterium szukgai genomic DNA, SEQ ID NO: 209 and SEQ ID NO: 222 are Mycobacterium simiae genomic DNA, SEQ ID NO: 210 and SEQ ID NO: 223 are Mycobacte
rium shimoidei genomic DNA, SEQ ID NO: 211 and SEQ ID NO: 224 are Mycobacterium scrofulanse genomic DNA, SEQ ID NO: 212 and SEQ ID NO: 225 are Mycobacterium nonchromog
en Genomic DNA, SEQ ID NO: 213 and SEQ ID NO: 226 are Mycob
acterium marinum genomic DNA, SEQ ID NO: 214 and SEQ ID NO: 227 are Mycobacterium malmoense genomic DNA, SEQ ID NO: 215 and SEQ ID NO: 228 are Mycobacterium intracellu
lar genomic DNA, SEQ ID NO: 216 and SEQ ID NO: 229 are Myco
The bases shown in bacterium gordonae genomic DNA, SEQ ID NO: 217 and SEQ ID NO: 230 are Mycobacterium fortuitum genomic DNA, and SEQ ID NO: 218 and SEQ ID NO: 231 are Mycobacteri
Using um chelonae genomic DNA as a template, the nucleic acid probes corresponding to these positive and negative controls
Prepared using primers 4 and 205.

The primer for preparing the nucleic acid probe can be included in a tuberculosis bacterium diagnostic kit together with the substrate on which the oligonucleotide is immobilized.

<5> Hybridization between oligonucleotide on substrate and nucleic acid probe Hybridization can be performed in the same manner as ordinary nucleic acid hybridization. Hereinafter, a specific method will be exemplified.

The nucleic acid probe was added to a fusion solution comprising a salt solution such as SSC (Standard Saline Citrate), a blocking solution such as sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and an additive for promoting the fusion reaction. Add. When the probe is double-stranded, denaturation is performed by heat or the like. After adding several μL of the nucleic acid probe solution to the substrate, a heating operation (usually at 37 to 50 ° C.) is performed for several hours to form a hybrid between the oligonucleotide immobilized on the substrate and the nucleic acid probe.

On the substrate, 5 × SSC or 3M tetramethylammonium chloride is added and heated (usually 37 to 50).
° C), those that do not form non-specific hybrids are peeled off the substrate, leaving only specific hybrids selectively on the substrate.

For detection of a hybrid, a fluorescent substance or a hapten introduced into a nucleic acid probe is used. When a hapten is used, a solution containing a hapten-recognizing protein or a protein binding thereto and a conjugate (enzyme conjugate) such as alkaline phosphatase or horseradish peroxidase is added to the substrate, and reacted at room temperature for several tens of minutes. Let it. Before performing the binding reaction between the hapten and the enzyme conjugate, by completely covering the region of the substrate other than the region where the oligonucleotide is immobilized with a protein such as casein, the non-specificity of the enzyme conjugate and the substrate is reduced. Can prevent the reactive adsorption reaction. In this treatment, after fixing the oligonucleotide, a solution of a protein such as casein is added onto the substrate,
It can be carried out by leaving at room temperature for several tens of minutes.

After the completion of the binding reaction between the enzyme conjugate and the hapten in the nucleic acid probe, the enzyme conjugate that did not bind to the hapten was washed away with a suitable buffer containing a surfactant to remove the conjugate. Only the enzyme conjugate bound to the hapten in the nucleic acid probe will remain.

To visualize the hybrid, a compound that becomes an insoluble compound only when only the hapten and the enzyme conjugate is present is added, and the production of the insoluble compound is amplified and visualized by an enzymatic reaction. As the compound used at this time, when the enzyme in the enzyme conjugate is alkaline phosphatase, nitro blue tetrazolium chloride (NBT) and BCIP (5-bromo-4-
Chloro-3-indolyl phosphate-p toluidine salt) is used. When the enzyme is horseradish peroxidase, TMB (3,3 ', 5,5'tetramethylbenzidine) or the like can be used.

[0102] The determination of drug resistance based on the obtained hybridization results is carried out by observing the pigmentation or fluorescence at the position where the capture oligo is immobilized. That is, the position where pigmentation or fluorescent coloration is present is the relevant gene. If a positive signal is observed at the position where the wild-type capture oligo is fixed,
If there is no point mutation in the gene involved in drug resistance and a positive signal is observed at the position where the mutant capture oligo is immobilized, it means that the test microorganism has the drug-resistant mutant sequence. .

When a positive signal is observed at the position of the positive control oligo of each of the Mycobacterium tuberculosis or the atypical acid-fast bacterium, it means that the test microorganism is a strain corresponding to the positive control, and If a positive signal is observed at the position of the negative control oligo of each atypical acid-fast bacterium, it means that hybridization was not performed under appropriate conditions. In addition, if a positive signal is observed at both the positions of the positive control oligo and the negative control oligo, it means that hybridization was performed under appropriate conditions.

The kit of the present invention includes a substrate having oligonucleotides immobilized thereon. In addition, the kit of the present invention can include primers or labeled nucleic acid probes for preparing nucleic acid probes, buffers, hybridization reagents such as enzyme conjugates that recognize haptens, and the like.

[0086]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

[0087]

Example 1 Synthesis of Oligonucleotide According to a conventional method, an oligonucleotide synthesizer (Perkin-elmer) was used.
An oligonucleotide having an amino group or a hydroxyl group at the 5 'end was synthesized using Applied Biosystems, deprotected, and dried. The dried oligonucleotide was dissolved using 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer to prepare a 100 pmol / μl oligonucleotide solution. This synthesis method is the same for any oligonucleotide used as a capture oligo. The nucleotide sequences of the synthesized oligonucleotides are shown in SEQ ID NOs: 1 to 145 and 160 to 203 in the sequence listing.

[0088]

Example 2 Spotting of Oligonucleotide to Substrate (When Using Oligonucleotide Having an Amino Group at the 5 'End) Oligonucleotide solution having an amino group at the 5' end 10
Microspotting solution (TeleChem Int
ernational Inc.) was mixed and dispensed on a microtiter plate (Greiner Labotechnik Ltd.). A silanized slide glass (Matsunami Glass Ind., Ltd.) was placed at a predetermined position of the spotting machine, and the spotting machine was operated. After the spotting was completed, steam from hot water was applied to the slide glass for several seconds, and then irradiated with 30 mJ of ultraviolet rays. After re-exposure to steam for a few seconds,
A slide glass was placed on a hot plate to remove moisture. The slide glass was rinsed with a 0.1% sodium dodecyl sulfate aqueous solution and then with distilled water. 3% slide glass
100 mM Tris-HCl containing BSA (bovine serum albumin) (pH 7.
5) The plate was immersed in 100 mM NaCl, 0.1% Triton X-100 at room temperature for 30 minutes for blocking. Then, after drying at room temperature, it was washed with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer. The slide glass was dried again at room temperature and stored dry in a cool, dark place until use.

[0089]

Example 3 Spotting of Oligonucleotide to Substrate (In the Case of Using Oligonucleotide Having a Hydroxyl Group at 5′-Terminal) Oligonucleotide Solution Having a Hydroxyl Group at 5′-End 10 μm
10 μl of a spotting solution (2 M aqueous sodium chloride solution) per 1 l
r Labotechnik Ltd.). A slide glass having a carbodiimide group was placed at a predetermined position of the spotting machine, and the spotting machine was operated.
After the spotting, the slide glass was placed in a dryer at 37 ° C. for 30 minutes. Slide glass 100 mM with 3% BSA
Blocking was performed by immersing in Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% Triton X-100 at room temperature for 30 minutes. Then, after drying at room temperature, it was washed with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer. The slide glass was dried again at room temperature and stored dry in a cool, dark place until use.

[0090]

Example 4 Preparation of Nucleic Acid Probe US Department of Health and Huma
According to the standard method introduced by N Services), bacteria were isolated from sputum by alkaline treatment, and the genome of the bacterial cells was disrupted using the method of Jacobs et al. to extract DNA (24, 25).

The first PCR amplification consisted of 1 unit of Z-Taq polymerase (Takara Shuzo Co., Ltd.), 100 μmol / μl of 5 ′ first primer and 3 ′ first primer, 1 μl each, × 10 reaction buffer 2 μl, 2.5 μl 2 μl of mM Dntp (Gibco BRL) and 0.2 μl of 10 ng / μl genomic DNA 1 μl were added to each tube, and sterile distilled water was added to make a total volume of 20 μl. Set the tubes in a thermal cycler (PTC-200, MJ Research Inc.), 98 ° C: 2 minutes, 98 ° C: 5 seconds, 55 ° C: 5 seconds, 72 ° C.
During a heating cycle at 10 ° C. for 10 seconds and at 72 ° C. for 2 minutes, a program was repeated in which 〜 was repeated 50 times.

In the second step of PCR amplification, 1 unit of Z-Taq polymerase (Takara Shuzo Co., Ltd.), 100 pmol / μl of the 5 ′ second primer and 3 ′ 2 ′ primer with biotin added to 5 ′ were respectively used. 1 μl, × 10 reaction buffer 2 μl, 2.5 mM dNTP (G
ibco BRL) and 0.2 ml of the first PCR reaction solution (1 μl) were added to tubes, respectively, and sterilized distilled water was added to make a total volume of 20 μl. Thermal cycler (PTC-200, MJ Research In
c.) Set the tube at 98 ° C: 2 minutes, 98 ° C: 5
During a heating cycle of seconds, 55 ° C .: 5 seconds, 72 ° C .: 10 seconds, and 72 ° C .: 2 minutes, a program was repeated, which was repeated 50 times.

The combinations of the first primer and the second primer used above are as shown in Table 3. In this example, electrophoresis using an agarose gel described below was performed as a confirmation test, but this is not necessary for actual clinical discrimination.

