JP3360736B2 - Probe specifically recognizing mycobacterial microorganism and method for detecting mycobacterial microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a mycobacterium (My
Cobacteria) a method for detecting a microorganism belonging to the probes specifically recognizing and Mycobacterium genus microorganism belonging to the genus.

[0002]

2. Description of the Related Art The number of mycobacterial infections including tuberculosis has been steadily reduced due to the development of effective chemotherapeutic agents, improvement of nutritional status and improvement of life. However, in recent years, the decreasing trend has slowed down. In 1988, the number of registered patients with tuberculosis exceeded 50,000, and atypical anti-tuberculosis such as Mycobacterium avium (M. avium) was used as an opportunistic infection during AIDS and immunosuppression. Many cases of infection with acid bacteria have been reported. Therefore, it is very important to separately detect various mycobacteria, detect them with high sensitivity, and quickly identify them, in determining a treatment policy.

[0003] Conventionally, the detection of acid-fast bacteria has been carried out mainly by smear staining and culture. Although smear staining is simple,
Low sensitivity, unable to identify bacteria. On the other hand, the culture method has an advantage that the acid-fast bacterium can be detected with high sensitivity, and the bacterium can be distinguished by a biochemical identification method. However, mycobacteria have a very slow rate of division, require a period of 3-8 weeks to culture, and take 2-4 weeks for final identification. Therefore,
This was inconvenient for early diagnosis.

On the other hand, as an early diagnosis method, Japanese Patent Application Laid-Open No. 63-180
No. 859 describes a measurement method in which a monoclonal antibody specific to M. tuberculosis is produced by a hybridoma cell line and the monoclonal antibody is used. However, in this method, the pretreatment of the test sample was complicated. Hybridization methods using I ¹²⁵ labeled DNA probes specific to various Mycobacterium tuberculosis have also been developed (for example, Diag).
n. Microbiol. 1987; 8: 69-7.
8). However, this method requires special equipment because it uses a radioactive substance, and it is not always possible to directly detect and identify M. tuberculosis from clinical samples.

[0005]

SUMMARY OF THE INVENTION The present inventor has made it possible to amplify DNAs of M. tuberculosis and atypical mycobacteria contained in clinical samples with specific DNA primers with high sensitivity, and to amplify the amplified DNA with specific DNA primers. A method for distinguishing acid-fast bacilli by digestion with restriction enzymes and the electrophoresis pattern has been devised.
No. 6,199,619. Subsequently, as a result of repeated studies to realize a more accurate and quick diagnosis, the entire nucleotide sequence of positions 1414 to 1609 in the DNA sequence encoding the dnaJ protein of 19 microorganisms belonging to the genus Mycobacteria was determined. Found that there are nucleotide sequences unique to 19 kinds of bacteria. And
Based on these nucleotide sequences, various probes that specifically recognize each bacterial species are prepared, and by using these probes, microorganisms belonging to the genus Mycobacterium can be detected and sorted with high sensitivity and speed. It became possible. Therefore, an object of the present invention is to detect microorganisms belonging to the genus Mycobacterium specifically, with high sensitivity, and quickly,
It is to provide means for discriminating.

[0006]

Accordingly, the present invention relates to a genus of Mycobacteria, which comprises using an oligonucleotide probe consisting of any one of the nucleotide sequences represented by SEQ ID NOS: 5 to 19. The present invention relates to a method for detecting a microorganism belonging thereto. The present invention also relates to an oligonucleotide probe consisting of any one of the nucleotide sequences represented by SEQ ID NOs: 5 to 19. Further, the present invention provides
The first oligonucleotide primer comprising the nucleotide sequence represented by SEQ ID NO: 3 and the second oligonucleotide primer comprising the nucleotide sequence represented by SEQ ID NO: 4; It also relates to primer sets.