After the probe synthesis reaction was completed, 1 μl of the reaction product was taken out, and 6 × migration dye (30% glycerol, 0.1%) was added.
1% of 25% bromophenol blue, 0.25% xylene cyanol), 10 mM Tris-HCl (pH 7.5), 1 mM EDTA buffer 4
mixed with μl. After mixing 100% on 25% agarose gel, 25
The electrophoresis was performed under the condition of minutes. After electrophoresis, the gel was immersed in distilled water containing ethidium bromide at 0.5 μg / ml for 15 minutes, and recorded on a photograph by ultraviolet irradiation.

[0095]

[Example 5] Hybridization 5 µl of the nucleic acid probe prepared in Example 3 was added to a 1.5 ml tube, and further added to UniHyb Hybridization Solutio.
20 μl of n (TeleChem International Inc.) was added and mixed to prepare a hybridization solution. After heat-treating this solution at 100 ° C. for 10 minutes, it was immersed in ice for 5 minutes. 10 μl of this nucleic acid probe solution was placed on the substrate on which the oligonucleotides prepared in Examples 2 and 3 were spotted, and a cover glass was placed. Hybrid cassette device (Tel
The substrate was placed in eChem International Inc.), placed in a water bath at 42 ° C., and left for 1 hour in the dark. The substrate is taken out from the hybridization cassette device, and 5 × SSC (0.75M NaCl, 0.
(075 M sodium citrate). The substrate was immersed twice in 5 × SSC at 4 ° C. for 30 minutes, rinsed twice with a 3M aqueous solution of tetramethylammonium chloride at room temperature, and then twice with a 3M aqueous solution of tetramethylammonium chloride at 45 ° C. Finally, the substrate was transferred to a container containing 2 × SSC.

[0096]

Example 6 A substrate was removed from a container for detecting chemical coloration, and the moisture of the portion where the oligonucleotide was not immobilized was wiped off with a paper towel.
Block Ace solution for oligonucleotide immobilization
(Dainippon Pharmaceutical Co., Ltd.) was placed in an amount of 200 μl. After standing at room temperature for 30 minutes, the solution was sucked off with a pipettor. 5 ml of TBST solution (50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 0.05% Tween 2
0) to avidin DH and biotinylated peroxidase (Ve
ctor Laboratories, Inc.) was placed on the nucleotide-immobilized portion on the substrate, and left at room temperature for 30 minutes. Aspirate the solution with a dispenser,
The substrate was transferred to a container containing a TBST solution and washed at room temperature for 5 minutes while allowing the substrate to permeate. Discard the solution and add a new TBST
The solution was added to the container and washed at room temperature for 5 minutes while being permeated. The substrate was taken out of the container, and the water on the portion where the oligonucleotide was not immobilized was wiped off with a paper towel. The TMB solution (Vect
or Laboratories, Inc.) and reacted at room temperature for 20 minutes, and then the substrate was washed with deionized water to stop the enzyme reaction.

[0097]

[Example 7] Judgment of drug resistance After chemical color detection, an OA scanner (NEC Multireader600)
SR) and Photoshop Ver5.0 (Adobe Systems Inc.) and saved as TIFF images in the computer. Based on this image, if there is pigmentation formed by chemical color detection, the oligonucleotide (capture oligo) immobilized on the substrate and the nucleic acid probe in the sample have perfect complementarity, and a double strand is formed. Indicates that it was formed.

Among the arrangements of all the drugs (five drugs) shown in FIG. 1, the test results of each drug resistance discrimination are shown in FIGS. 2 to 21. Other capture oligos related to the resistance are also subjected to the same method. Can be determined.

Each number among the markers in the photograph corresponds to the sequence number. If pigmentation is observed in all of the capture oligos of 67 to 73, and no pigmentation is observed in any of 1 to 19, the nucleic acid flow derived from the sample DNA is diagnosed as sensitive to rifampicin. On the other hand, capture oligos 67-73 derived from M. tuberculosis
If no pigmentation is seen in any of the above and any of the capture oligos 1 to 19 containing the mutant sequence are found to be pigmented, it is diagnosed as rifampicin resistance. For kanamycin, 80, 81 are the wild-type sequence, 34-37
Corresponds to the mutant sequence. In the case of streptomycin, 74 to 79 correspond to the wild type sequence and 20 to 33 correspond to the mutant sequence. In the case of ethambutol, 100 and 101 correspond to the wild type sequence, and 61 to 66 correspond to the mutant sequence. In the case of isoniazid, 82 to 99 correspond to the wild type sequence, and 38 to 60 correspond to the mutant sequence.

FIGS. 2 to 21 show the results of experiments using 21 clinically separated samples. In the case of FIG. 2, pigmentation was observed in all of 67 to 73, and all of 1 to 19 were rifampicin sensitive without deposition.

In the case of FIG. 3, only W1 out of 67 to 73 showed no pigmentation, and only 1 out of 1 to 19 showed pigmentation, which was determined to be rifampicin resistance. The case in FIG.
No pigmentation was observed in only 67 of 7 to 73, and pigmentation was observed in only 3 of 1 to 19, which was determined to be rifampicin resistance.

Hereinafter, 4, 5, 10, 11,
Pigmentation was observed only in 12, 17, and 18, and no or no weak pigmentation was observed in the corresponding wild-type sequences (68, 71, and 72), indicating rifampicin resistance. In FIG. 12, deposition is observed only in the wild type sequences 80 and 81, and no deposition is observed in the corresponding mutant sequence. Therefore, it is determined to be sensitive to kanamycin. 13 to 15 are 34,
Pigmentation is observed only in any of 35, 37, 36,
No pigmentation was observed in the corresponding wild-type sequence (80, 81), indicating that it was kanamycin resistant. In FIGS. 16 to 19, the determination was performed using the sequence derived from the sample DNA amplified by the primers of SEQ ID NOS: 108 and 109, 110, 111, 112 and 113 as probes. In the case of FIGS.
Pigmentation was observed only in sample No. 79, and it was determined that the sample was susceptible to streptomycin.
Is determined to be streptomycin-resistant because no pigmentation is observed in the corresponding wild-type sequence (75). In the figures shown here, the determination is performed individually, but the sequences amplified by the above primers 108 to 113 can be mixed in advance and determined at once. FIG.
In the case of 0, pigmentation was observed only in the wild-type sequences 100 and 101, and no pigmentation was observed in the corresponding mutant sequence. Thus, it was determined that ethambutol was sensitive. In the case of FIG.
Pigmentation was observed at 82 to 99, and the corresponding mutant sequence (38 to
Since no pigmentation was observed in 60), it can be determined that it is susceptible to isoniazid. These image data are 0.24cm on the board
What was observed in a ² (0.4 × 0.6 cm) area was taken into a computer using a scanner and enlarged.

Further, the TIFF image is converted to SCNImage (scion corpor
ation), and the intensity of each spot is measured to make a judgment. Table 4 summarizes the measurement results in this case of FIGS. In Table 4, the intensity of the mutant sequence is higher than the intensity of the corresponding wild-type sequence, and it can be determined that rifampicin resistance is obtained.

[0104]

[Table 4]

[0105]

Example 8 Determination of Drug Resistance In the present invention, in order to demonstrate that the present invention can be carried out even with a sequence shortened or extended on the 5 ′ side and / or 3 ′ side of the sequence shown in SEQ ID NOs: 1 to 66, The oligomers shown in the correspondence table (Table 5) were synthesized according to the method of Example 1.

[0106]

[Table 5]

Sequence numbers 166 to 203 were spotted on a substrate in the same manner as in Example 2 or 3. Further, a nucleic acid probe was prepared in the same manner as in Example 4. Further, hybridization was performed in the same manner as in Example 5,
Chemical color detection was performed in the same manner as in Example 6. The method of Example 7 was used to determine drug resistance. 22 and 23 show the results of rifampicin determination, FIG. 24 shows the results of streptomycin resistance determination, and FIG. 25 shows the results of kanamycin resistance determination. From the determination results of FIGS.
It can be seen that a sequence shortened or extended on the 5 ′ side and / or 3 ′ side of the sequence shown in FIG. 66 can be used.

<Reference Documents> Suzuki Sadahiko et al., "Testing for Drug Resistance of Mycobacterium tuberculosis: Genes Involved in Drug Resistance and Possibility of Rapid Identification", Clinical and Microbial Studies 125: 795-801, 1998 2. Hirano, K, et al., "Resistance to anti-tuberc
ulosis drugs in Japan "Tuber. Lung. Dis., 77: 130-13
5, 1996 3. 3. Toru Mori, "Current Situation and Measures of Tuberculosis in Japan" History of Medicine 189: 869-872,1999 Izuo Roguchi, “Tuberculosis-From Infection to Disease Onset”, Medical History 189: 873-876, 1999 5. 5. Chiyoji Abe, "Advances in Detection of Mycobacterium tuberculosis", History of Medicine 189: 877-880, 1999 Abe, C. et al., "Detection of micobacterium t
uberculosis in clinical specimens by polymerase ch
ain reaction and Gen-Probe Amplified Mycobacterium
Tuberculosis Direct Test ", J. Clin. Microbial., 3
1: 3270-3274, 1993 7. 7. Chiyoharu Abe et al., “Collaborative Study on MTD Evaluation for Rapid Detection of Mycobacterium tuberculosis”, Tuberculosis 70: 467-472, 1993 8. Telenti, A. et al., "Detection of rifampicin-r
esistance mutations inMycobacterium tuberculosis ",
Lancet, 341: 647-650, 1993 9. Suzuki, Y. et al., "Mutations in rpoB gene of
rifampicin resistant clinical isolates of Mycobact
erium tuberculosis in Japan "Journal of Infectious Diseases 69: 413,
1995 10. Finken, M. et al., "Molecular basis of stre
ptomycin resistance inMycobacterium tuberculosis:
alternation of the ribosomal protein S12 gene and
point mutations within a function 16S ribosomal RN
A psudoknot "Mol. Microbiol., 9: 1293, 1993 11. Katsukawa. C. et al.," Characterization of
the rpsL and rrs genesof streptomycin-resistant cl
inical isolates of Mycrobacterium tuberculosis in
Japan "J. Appl. Microbiol., 9: 1239, 1993 12. Sreevastan, S. et al.," Ethambutol registan
ce in Mycrobacterium tuberculosis: clinical role o
f embB mutations "Antimicrob. Chemothera. 41: 1677,
1997 13. Banerjee, A. et al., "IhnA, a gene encoding
a target for isoniazid and ethinamide in Mycobact
erium tuberculosis "Science 263: 227, 1994 14. Hirano, K. et al.," Mutation in pncA is am
ajor mechanism of pyrazinamide resistance in Mycob
actrium tuberculosis "Tuber. Lung Dis. 78: 117, 199
7 15. Hideaki Ohno et al., “2. Expression Mechanism of Resistant Bacteria” Kekkaku.
73: 657-663, 1998 Suzuki. Y. et al., "Detection of Kanamycin-
resistant Mycobacterium tuberculosis by identifyin
g mutations in the 16S rRNA gene "J. Clin. Microbi
ol., 36: 1220-1225 Telenti, A. et al., Antimicrob. Chemother.,
37: 2054-2058, 1993 18. Nair, J. et al., Mol. Microbiol., 10: 521-52.
7, 1993 19. Piatek, AS et al., "Molecular beacon seq
uence analysis for detecting drug-resistance in My
crobacterial tuberculosis "Nat. Biotecnol., 16: 359,
1998 20. Gingeras, TA et al., ”CHIP-BASED SPECIAT
ION AND PHENOTYPIC CHARCTERIZATION OF MICROORGANIS
MS ”WO97 / 29212 21. Zang, Y. et al.,“ Molecular basis for the e
xquisite sensitivity of Mycrobacterium tuberculosi
s to isoniazide "Proc. Natl. Sci. USA, 93: 13212, 1
996 22. Taniguchi, H. et al., "Molecular analysis o
f kanamycin and viomycin resistance in Mycrobacter
ium tuberculosis by use of the conjugation system "
J. Bacteteriol., 179: 4795, 1997 23. Telenti. A. et al., "The emb operon, a gene
cluster of Mycrobacterium tuberculosis involved i
n resistance to ethambutol "Nat. Med., 3: 567, 1997 24. Jacobs, WR Jr. et al.," Genetic system f
or Mycobacteria "Methods in Enzymology, 204: 537,
1991 25. Kent, PT et al., Mycobacteriology. A guid
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PROBES ON BIOLOGICALCHIPS ”USP 5,837,832

[0109]

Industrial Applicability According to the present invention, there is provided a kit capable of rapidly identifying drug resistance of M. tuberculosis and determining infection of M. tuberculosis. By using the kit of the present invention, resistance to a plurality of drugs can be distinguished. In addition, at the same time as drug identification, M. tuberculosis infection can be diagnosed separately from infection with other Mycobacterium bacteria. The time required for the test is about 6 hours after receiving the test sample from the patient, which is a rapid diagnostic method completely different from the prior art. Also,
Since the drug resistance positive data does not include false positives, there is no concern that therapeutic opportunities will be lost for a particular drug. By using this technique, a drug suitable for treatment can be selected, the appearance of tuberculosis bacteria resistant to a plurality of drugs is suppressed, and effective treatment of tuberculosis becomes possible.

[0110]