According to the present invention, microorganisms belonging to 19 kinds of Mycobacteria whose DNA sequences encoding the dnaJ protein have been determined are shown in the following list 1. In addition, the names of the standard strains used in the present invention are shown after the scientific names of these 19 microorganisms (see Examples 2 and 4). NIHJ is the National Institute of Health.
Institute of Health, Japan), ATCC is American
Type Culrture Collection, NCTC is National Col
lection of Type Cultures, and KK are the Tuberculosis Research Institute. List 1 (1) Mycobacterium tuberculosis (M. tuberculosis; M. tuberculosis; NIHJ1633) (2) Mycobacterium bovis BCG (M. bovis BCG: M. bovis BCG; KK 12-02) 3) Mycobacterium bovis: M.bo
vis; NIHJ 1607) (4) Mycobacterium africanum (M. africanum;
NIHJ 1602) (5) M.microti; KK 14
-01) (6) M.marinum; NIH
J 1620) (7) M. simiae; NIHJ 1
627) (8) Mycobacterium scrofuraseum
laceum; NIHJ 1626) (9) M. szulgai; NCT
C 10831) (10) Mycobacterium gordona (M.gordona)
e; NIHJ 1617) (11) Mycobacterium avium:
M.avium; NIHJ 1605) (12) Mycobacterium intracellulare (M.in
tracellulare; ATCC 13950) (13) M. xenopi; NIHJ 1
638) (14) M. gastri; NIHJ
1616) (15) Mycobacterium kansasii (M. kansasii;
ATCC 12478) (16) Mycobacterium fortutam (M.fort
uitum; NIHJ 1615) (17) M. chelonei;
NIHJ 1611) (18) M. hemophil
um; KK 49-01) (19) Mycobacterium paratuberculosis (M.
paratuberculosis; ATCC19698).

[0008] Each of the 19 types of acid-fast bacilli has a specific protein, dnaJ protein,
It is considered that their amino acid sequences are almost common. Therefore, the present inventors have focused on the DNA encoding the dnaJ protein of each acid-fast bacterium, and decided to determine the nucleotide sequence of the DNA encoding the protein. First, the D
To amplify NA, PCR (Polymerase)
A Chain Reaction method was used. By utilizing the PCR method, a desired DN can be obtained from a very small amount of cellular DNA.
Only the A region can be automatically amplified to about 1,000,000 times (Science, 239: 487-491,
1988). In the PCR method, an amplification cycle is repeated using two types of DNA primers for a + strand and a − strand and a heat-resistant DNA polymerase across a DNA region to be amplified.

As primers for amplifying DNA encoding the dnaJ protein, Japanese Patent Application Laid-Open No. 4-361
The primer disclosed in JP-A-99-99 can be used. That is, a DNA primer containing an oligonucleotide portion of at least 10 bases of the nucleotide sequence described in SEQ ID NO: 1 in the Sequence Listing is used as the primer 1, and the nucleotide sequence described in SEQ ID NO: 2 in the Sequence Listing is used as the primer 2. DNA encoding the dnaJ protein of each acid-fast bacterium by using a DNA primer containing an oligonucleotide portion of at least 10 bases
196 bp from the 1414th base to the 1609th base (as a whole, including a primer portion before and after the base, if each primer consisting of 20 bases is used, the base from the 1394th base to the 1629th base is used) 2
36 bp) can be specifically amplified.

However, for Mycobacterium chelonei, amplification is not carried out with the above-mentioned primer set, so that another primer set, that is, primer 3 has the sequence of SEQ ID NO: 3 in the sequence listing. A DNA primer containing at least a 10-base oligonucleotide portion of the base sequence described in SEQ ID NO: 4 in the Sequence Listing as a primer 4, using a DNA primer containing at least a 10-base oligonucleotide portion of the base sequence described in By using
5 bp (as a whole, including a primer portion before and after, if each primer consisting of 20 bases is used, 225
bp) can be specifically amplified. As a result of the base sequence analysis, d.
Since a deletion occurs between positions 1409 and 1418 of the naJ gene, it is considered that amplification is not performed by the combination of the primer 1 and the primer 2.