[Sequence List] SEQUENCE LISTING <110> System research Inc. Nisshinbo Industry Inc. <120> Tuberculosis diagnostic kit <130> P-6959 <140> <141> 2000- 07-26 <150> JP 11-220357 <151> 1999-08-03 <160> 231 <170> PatentIn Ver. 2.0 <210> 1 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> < 223> capture <400> 1 gctgagcaaa ttcatgg 17 <210> 2 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 2 gctgagcgaa ttcatgg 17 <210> 3 <211> 17 < 212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 3 gctgagctaa ttcatgg 17 <210> 4 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 4 gctgagccca ttcatgg 17 <210> 5 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <400> 5 aattcatggt ccagaacaa 19 <210> 6 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 6 aattcatgga acagaacaa 19 <210> 7 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <40 0> 7 gaacaactcg ctgtcgg 17 <210> 8 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 8 ccgctgttgg ggttg 15 <210> 9 <211> 17 <212> DNA < 213> Mycobacterium tuberculosis <220> <223> capture <400> 9 ggttgacgcc ccagcgc 17 <210> 10 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 10 ggttgaccaa caagcgc 17 < 210> 11 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 11 ggttgaccga caagcgc 17 <210> 12 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 12 ggttgaccta caagcgc 17 <210> 13 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 13 ggttgacccc caagcgc 17 <210> 14 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 14 ggttgacccg caagcgc 17 <210> 15 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 15 ggttgaccca aaagcgc 17 <210> 16 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <4 00> 16 ggttgaccca gaagcgc 17 <210> 17 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 17 cgactgtggg cgctg 15 <210> 18 <211> 15 <212> DNA < 213> Mycobacterium tuberculosis <220> <223> capture <400> 18 cgactgttgg cgctg 15 <210> 19 <211> 14 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 19 tcggcgccgg ggcc 14 < 210> 20 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 20 gaagaagtac cggcca 16 <210> 21 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 21 ccagtagccg cggta 15 <210> 22 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 22 ccagtagccg cggta 15 <210> 23 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 23 ccagctgccc gcggta 16 <210> 24 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 24 ccagcagctg cggta 15 <210> 25 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 25 ggctaaaact aaaaggaat 19 <210> 26 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 26 ggctaaaact aaaaggaat 19 <210> 27 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 27 ggctaaaact cgaaggaat 19 <210> 28 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 28 ccgcctaggg agtac 15 <210> 29 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 29 cactccgagg aagccg 16 <210> 30 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 30 cactccgacg aagccg 16 <210> 31 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 31 ggtgcaggac ctgcct 16 <210> 32 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <400> 32 ggtgagggac ctgcct 16 <210> 33 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 33 ggtgatggac ctgcct 16 <210> 34 <211 > 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 34 gcccgtcgcg tc atga 16 <210> 35 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <400> 35 gcccgtcaag tcatga 16 <210> 36 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <220> <223> capture <400> 36 gcccgtcatg tcatga 16 <210> 37 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 37 gattgggact aagtcgt 17 <210> 38 < 211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 38 gcggcgaggc gataggt 17 <210> 39 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 39 gcggcgagat gataggt 17 <210> 40 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 40 acgataggat gtcgggg 17 <210> 41 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 41 acgatagggt gtcgggg 17 <210> 42 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 42 gcccgccgcc tacggcc 17 <210> 43 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 43 ggcgtggg ag gctgccg 17 <210> 44 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 44 ggcgccggcg gcggcat 17 <210> 45 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 45 gcccgaccac gccagct 17 <210> 46 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 46 gcccgacagc gccagct 17 <210> 47 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 47 caacgccaac ttggaca 17 <210> 48 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 48 ccagcttggc caaggcg 17 <210> 49 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 49 gaagctctta tgggcgg 17 <210> 50 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 50 gcaactgcac gctggaa 17 <210> 51 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 51 cttcaagaag ttcgggt 17 <210> 52 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 52 tcggtaaggc ccatggc 17 <210> 53 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 53 gatcaccaac ggcatcg 17 <210> 54 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 54 gatcaccacc ggcatcg 17 <210> 55 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 55 gatcaccaca ggcatcg 17 <210> 56 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 56 cgacgaaagg ggacaac 17 <210> 57 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 57 ccagatcctg gcatcgg 17 <210> 58 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 58 accgcatgag cggcggc 17 <210> 59 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 59 ggtgccctcc accccgg 17 <210> 60 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 60 ggcagatgac ttccgaa 17 <210> 61 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture < 400> 61 cctgggcgtg gcccgag 17 <210> 62 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 62 cctgggcctg gcccgag 17 <210> 63 <211> 17 <212> DNA < 213> Mycobacterium tuberculosis <220> <223> capture <400> 63 cctgggcata gcccgag 17 <210> 64 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 64 cctgggcatt gcccgag 17 < 210> 65 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 65 cctgggcatc gcccgag 17 <210> 66 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 66 aggatcccgt cggctgg 17 <210> 67 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 67 gctgagccaa ttcatgg 17 <210> 68 <211> 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 68 aattcatgga ccagaacaa 19 <210> 69 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 69 gaacaacccg ctgtcgg 17 <210> 70 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 70 ccgctgtcgg ggttg 15 <210> 71 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 71 ggttgaccca caagcgc 17 <210> 72 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 72 cgactgtcgg cgctg 15 <210> 73 <211> 14 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 73 tcggcgctgg ggcc 14 <210> 74 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 74 gaagaagcac cggcca 16 <210> 75 <211> 15 <212> DNA <213> Mycobacterium tuberculosis < 220> <223> capture <400> 75 ccagcagccg cggta 15 <210> 76 <211> 15 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 76 ccgcctgggg agtac 15 <210> 77 <211 > 19 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 77 ggctaaaact caaaggaat 19 <210> 78 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture < 400> 78 cactccgaag aagccg 16 <210> 79 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> ca pture <400> 79 ggtgaaggac ctgcct 16 <210> 80 <211> 16 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 80 gcccgtcacg tcatga 16 <210> 81 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 81 gattgggacg aagtcgt 17 <210> 82 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 82 gcggcgagac gataggt 17 <210> 83 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 83 acgataggtt gtcgggg 17 <210> 84 <211> 17 <212> DNA <213> Mycobacterium tuberculosis < 220> <223> capture <400> 84 gcccgccgac tacggcc 17 <210> 85 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 85 ggcgtggcac gctgccg 17 <210> 86 <211 > 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 86 ggcgccgggg gcggcat 17 <210> 87 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture < 400> 87 gcccgacaac gccagct 17 <210> 88 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <2 23> capture <400> 88 caacgccagc ttggaca 17 <210> 89 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 89 ccagcttgga caaggcg 17 <210> 90 <211> 17 < 212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 90 gaagctctca tgggcgg 17 <210> 91 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 91 gcaactgcgc gctggaa 17 <210> 92 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 92 cttcaagacg ttcgggt 17 <210> 93 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 93 tcggtaagac ccatggc 17 <210> 94 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 94 gatcaccagc ggcatcg 17 <210> 95 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 95 cgacgaaatg ggacaac 17 <210> 96 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 96 ccagatccgg gcatcgg 17 <210> 97 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 97 accgcatggg cggcggc 17 <210> 98 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 98 ggtgcccttc accccgg 17 <210> 99 < 211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 99 ggcagatggc ttccgaa 17 <210> 100 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 100 cctgggcatg gcccgag 17 <210> 101 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 101 aggatccctt cggctgg 17 <210> 102 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 102 cacgggatgc atgtctt 17 <210> 103 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> capture <400> 103 cacggggcgc atgtctt 17 <210> 104 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify M. tuberculosis rrs gene <400> 104 tgggaaactg ggtctaatac 20 <210> 105 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs gene <220> <223> biotin a t 5 'terminus <400> 105 gcgggctcat cccacaccgc 20 <210> 106 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpoB gene <400> 106 gccgcgatca aggagtt 17 <210> 107 <211> 17 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpoB gene <220> <223> biotin at 5 'terminus <400> 107 cacgtgacag accgccg 17 <210> 108 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs 530 region <400> 108 tctctcggat tgacggtagg 20 <210> 109 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223 > primer: amplify rrs530 region <220> <223> biotin at 5 'terminus <400> 109 cctacgagct ctttacgccc 20 <210> 110 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs 915 region <400> 110 gatccgtgcc gtagctaacg 20 <210> 111 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs 915 region <220> <223> biotin at 5 'terminus <400> 111 cgcttgtgcg ggcccccgtc 20 <210> 112 <211> 20 <212> DNA <2 13> Mycobacterium tuberculosis <220> <223> primer: amplify rpsL gene <400> 112 gcacccgcgt gtacaccacc 20 <210> 113 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpsL gene <220> <223> biotin at 5 'terminus <400> 113 ttgtagcgca caccaggcag 20 <210> 114 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs 1400 region <400> 114 tcccgggcct tgtacacacc 20 <210> 115 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs 1400 region <220> <223> biotin at 5 'terminus <400> 115 cttccggtac ggctaccttg 20 <210> 116 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify inhA gene <400> 116 gaaagttccc gccggaaatc 20 <210> 117 <211> 20 <212> DNA <213 > Mycobacterium tuberculosis <220> <223> primer: amplify inhA gene <220> <223> biotin at 5 'terminus <400> 117 actgaacggg atacgaatgg 20 <210> 118 <211> 20 <212> DNA <213> Mycobacterium tuberculosis < 220> <223> primer: amplify katG gene <400> 118 gg tcgcgacc atcgacgttg 20 <210> 119 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 119 tcgtggatgc ggtaggtgcc 20 < 210> 120 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 120 ggcacctacc gcatccacga 20 <210> 121 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 121 tgagagcttc ttgccgtact 20 <210> 122 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 122 gctgtggccg gtcaagaaga 20 <210> 123 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 123 tagacctcat cgggctccca 20 <210> 124 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 124 cattcgcgag acgtttcggc 20 <210> 125 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 125 cccatctgct ccagcggagc 20 <210> 126 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer : amplify katG gene <400> 126 aagagctcgt atggcaccgg 20 <210> 127 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 127 agcgccagca gggctcttcg 20 <210> 128 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 128 ggcgaagccg agattgccag 20 <210> 129 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 129 ctgcaggcgg atgcgaccac 20 <210> 130 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 130 aagcagcaaa ggcggctggc 20 <210> 131 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <220> <223> biotin at 5 'terminus <400> 131 acgggttgcc c tttccgagg 20 <210> 132 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify embB gene <400> 132 gcgcgaattc gtcggacgac 20 <210> 133 <211> 20 <212> DNA < 213> Mycobacterium tuberculosis <220> <223> primer: amplify embB gene <220> <223> biotin at 5 'terminus <400> 133 tgacatgggt catcagcgcc 20 <210> 134 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpoB gene <400> 134 cgttgatcaa catccggccg 20 <210> 135 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpoB gene <400> 135 tgcacgtcgc ggacctccag 20 <210> 136 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs gene <400> 136 agagtttgat cctggctcag 20 <210> 137 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rrs gene <400> 137 tacggctacc ttgttacgac 20 <210> 138 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpsL gene <400> 138 gcggctctga agggcagccc 20 <210> 139 <2 11> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify rpsL gene <400> 139 gtttgcggtt cttgacaccc 20 <210> 140 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: ampify inhA gene <400> 140 caccgacaaa cgtcacgagc 20 <210> 141 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify inhA gene <400> 141 gccgctgtgc gatcgccagc 20 <210> 142 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 142 cgatccctgc ggcgttcgac 20 <210> 143 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify katG gene <400> 143 ccttgtcgag cagcatgtac 20 <210> 144 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify embB gene <400 > 144 tgatattcgg cttcctgctc 20 <210> 145 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <220> <223> primer: amplify embB gene <400> 145 gcggccaggt ctggcaggcg 20 <210> 146 <211> 124 <212 > DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis rpoB gene <400> 146 gccgcgatca aggagttctt cggcaccagc cagctgagcc aattcatgga ccagaacaac 60 ccgctgtcgg ggttgaccca caagcggccga ctgtcggcgc tggggctract <120> <c> cgtcgt <120> <c> cgtcgt <120> <220> <223> nucleotide probe: amplified from M. tuberculosis rrs gene <400> 147 tctctcggat tgacggtagg tggagaagaa gcaccggcca actacgtgcc agcagccgcg 60 gtaatacgta gggtgcgagc gttgtccgga <tactgggc gtaaagagcct gtaaagagctcag gtaaagagctcag gtaaagagctcg> tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis rrs gene <400> 148 gatccgtgcc gtagctaacg cattaagtac cccgcctggg gagtacggcc gcaaggctaa 60 aactcaaagg aattgacggg ggcccgcaca agcg 94 <210> tract 1492 <220> <223> nucleotide probe: amplified from M. tuberculosis rpsL gene <400> 149 gcacccgcgt gtacaccacc actccgaaga agccgaactc ggcgcttcgg aaggttgccc 60 gcgtgaagtt gacgagtcag gtcgaggtca c ggcgtacat tcccggcgag ggccacaacc 120 tgcaggagca ctcgatggtg ctggtgcgcg gcggccgggt gaaggacctg cctggtgtgc 180 gctacaa 187 <210> 150 <211> 140 <212> DNA <213> Mycobacterium tuberculosis <220> from <220> tcccgggcct tgtacacacc gcccgtcacg tcatgaaagt cggtaacacc cgaagccagt 60 ggcctaaccc tcgggaggga gctgtcgaag gtgggatcgg cgattgggac gaagtcgtaa 120 caaggtagcc gtaccggaag 140 <210> 151 <2> <400> 151 gaaagttccc gccggaaatc gcagccacgt tacgctcgtg gacataccga tttcggcccg 60 gccgcggcga gacgataggt tgtcggggtg actgccacag ccactgaagg ggccaaaccc 120 ccattcgtat cccgttcagt 140 <210> 122 <220> tuberculosis katG gene <400> 152 ggtcgcgacc atcgacgttg acgccctgac gcgggacatc gaggaagtga tgaccacctc 60 gcagccgtgg tggcccgccg actacggcca ctacgggccg ctgtttat cc ggatggcgtg 120 gcacgctgcc ggcacctacc gcatccacga 150 <210> 153 <211> 150 <212> DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis katG gene <400> 153 ggcacctacc gcatcccgg cggccgcgggcggcggcgg 60 ccgcttaaca gctggcccga caacgccagc ttggacaagg cgcgccggct gctgtggccg 120 gtcaagaaga agtacggcaa gaagctctca 150 <210> 154 <211> 150 <212> DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe <RTIgt; g. gtcaagaaga agtacggcaa gaagctctca tgggcggacc tgattgtttt 60 cgccggcaac tgcgcgctgg aatcgatggg cttcaagacg ttcgggttcg gcttcggccg 120 ggtcgaccaccag tgggagcccg atgaggtcta 150 <220> <r> <r> <br> <br> <br> <br> <400> 155 cattcgcgag acgtttcggc gcatggccat gaacgacgtc gaaacagcgg cgctgatcgt 60 cggcggtcac actttcggta agacccatgg cgccggcccg gccgatctgg tcggccccga 120 acccgaggct gctccgctgg agcagatggg 150 <210> 156 <211> 150 <212> DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis katG gene <400> 156 aagagctcgt atggcaccgg aaccggtaag gacgcgatca ccagcggcat cgaggtcgacatccc tacgac tacgcc agatcctgta cggctacgag 120 tgggagctga cgaagagccc tgctggcgct 150 <210> 157 <211> 150 <212> DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis katG gene <400> 157 ggcgagcgaggattcag gagattcgaggattcag aggattgag 60 cagctagttt cgaccgcatg ggcggcggcg tcgtcgttcc gtggtagcga caagcgcggc 120 ggcgccaacg gtggtcgcat ccgcctgcag 150 <210> 158 <211> 150 <212> DNA <213> Mycobacterium tuberculosis <220> ag <ag> ag. ggcggctggc cacaacatca cggtgccctt caccccgggc cgcacggatg 60 cgtcgcagga acaaaccgac gtggaatcct ttgccgtgct ggagcccaag gcagatggct 120 tccgaaacta cctcggaaag ggcaacccgt 150 <210> 159 DNA <213> Mycobacterium tuberculosis <220> <223> nucleotide probe: amplified from M. tuberculosis embB gene <400> 159 gcgcgaattc gtcggacgac ggctacatcc tgggcatggc ccgagtcgcc gaccacgcc gcaccat gcaccatgc gcaccattgc cagattcg cgctggccctgcgccctgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgccgcgcgcgcgcgccgcgcgcgcggcGcGc > 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 160 accacgggat gcatgtct 18 <210> 161 <211> 18 <213> artificial sequence <220> <223> capture <400> 161 accacgaaat gcatgtct 18 <210 > 162 <211> 18 <213> Mycobacterium avium complex <220> <223> capture <400> 162 taggacctca agacgcat 18 <210> 163 <211> 18 <213> artificial sequence <220> <223> capture <400> 163 cacgtaacaa tctgccct 18 <210> 164 <211> 18 <213> Mycobacterium kansasii <220> <223> capture <400> 164 cacgtggcaa tctgccct 18 <210> 165 <211> 18 <213> artificial sequence <220> <223> capture <400> 165 taggaaatca agacgcat 18 <210> 166 <211> 19 <213> Mycobacterium tuberculosis <220> <223> capture <400> 166 agctgagcca attcat gga 19 <210> 167 <211> 16 <213> Mycobacterium tuberculosis <220> <223> capture <400> 167 gctgagcaaa ttcatg 16 <210> 168 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture < 400> 168 gctgagctaa ttcatgg 17 <210> 169 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 169 agctgagccc attcatgg 18 <210> 170 <211> 18 <213> Mycobacterium tuberculosis <220> < 223> capture <400> 170 aattcatggt ccagaaca 18 <210> 171 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 171 attcatggaa cagaaca 17 <210> 172 <211> 21 <213> Mycobacterium tuberculosis <220> <223> capture <400> 172 acagaacaac ccgctgtcggg 21 <210> 173 <400> 173 000 <210> 174 <211> 15 <213> Mycobacterium tuberculosis <220> <223> capture <400> 174 ccgctgttgg ggttg 15 <210> 175 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 175 ggttgaccca caagcgcc 18 <210> 176 <211> 16 <213> Mycobacterium tuberculosis <220> <223> capture <400> 176 gttgacgccc cagcgc 16 <210> 177 <211> 16 <213> Mycob acterium tuberculosis <220> <223> capture <400> 177 ggttgaccaa caagcg 16 <210> 178 <211> 16 <213> Mycobacterium tuberculosis <220> <223> capture <400> 178 ggttgaccga caagcg 16 <210> 179 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 179 ggttgaccta caagcgc 17 <210> 180 <211> 20 <213> Mycobacterium tuberculosis <220> <223> capture <400> 180 gggttgaccc ccaagcgccg 20 <210> 181 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 181 ggttgacccg caagcgcc 18 <210> 182 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 182 ggttgaccca aaagcgc 17 <210> 183 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 183 ggttgaccca gaagcgc 17 <210> 184 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400 > 184 ccgactgtcg gcgctgg 17 <210> 185 <211> 14 <213> Mycobacterium tuberculosis <220> <223> capture <400> 185 gactgtgggc gctg 14 <210> 186 <211> 14 <213> Mycobacterium tuberculosis <220> <223 > capture <400> 186 cgactgttgg cg ct 14 <210> 187 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 187 tgtcggcgct ggggccc 17 <210> 188 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture < 400> 188 agaagaagca ccggccaa 18 <210> 189 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 189 tgccagccgc cgcggta 17 <210> 190 <211> 16 <213> Mycobacterium tuberculosis <220> < 223> capture <400> 190 gctaaaacta aaagga 16 <210> 191 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 191 gctaaaactg aaaggaa 17 <210> 192 <211> 15 <213> Mycobacterium tuberculosis <220> <223> capture <400> 192 ggtgatggac ctgcc 15 <210> 193 <211> 17 <213> Mycobacterium tuberculosis <220> <223> capture <400> 193 cccgcctggg gagtacg 17 <210> 194 <211> 23 < 213> Mycobacterium tuberculosis <220> <223> capture <400> 194 caccactccg aagaagccga act 23 <210> 195 <211> 15 <213> Mycobacterium tuberculosis <220> <223> capture <400> 195 ggtgcaggac ctgcc 15 <210> 196 <211> 15 <213> Mycobacterium tuberculosis <22 0> <223> capture <400> 196 ggtgagggac ctgcc 15 <210> 197 <211> 14 <213> Mycobacterium tuberculosis <220> <223> capture <400> 197 gtgatggacc tgcc 14 <210> 198 <211> 18 <213 > Mycobacterium tuberculosis <220> <223> capture <400> 198 cccgtcacgt catgaaag 18 <210> 199 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 199 cccgtcgcgt catgaaag 18 <210> 200 <211 > 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 200 accgcccgtc aagtcatg 18 <210> 201 <211> 18 <213> Mycobacterium tuberculosis <220> <223> capture <400> 201 accgcccgtc atgtcatg 18 <210 > 202 <211> 16 <213> Mycobacterium tuberculosis <220> <223> capture <400> 202 attgggacga agtcgt 16 <210> 203 <211> 16 <213> Mycobacterium tuberculosis <220> <223> capture <400> 203 attgggacta agtcgt 16 <210> 204 <211> 18 <213> Mycobacteriae <220> <223> primer amplify rrs gene <400> 204 ctcgagtggc gaacgggt 18 <210> 205 <211> 16 <213> Mycobacteriae <220> <223> primer amplify rrs gene <400> 205 agctgatagg ccgcgggc 16 <210> 206 <211> 18 <213> Mycobacterium xenopi <220> <223> capture <400> 206 taggaccatt ctgcgcat 18 <210> 207 <211> 18 <213> Mycobacterium ulcerans <220> <223> capture <400> 207 accacgggat tcatgtcc 18 <210> 208 <211> 18 <213> Mycobacterium szukgai <220> <223> capture <400> 208 taggaccccg aggcgcat 18 <210> 209 <211> 18 <213> Mycobacterium simiae <220> <223> capture <400> 209 cacgtgggta atctgccc 18 <210> 210 <211> 18 <213> Mycobacterium shimoidei <220> <223> capture <400> 210 accacatgtc gcatggtg 18 <210> 211 <211> 18 <213> Mycobacterium scrofulaceum <220> <223> capture <400> 211 cacgtgggca atctgccc 18 <210> 212 <211> 18 <213> Mycobacterium nonchromogen <220> <223> capture <400> 212 accgcatgct gcatggtg 18 <210> 213 <211> 18 <213> Mycobacterium marinum <220 > <223> capture <400> 213 accacgggat tcatgtcc 18 <210> 214 <211> 18 <213> Mycobacterium malmoense <220> <223> capture <400> 214 accccgaggc gcatgcct 18 <210> 215 <211> 18 <213> Mycobacterium intracellular <220> <223> capture <400> 215 taggaccttt agg cgcat 18 <210> 216 <211> 18 <213> Mycobacterium gordonae <220> <223> capture <400> 216 taggaccaca ggacacat 18 <210> 217 <211> 18 <213> Mycobacterium fortuitum <220> <223> capture < 400> 217 tatgaccgcg cacttcct 18 <210> 218 <211> 18 <213> Mycobacterium chelonae <220> <223> capture <400> 218 taggaccaca cacttcat 18 <210> 219 <211> 18 <213> artificial sequence <220> < 223> capture <400> 219 taggaccatt ctaagcat 18 <210> 220 <211> 18 <213> artificial sequence <220> <223> capture <400> 220 accacgaaat tcatgtcc 18 <210> 221 <211> 18 <213> artificial sequence <220> <223> capture <400> 221 taggaccccg aaacgcat 18 <210> 222 <211> 18 <213> artificial sequence <220> <223> capture <400> 222 cacgtgaata atctgccc 18 <210> 223 <211> 18 < 213> artificial sequence <220> <223> capture <400> 223 accacatata gcatggtg 18 <210> 224 <211> 18 <213> artificial sequence <220> <223> capture <400> 224 cacgtgaaca atctgccc 18 <210> 225 < 211> 18 <213> artificial sequence <220> <223> capture <400> 225 accgcatgct aaatggtg 18 < 210> 226 <211> 18 <213> artificial sequence <220> <223> capture <400> 226 accacgaaat tcatgtcc 18 <210> 227 <211> 18 <213> artificial sequence <220> <223> capture <400> 227 accccgaggc aaatgcct 18 <210> 228 <211> 18 <213> artificial sequence <220> <223> capture <400> 228 taggaccttt aggcaaat 18 <210> 229 <211> 18 <213> artificial sequence <220> <223> capture <400> 229 taggaccaca aaacacat 18 <210> 230 <211> 18 <213> artificial sequence <220> <223> capture <400> 230 tatgaccgca aacttcct 18 <210> 231 <211> 18 <213> artificial sequence <220> <223> capture <400> 231 taggacaaaa cacttcat 18