[0011] These primers 1 to 4 can be used
A It can be prepared by a known DNA synthesis method (for example, a phosphoramidite method) using an automatic synthesizer (for example, an automatic synthesizer manufactured by Applied Biosystems). In the present invention, a heat-resistant D
An NA polymerase, particularly a thermostable DNA polymerase capable of maintaining its activity at a temperature up to about 95 ° C., for example, a commercially available Taq polymerase can be used.

More specifically, in the same manner as in the method described in JP-A-4-36199, 19
DNA is extracted from the cultured cells of each type of acid-fast bacterium by a known technique, and the primer, DNA polymerase, or a buffer (eg, Tris-HCl buffer), a stabilizer (eg, gelatin), Alternatively, a salt (for example, sodium chloride) is contained, and the amplification cycle of the PCR method is performed using this mixture. For the dnaJ gene of each acid-fast bacterium thus amplified, the conventionally known Maxam-Gilbert method (MG method), dideoxy method, genomic
The DNA base sequence is determined by a sequencing method or the like.

From the determined base sequences, specific portions were selected, and oligonucleotide probes consisting of 15 to 40 bases were prepared. Suitable probes are, for example, as shown in Listing 2 below. List 2 (1) Oligonucleotide consisting of at least 15 bases of the nucleotide sequence described in SEQ ID NO: 5 in the sequence listing (2) Mycobacterium kansasii Sequence Listing Oligonucleotide consisting of at least 15 bases of the base sequence described in SEQ ID NO: 6 (3) M.avium consisting of at least 15 bases of the base sequence described in SEQ ID NO: 7 in the sequence listing Oligonucleotides (4) Mycobacterium intracellulare (M.intr
acellulare) an oligonucleotide consisting of at least 15 bases of the nucleotide sequence described in SEQ ID NO: 8 in the sequence listing (5) Mycobacterium marinum (M. marinum) the oligonucleotide described in SEQ ID NO: 9 in the sequence listing Oligonucleotide consisting of at least 15 bases (6) Oligonucleotide consisting of at least 15 bases of the base sequence described in the sequence of SEQ ID NO: 10 in M. gastri (M. gastri) M.scrofu
laceum) Oligonucleotide consisting of at least 15 bases of the nucleotide sequence of SEQ ID NO: 11 in the sequence listing (8) Mycobacterium sullgai (M. szulgai) The oligonucleotide of the nucleotide sequence of SEQ ID NO: 12 in the sequence listing Oligonucleotide consisting of at least 15 bases (9) Mycobacterium gordonae (M. gordonae) Oligonucleotide consisting of at least 15 bases of the base sequence described in SEQ ID NO: 13 in the sequence listing (10) Mycobacterium zenopi ( M. xenopi) Oligonucleotide consisting of at least 15 bases of the base sequence described in SEQ ID NO: 14 in the sequence listing (11) Mycobacterium simiae (M. simiae) Base described in SEQ ID NO: 15 in the sequence listing Oligonucleotide consisting of at least 15 bases in sequence (12) Mycobacterium fortuy Tom (M.fort
uitum) an oligonucleotide consisting of at least 15 bases of the base sequence described in SEQ ID NO: 16 in the sequence listing (13) Mycobacterium chelonii (M. chelonei) The oligonucleotide having the base sequence described in SEQ ID NO: 17 in the sequence listing Oligonucleotide consisting of at least 15 bases (14) M. hemophil
um) Oligonucleotide consisting of at least 15 bases of the base sequence described in SEQ ID NO: 18 in the sequence listing. (15) Mycobacterium paratuberculosis (M.
paratuberculosis) Oligonucleotide consisting of at least 15 bases in the nucleotide sequence of SEQ ID NO: 19

[0014] M. bovis BCG
), Mycobacterium bovis, M. bovis
African Num (M. africanum) and Mycobacterium
The nucleotide sequence of the dnaJ gene of M. microti is completely identical to the nucleotide sequence of the dnaJ gene of M. tuberculosis. The same probe can be used. The probes described in List 2 can be synthesized by conventionally known means in the same manner as in the primers 1 to 4.