[Brief description of the drawings]

FIG. 1 is a diagram showing the arrangement of oligonucleotides immobilized on a substrate. The symbols in the figure are as follows. RFP: Indicates an oligonucleotide-immobilized region for determining rifampicin resistance Sm: Indicates an oligonucleotide-immobilized region for determining streptomycin resistance Km: Indicates an oligonucleotide-immobilized region for determining kanamycin resistance INH: Indicates an isoniazid oligonucleotide-immobilized region : Indicates the immobilized region of ethambutol resistance-determining oligonucleotide B: Indicates the position where the biotinylated oligonucleotide is immobilized

FIG. 2 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-sensitive tuberculosis using the kit. The symbols in the figure are as follows. The number in ● or ○ indicates the oligonucleotide spot position (the same applies to the following figures). ●: Indicates a spot which was recognized as positive by pigmentation after chemical coloring. ：: A spot which was recognized as negative by pigmentation after chemical coloring was shown.

FIG. 3 is a schematic diagram of a kit of the present invention and a diagram showing a detection result of rifampicin-sensitive tuberculosis bacterium using the kit (detection result in which a signal was obtained at position M2).

FIG. 4 is a schematic diagram of the kit of the present invention and a diagram showing a detection result of rifampicin-resistant tuberculosis bacterium using the kit (detection result in which a signal was obtained at the position of M3).

FIG. 5 is a schematic diagram of a kit of the present invention and a diagram showing a detection result of rifampicin-resistant tuberculosis bacterium using the kit (detection result in which a signal was obtained at position M4).

FIG. 6 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacteria using the kit (detection results in which a signal was obtained at the position of M5).

FIG. 7 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacteria using the kit (detection results in which a signal was obtained at M10).

FIG. 8 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacterium using the kit (detection result of signal obtained at position M11).

FIG. 9 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of rifampicin-resistant tuberculosis bacterium using the kit (detection results in which a signal was obtained at the position of M12).

FIG. 10 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacteria using the kit (M17).
(A detection result in which a signal was obtained at the position of).

FIG. 11 is a schematic diagram showing the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacteria using the kit (M18).
(A detection result in which a signal was obtained at the position of).

FIG. 12 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of kanamycin-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 80 and 81)

FIG. 13 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of kanamycin-resistant Mycobacterium tuberculosis using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 34)

FIG. 14 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of kanamycin-resistant Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOS: 35 and 37).

FIG. 15 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of kanamycin-resistant Mycobacterium tuberculosis using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 36)

FIG. 16 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of streptomycin-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 74 and 75).

FIG. 17 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of streptomycin-resistant Mycobacterium tuberculosis using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 22)

FIG. 18 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of streptomycin-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 76 and 77).

FIG. 19 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of streptomycin-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 78 and 79).

FIG. 20 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of ethambutol-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOS: 100 and 101).

FIG. 21 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of isoniazid-sensitive Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 82 to 89).

FIG. 22 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of rifampicin-resistant tuberculosis bacteria using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 181)

FIG. 23 is a schematic diagram of the kit of the present invention and a diagram showing the detection results of rifampicin-resistant tuberculosis bacteria using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 171)

FIG. 24 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of streptomycin-resistant Mycobacterium tuberculosis using the kit (detection results in which a signal was obtained at the position of SEQ ID NO: 189)

FIG. 25 is a schematic diagram of the kit of the present invention and a diagram showing the results of detection of kanamycin-resistant Mycobacterium tuberculosis using the kit (detection results in which signals were obtained at positions of SEQ ID NOs: 189 and 190).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:32) C12R 1: 325) (C12Q 1/68 (C12Q 1/68 A C12R 1: 325) C12R 1:33) (C12Q 1/68 C12N 15/00 ZNAA C12R 1:33) F (72) Inventor Soichiro Takenishi 1-18-1 Nishiarai Sakaemachi, Adachi-ku, Tokyo Nisshin Spinning Co., Ltd. Tokyo Research Center

Claims (62)

[Claims] 1. A wild-type tuberculosis bacterium which is susceptible to said drug and a mutant which has resistance to any drug derived from a gene on the genome of M. tuberculosis which is involved in resistance to a drug used for tuberculosis treatment An M. tuberculosis diagnostic kit comprising a substrate on which an oligonucleotide synthesized based on a base sequence obtained from each of M. tuberculosis is immobilized by a covalent bond, the M. tuberculosis being hybridized with a nucleic acid derived from a test microorganism. A diagnostic kit for identifying drug resistance of bacteria or determining infection with Mycobacterium tuberculosis. 2. A carbodiimide group or an isocyanate group is coated on the surface of a substrate, and a covalent bond is formed by a reaction between the carbodiimide group or the isocyanate group and an amino group of the oligonucleotide or a linker added to the terminal of the oligonucleotide. The kit according to claim 1. 3. The kit according to claim 1, wherein the oligonucleotide is fixed on the surface of the substrate to a size of 10 to 1,000 μm in diameter. 4. The kit according to claim 1, wherein the gene relating to drug resistance is any one or more genes selected from genes relating to resistance to rifampicin, streptomycin, kanamycin, isoniazid or ethambutol. 5. The kit according to claim 4, wherein the gene relating to drug resistance is a gene relating to resistance to rifampicin and isoniazid. 6. The kit according to claim 4, wherein the genes involved in drug resistance are all genes involved in resistance to rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol. 7. Rifampicin, streptomycin,
Mutant Mycobacterium tuberculosis resistant to any of the drugs kanamycin, isoniazid and ethambutol, derived from a mutant gene having a point mutation related to the resistance, and containing 12 to 24 bases including the mutation site Oligonucleotides designed and synthesized to base length. 8. Rp of rifampicin-resistant mutant M. tuberculosis
The oligonucleotide according to claim 7, which is derived from a sequence containing the mutation site of the oB gene and comprises any of the nucleotide sequences shown in SEQ ID NOs: 1 to 19. 9. 5 ′ of the base sequence shown in SEQ ID NOs: 1 to 19
A nucleotide sequence obtained by extending or shortening the gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the side or the 3 'side or both, The oligonucleotide according to claim 7, wherein the mutation site contained in the original nucleotide sequence of (1) is not substituted. 10. The base sequence shown in SEQ ID NOs: 20 to 28 derived from the sequence containing the above mutation site of the rrs gene of streptomycin-resistant mutant Mycobacterium tuberculosis, or the aforementioned mutation site of the rpsL gene of streptomycin-resistant mutant Mycobacterium tuberculosis 8. The oligonucleotide according to claim 7, comprising any one of the nucleotide sequences shown in SEQ ID NOS: 29 to 33 derived from a sequence containing 11. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NOs: 20 to 33 based on each nucleotide sequence 8. The oligonucleotide according to claim 7, which is a nucleotide sequence obtained by extending or shortening, wherein the mutation site contained in each original nucleotide sequence is not substituted. 12. The rr of kanamycin resistant mutant M. tuberculosis
The oligonucleotide according to claim 7, which is derived from a sequence containing the mutation site of the s gene and comprises any of the nucleotide sequences shown in SEQ ID NOs: 34 to 37. 13. The gene sequence of wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NOs: 34 to 37 based on each nucleotide sequence. 8. The oligonucleotide according to claim 7, which is a nucleotide sequence obtained by extending or shortening, wherein the mutation site contained in each original nucleotide sequence is not substituted. 14. An inoniazid-resistant mutant of M. tuberculosis
The nucleotide sequences shown in SEQ ID NOs: 38 to 41 derived from the sequence containing the mutation site of the hA gene, or the nucleotide sequences shown in SEQ ID NOs: 42 to 60 derived from the sequence containing the mutation site in the katG gene of the isoniazid-resistant mutant Mycobacterium tuberculosis The oligonucleotide of claim 7, comprising any of the sequences. 15. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NOs: 38 to 60 based on each nucleotide sequence. 8. The oligonucleotide according to claim 7, which is a nucleotide sequence obtained by extending or shortening, wherein the mutation site contained in each original nucleotide sequence is not substituted. 16. The e ethambutol-resistant mutant tubercle bacillus e
The oligonucleotide according to claim 7, wherein the oligonucleotide is derived from a sequence containing the mutation site of the mbB gene and comprises any of the nucleotide sequences shown in SEQ ID NOs: 61 to 66. 17. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 ′ side and / or 3 ′ side of the nucleotide sequence shown in SEQ ID NOs: 61 to 66 based on each nucleotide sequence 8. The oligonucleotide according to claim 7, which is a nucleotide sequence obtained by extending or shortening, wherein the mutation site contained in each original nucleotide sequence is not substituted. 18. A nucleotide sequence of an oligonucleotide derived from a wild-type gene of a wild-type Mycobacterium tuberculosis susceptible to rifampicin, streptomycin, kanamycin, isoniazid and ethambutol and derived from a mutant gene involved in resistance of the drug. 12 bases obtained from the gene sequence corresponding to the position on the M. tuberculosis genome
Oligonucleotides designed and synthesized to be 24 bases in length. 19. A r-free strain of wild-type Mycobacterium tuberculosis
The oligonucleotide according to claim 18, which is derived from the poB gene and comprises any of the nucleotide sequences shown in SEQ ID NOs: 67 to 73. 20. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the base sequence shown in SEQ ID NOs: 67 to 73 based on each base sequence 19. A base sequence obtained by extending or shortening
The oligonucleotide as described. 21. An R-free strain of a wild-type Mycobacterium tuberculosis
The nucleotide sequences shown in SEQ ID NOs: 74 to 77 derived from the rs gene, or SEQ ID NOs: 78 to 7 derived from the rpsL gene
19. The oligonucleotide according to claim 18, comprising any one of the nucleotide sequences shown in 9. 22. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NOs: 74 to 79 based on each nucleotide sequence 19. A base sequence obtained by extending or shortening
The oligonucleotide as described. 23. An r-free strain of a M. tuberculosis wild-type
19. The oligonucleotide according to claim 18, which is derived from the rs gene and comprises the nucleotide sequence shown in SEQ ID NO: 80 or 81. 24. A gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NO: 80 or 81 based on each nucleotide sequence 2. A nucleotide sequence obtained by extending or shortening.
9. The oligonucleotide according to 8. 25. i which does not contain a mutation of a wild-type Mycobacterium tuberculosis
19. The oligonucleotide according to claim 18, comprising any one of the nucleotide sequences shown in SEQ ID NOs: 82 and 83 derived from the nhA gene or the nucleotide sequences shown in SEQ ID NOs: 84 to 99 derived from the katG gene. 26. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the nucleotide sequence shown in SEQ ID NOS: 83 to 99 based on each nucleotide sequence 2. A nucleotide sequence obtained by extending or shortening the nucleotide sequence.
9. The oligonucleotide according to 8. 27. e which does not contain a mutation of M. tuberculosis
The oligonucleotide according to claim 18, which is derived from the mbB gene and comprises the nucleotide sequence shown in SEQ ID NO: 100 or 101. 28. The gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the base sequence shown in SEQ ID NO: 100 or 101 based on each base sequence 19. The oligonucleotide according to claim 18, which is a nucleotide sequence obtained by extending or shortening of the oligonucleotide. 29. The synthetic oligonucleotide comprises a combination of the following (a) and (b):
The base sequence of the oligonucleotide selected from (a) and the base sequence of the oligonucleotide selected from (b) include those whose gene positions on the genome of M. tuberculosis correspond to each other. The kit according to any one of 1 to 6, (a) a point mutation associated with the resistance of a mutant tubercle bacillus having resistance to any of the drugs rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol; The nucleotide sequence shown in SEQ ID NOs: 1 to 66, or 5 ′ or 3 ′ of each of the above nucleotide sequences
A base sequence obtained by extending or shortening the wild-type Mycobacterium tuberculosis gene sequence whose position on the M. tuberculosis bacterium corresponds to each base sequence on either or both sides, wherein each original base Wherein the mutation site contained in the sequence is not substituted;
(B) a wild-type gene of a wild-type Mycobacterium tuberculosis susceptible to rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol; 67 to 101, or 12 to 24 nucleotides obtained from a gene sequence whose position on the genome of M. tuberculosis corresponds to the nucleotide sequence described in (a) above.
An oligonucleotide comprising any of a base sequence designed to have a base length. 30. The kit according to claim 29, wherein the kit is a kit for distinguishing resistance to rifampicin and isoniazid, wherein the oligonucleotide (a) comprises a group consisting of SEQ ID NOS: 1 to 19 and a SEQ ID NO. 38
Or an oligonucleotide selected from each of the groups consisting of the following nucleotides: or at the 5′-end and / or 3′-end of the nucleotide sequence of each of the oligonucleotides, A nucleotide sequence obtained by extending or shortening the corresponding wild-type Mycobacterium tuberculosis gene sequence, wherein the mutation site contained in each original nucleotide sequence is not substituted. Oligonucleotides selected from those derived from the group consisting of SEQ ID NOS: 1 to 19, which are nucleotide sequences designed to have a length of 12 to 24 bases, and those derived from the group consisting of SEQ ID NOS: 38 to 60 And the oligonucleotide of (b) comprises a group consisting of SEQ ID NOs: 61 to 66 and Column number 8
An oligonucleotide selected from each of the group consisting of 2 to 89, or a nucleotide sequence at the 5 ′ side and / or 3 ′ side of the nucleotide sequence of each of the oligonucleotides, on the M. tuberculosis genome. Is a nucleotide sequence obtained by extending or shortening the corresponding wild-type Mycobacterium tuberculosis gene sequence, wherein the mutation site contained in each original nucleotide sequence is not substituted. Selected from the group consisting of SEQ ID NOS: 61 to 66, which is a nucleotide sequence designed to have a length of 12 to 24 bases, and the group consisting of SEQ ID NOs: 82 to 89. A kit comprising an oligonucleotide. 31. The kit according to claim 29, wherein the kit is a kit for discriminating multidrug resistance discrimination, wherein the oligonucleotide (a) has SEQ ID NOS: 1 to 19.
A group consisting of SEQ ID NOS: 20 to 28 and a group consisting of SEQ ID NOS: 29 to 33; a group consisting of SEQ ID NOS: 34 to 37; a group consisting of SEQ ID NOs: 38 to 41; a group consisting of SEQ ID NOS: 42 to 60; It is an oligonucleotide selected from each of the groups consisting of SEQ ID NOs: 61 to 66, or at the 5 ′ side and / or 3 ′ side of the nucleotide sequence of the oligonucleotide, each nucleotide sequence has A nucleotide sequence obtained by extending or shortening the corresponding wild-type Mycobacterium tuberculosis gene sequence, wherein the mutation site contained in each original nucleotide sequence is not substituted. A nucleotide sequence designed to have a length of 12 to 24 bases, Those derived from the group consisting of SEQ ID NOS: 20 to 28, those derived from the group consisting of SEQ ID NOS: 29 to 33, those derived from the group consisting of SEQ ID NOS: 34 to 37, Oligonucleotides selected from those derived from the group consisting of SEQ ID NOs: 38 to 41 and those derived from the group consisting of SEQ ID NOs: 42 to 60, and those derived from the group consisting of SEQ ID NOs: 61 to 66, And the oligonucleotide of (b) is a group consisting of SEQ ID NOS: 67 to 73, SEQ ID NOs: 74 to 77, and SEQ ID NOs: 78 to 79;
An oligonucleotide selected from the group consisting of 0 to 81, the group consisting of SEQ ID NOs: 82 to 83 and the group consisting of SEQ ID NOs: 84 to 99, and the group consisting of SEQ ID NOS: 100 to 101; A nucleotide sequence obtained by extending or shortening the gene sequence of a wild-type Mycobacterium tuberculosis corresponding to the position on the genome of Mycobacterium tuberculosis on the 5 'side and / or 3' side of the base sequence, respectively. Wherein the mutation site contained in each of the original nucleotide sequences is a nucleotide sequence designed to be 12 to 24 nucleotides in length that is not substituted.
Derived from the group consisting of SEQ ID NOS: 20 to 2
From the group consisting of 8 and SEQ ID NO: 2
Those derived from the group consisting of 9 to 33, those derived from the group consisting of SEQ ID NOs: 34 to 37, those derived from the group consisting of SEQ ID NOs: 38 to 41, and those derived from the group consisting of SEQ ID NOS: 42 to 60 And a kit derived from the group consisting of SEQ ID NOs: 61 to 66. 32. In addition to the synthetic oligonucleotide, an oligonucleotide synthesized from the following (c) and (d) is immobilized on a substrate, and the oligonucleotide (c) is specific to M. tuberculosis DNA. The oligonucleotide of (d) functions as a negative control that does not cause sequence-specific hybridization to M. tuberculosis DNA, and functions as a positive control. (C) having a nucleotide sequence that is identical between drug-sensitive wild-type M. tuberculosis and drug-resistant M. tuberculosis, but is not genetically conserved among Mycobacterium species An oligonucleotide obtained from the gene region; and (d) at least one base relative to the nucleotide sequence of the oligonucleotide of (c), , Oligonucleotides obtained by replacing the base in the range of less than 20% of the number of bases having a nucleotide sequence. 33. The oligonucleotide according to (c),
33. The kit according to claim 32, having the nucleotide sequence of SEQ ID NO: 102 derived from the rrs gene. 34. The oligonucleotide of (c) has the nucleotide sequence of SEQ ID NO: 102, and the oligonucleotide of (d)
33. The kit according to claim 32, wherein the oligonucleotide has the nucleotide sequence shown in SEQ ID NO: 103. 35. A labeled nucleic acid probe derived from a nucleic acid of a test microorganism, which is used for identifying drug resistance of M. tuberculosis or determining M. tuberculosis infection using the kit according to claim 1. Extracting DNA from cells of a test microorganism isolated from sputum or a test microorganism obtained from a test microorganism obtained by culture; and using the DNA as a template, 5 ′ side or 3 ′ A method comprising the step of performing nucleic acid amplification using a primer labeled with a fluorescent substance or a hapten at either end on the 'side. 36. Fluorescein (FITC), rhodamine (Rod) obtained by the method of claim 35.
amine), phycoerythrin (PE), Texas
A nucleic acid probe labeled with a fluorescent substance of either Red or a cyanine fluorescent dye. 37. Biotin (BIOTIN), digoxigenin (Di) obtained by the method according to claim 35.
g) A nucleic acid probe labeled with a hapten, either dinitrophenyl (DNP) or fluorescein. 38. The method according to claim 35, wherein
An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOS: 106 and 107 corresponding to a part of the rpoB gene sequence related to rifampicin resistance, SEQ ID NOs: 108 and 109 or 110 corresponding to a part of the rrs gene sequence related to streptomycin resistance An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NO: 111, an oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOS: 112 and 113 corresponding to a part of the rpsL gene sequence related to streptomycin resistance, and a part of the rrs gene sequence related to kanamycin resistance An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOS: 114 and 115, an oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOs: 116 and 117 corresponding to a part of the inhA gene sequence involved in isoniazid resistance, SEQ ID NO: 118 corresponding to a part of the katG gene sequence involved in resistance between 119 and 120 and 121,1
22 and 123, 124 and 125, 126 and 127, 12
And oligonucleotide pairs having the nucleotide sequences shown in SEQ ID NOs: 132 and 133 corresponding to a part of the embB gene sequence involved in ethambutol resistance. Or any combination thereof. 39. The method of claim 35, wherein the primary nucleic acid amplification is performed using a primary primer that includes a region of the M. tuberculosis DNA that is amplified using the labeled primer and that amplifies a wider region, A method for performing nucleic acid amplification using the amplified DNA as a template and the labeled primer. 40. The sequence amplified by the primary nucleic acid amplification comprises rpoB gene, rrs gene, rpsL gene,
a region containing a point mutation relating to drug resistance on the entire gene or any one of the inhA gene, katG gene and embB gene, and containing the point mutation by nucleic acid amplification using the labeled primer. 40. The method of claim 39, wherein the region is more specifically amplified. 41. The method according to claim 40, wherein the rp
An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOS: 134 and 135 for amplifying the oB gene, SEQ ID NOs 136 and 137 for amplifying the rrs gene
Oligonucleotide pairs having the nucleotide sequences shown in SEQ ID NOs: 138 and 13 for amplifying the rpsL gene
An oligonucleotide pair having the nucleotide sequence shown in 9, SEQ ID NOS: 140 and 14 for amplifying the inhA gene
An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NOs: 142 and 14 for amplifying the katG gene
An oligonucleotide pair having the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NOs: 144 and 1 for amplifying the embB gene
45. A primary primer, which is any one or any combination selected from oligonucleotides having the nucleotide sequence shown in No. 45. 42. A substrate, wherein the nucleic acid probe obtained by the method according to claim 35, 39 or 40 is contained in the kit according to any one of claims 1 to 6 or 29 to 34. Hybridizing to the oligonucleotide immobilized thereon, identifying an oligonucleotide derived from a gene involved in drug resistance of mutant M. tuberculosis having a nucleotide sequence completely matching the nucleic acid probe, tuberculosis, A method for identifying drug resistance of bacteria. 43. The method according to claim 35, wherein there is a match between the drug-sensitive wild-type Mycobacterium tuberculosis and the drug-resistant mutant Mycobacterium tuberculosis, but genetically conserved between Mycobacterium species. A method wherein an oligonucleotide pair obtained from a gene region which has not been used is used as the primer. 44. The method according to claim 44, wherein the pair of oligonucleotides is rrs
44. The method according to claim 43, which has the nucleotide sequence of SEQ ID NO: 102 derived from a gene. 45. SEQ ID NO: 1 for amplifying the rrs gene prior to nucleic acid amplification using an oligonucleotide pair having the nucleotide sequence shown in SEQ ID NOs: 104 and 105
44. The method according to claim 43, wherein the primary nucleic acid amplification is performed using an oligonucleotide pair having the nucleotide sequences shown in 36 and 137. 46. A nucleic acid probe obtained by the method according to claim 43 or 45, which is hybridized to an oligonucleotide immobilized on a substrate contained in the kit according to claim 32, to thereby obtain a positive control and a negative control. A method for judging M. tuberculosis infection by comparing binding to a control. 47. The kit according to claim 1, which has a combination of the oligonucleotides (a) and (b) described below and a combination of the oligonucleotides (c) and (d) described below. , 39 or 40, and a nucleic acid probe obtained by the method according to any one of claims 43 to 45, simultaneously hybridizing on a substrate, wherein the drug resistance of M. tuberculosis is identified. Or a method of simultaneously performing infection determination: (a) a mutant gene having a point mutation related to the resistance, which is possessed by a mutant Mycobacterium tuberculosis having resistance to any of rifampicin, streptomycin, kanamycin, isoniazid and ethambutol; And the base sequence shown in SEQ ID NOs: 1 to 66, or the 5 ′ side or 3 ′ of each of the above base sequences
A base sequence obtained by extending or shortening the wild-type Mycobacterium tuberculosis gene sequence whose position on the M. tuberculosis bacterium corresponds to each base sequence on either or both sides, wherein each original base Wherein the mutation site contained in the sequence is not substituted;
(B) a wild-type gene of a wild-type Mycobacterium tuberculosis susceptible to rifampicin, streptomycin, kanamycin, isoniazid, and ethambutol; 67 to 101, or 12 to 24 nucleotides obtained from a gene sequence whose position on the genome of M. tuberculosis corresponds to the nucleotide sequence described in (a) above.
An oligonucleotide comprising any of a base sequence designed to have a base length. (C) a region of a gene having a nucleotide sequence that is identical between the drug-sensitive wild-type M. tuberculosis and the drug-resistant M. tuberculosis, but is not genetically conserved among Mycobacterium species; (D) in the base sequence of the oligonucleotide of (c), at least one base, and replacing a base within a range of less than 20% of the number of bases of the base sequence An oligonucleotide obtained by performing 48. The oligonucleotide according to (c),
48. The method according to claim 47, which has the nucleotide sequence of SEQ ID NO: 102 derived from the rrs gene. 49. A substrate comprising a substrate on which oligonucleotides synthesized based on nucleotide sequences obtained from genes on the genomes of bacteria belonging to Mycobacterium tuberculosis and atypical mycobacteria are covalently immobilized. A kit for identifying Mycobacterium infection by hybridization with a nucleic acid derived from a test bacterium. 50. A carbodiimide group or an isocyanate group coated on the surface, and a covalent bond is formed by a reaction between the carbodiimide group or the isocyanate group and an amino group of the oligonucleotide or a linker added to the terminal of the oligonucleotide. 50. The kit according to 49. 51. The kit according to claim 49, wherein said oligonucleotide is immobilized on the surface of said substrate to a size of 10 to 1,000 μm in diameter. 52. Mycobacterium xenopi, Mycobacterium ulcerans (Mycobacterium ulcerans) as bacteria belonging to the atypical mycobacteria
cerans), Mycobacterium szuk
gai), Mycobacterium simiae
e), Mycobacterium shim
oidei), Mycobacterium scrofuraseum (Mycobacteriu)
m scrofulaceum), Mycobacterium non-chromogen
nonchromogen), Mycobacterium marinu
m), Mycobacterium malmoense (Mycobacterium ma
lmoense), Mycobacterium intracellulare (Mycobacteriu)
m intracellular), Mycobacterium gordnae
onae), Mycobacterium fortuitum (Mycobacteriu)
m fortuitum), Mycobacterium chelone
ae), Mycobacterium avium
complex), Mycobacterium kansas
5. The method according to claim 4, wherein at least one of (ii) is included.
9. The kit according to 9. 53. The kit according to claim 49, wherein the gene on the genome is a 16S ribosomal gene. 54. The method according to claim 54, wherein the oligonucleotide is synthesized based on a base sequence specific to each of the bacteria belonging to the tuberculosis bacterium or the atypical mycobacterium present in the 16S ribosomal gene. 50. The kit of claim 49, wherein 55. An oligonucleotide as a positive control having the sequence of claim 54, wherein the oligonucleotide as a negative sequence has a sequence corresponding to the positive control and does not form sequence-specific hybridization. It is characterized by comprising an oligonucleotide in which a part of the base sequence of the positive control has been substituted,
50. The kit according to claim 49, wherein the result of the hybridization with the nucleic acid derived from the test bacterium is compared. 56. The positive control sequence and the negative control sequence are respectively SEQ ID NO: 160 and SEQ ID NO: 161 for M. tuberculosis, and SEQ ID NO: 162 for Mycobacterium avium.
And SEQ ID NO: 165, SEQ ID NO: 164 for Mycobacterium kansai
And SEQ ID NO: 163, SEQ ID NO: 206 and SEQ ID NO: 219 for Mycobacterium xenopi, SEQ ID NO: 2 for Mycobacterium ulcerans
07 and SEQ ID NO: 220, SEQ ID NO: 20 for Mycobacterium
8 and SEQ ID NO: 221, SEQ ID NO: 209 for Mycobacterium simiae
And SEQ ID NO: 222, SEQ ID NO: 21 for Mycobacterium simoydei
0 and SEQ ID NO: 223; SEQ ID NO: 211 and SEQ ID NO: 224 for Mycobacterium scroflaseum; SEQ ID NO: 212 and SEQ ID NO: 225 for Mycobacterium nonchromogen; SEQ ID NO: 213 for Mycobacterium marinum
And SEQ ID NO: 226, SEQ ID NO: 2 against Mycobacterium marmoense
14 and SEQ ID NO: 227, SEQ ID NO: 215 and SEQ ID NO: 228 for Mycobacterium intracellulare, SEQ ID NO: 21 for Mycobacterium gordonae
6, SEQ ID NO: 229; SEQ ID NO: 217 and SEQ ID NO: 230 for Mycobacterium fortuitum; SEQ ID NO: 218 for Mycobacterium keronae
56. The kit of claim 55, which is SEQ ID NO: 231. 57. A substrate comprising a substrate on which oligonucleotides synthesized based on nucleotide sequences obtained from genes on the genomes of bacteria belonging to Mycobacterium tuberculosis and atypical mycobacteria are covalently immobilized. A kit for simultaneously performing drug resistance discrimination of Mycobacterium tuberculosis and identification of bacterial infection belonging to the genus Mycobacterium including Mycobacterium tuberculosis and atypical acid-fast bacterium by hybridization with nucleic acid derived from a test bacterium. 58. The kit according to claim 1, 49 or 57, wherein the nucleic acid derived from the test bacterium is RNA. 59. The kit according to claim 1, 49 or 57, wherein the oligonucleotide to be synthesized is RNA or PNA (peptide nucleic acid). 60. When the oligonucleotide has a hairpin structure, a loop structure, or a three-dimensional structure that prevents hybridization with a test nucleic acid, one or more nucleotides constituting the oligonucleotide may be inosine or any one of them. An oligonucleotide whose tertiary structure has been released by substituting with a nucleic acid that does not also pair with the nucleotide of (1) to (6) or (3).
The kit according to any one of 4, 49, 57. 61. The kit according to claim 2, wherein ultraviolet light is irradiated during the reaction between the carbodiimide group or the isocyanate group on the substrate and the oligonucleotide. 62. A mutant gene which is derived from a mutant gene having a point mutation, a deletion mutation or an insertion mutation related to drug resistance of drug-resistant M. tuberculosis and containing the mutation site.
An oligonucleotide designed using a database containing the genes on the M. tuberculosis genome, in base length, with the M. tuberculosis genome in each nucleotide sequence 5 'and / or 3' to the nucleotide sequence. The above position is a nucleotide sequence obtained by extending or shortening the corresponding wild-type Mycobacterium tuberculosis gene sequence,
A method for designing an oligonucleotide, wherein the mutation site contained in each original base sequence is not substituted.