Each probe of the present invention can specifically recognize 15 types of acid-fast bacterium from the 19 types of acid-fast bacterium described in List 1. Although the dnaJ gene base sequences of M. tuberculosis, M. bovis BCG, M. bovis, Mycobacterium africanum and Mycobacterium microti are completely identical, they cannot be distinguished from each other. There is no clinical problem because they form a group. That is, in the present invention, the probe specifically recognizing one specific acid-fast bacterium contains at least 15 bases that match the nucleotide sequence of the DNA encoding the dnaJ protein of the specific acid-fast bacterium, And 15
It contains an oligonucleotide whose base sequence differs from the base sequence of the DNA encoding the dnaJ protein of the other 14 acid-fast bacteria by at least two. By appropriately adjusting the hybridization conditions, it is possible to prevent hybridization between the probe and DNA only by changing one base in the base sequence of the probe. However, from the viewpoint of reliability and reproducibility, a base sequence region having at least two bases different from the base sequence of the DNA encoding the dnaJ protein of the other 14 mycobacteria is used as the specific probe region. Therefore, the base sequence shown in List 2 is one of typical examples, and is not limited to this region.

By using the probe, 15 kinds of microorganisms belonging to the genus Mycobacteria described in List 1 can be specifically detected. That is, by attaching an appropriate label to the probe, bringing the probe into contact with a test sample, and detecting a signal from the label, a target microorganism can be specifically detected. As the label, a radioactive substance, more preferably a non-radioactive substance (for example, an enzyme, biotin, a fluorescent substance, a luminescent substance) can be used.

The specimen is not particularly limited as long as it may contain a microorganism belonging to the genus Mycobacteria. Particularly, clinical specimens such as sputum, gastric aspirate, bronchoalveolar linear fluid, stool, urine , Cerebrospinal fluid or blood. From these samples, DNA encoding the dnaJ protein of the acid-fast bacterium
Is removed by a known method to prepare a test sample that can be hybridized with each of the above-described probes. DNA
To remove SDS, for example, SDS containing protease K
After treating the sample with the solution, it is further treated with chloroform / phenol to precipitate the DNA with ethanol. The collected DNA is directly fixed on a suitable carrier (for example, a nylon filter) and a test sample for dot blot hybridization is prepared, or digested with a suitable restriction enzyme, and a test sample is prepared by Southern blotting. be able to. If necessary, DNA in the sample
Can be amplified by, for example, the PCR method using the above-mentioned primers 1 to 4.

In the method of the present invention, the contact between the label-carrying probe and the DNA in the test sample can be carried out by a conventionally known method, for example, by combining them on a filter after electrophoresis or on a slide glass on which cells are fixed. It can be carried out. That is, when both chains meet with a complementary chain during molecular movement in a solution, a double chain is formed by hydrogen bonding between the bases.

As a method for generating a signal from the label bound to the probe and measuring the signal, any conventionally known method can be used. For example, when a radioisotope is used as a label, after hybridization, it is exposed on a radiation emulsion or an X-ray film, developed, and its signal is detected. When biotin is used as a label, after hybridization, peroxidase-labeled avidin is reacted to form a biotin-avidin complex, and the color development of peroxidase is detected. In this way, it is possible to detect the Mycobacterium tuberculosis group, the atypical mycobacteria, and the atypical mycobacteria by type.

[0020]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Synthesis of primers A 20-base oligonucleotide represented by the sequence of SEQ ID NO : 1 in the sequence listing (primer 1), and a 20-base oligonucleotide represented by the sequence of SEQ ID NO: 2 in the sequence listing (primer 2) The oligonucleotide of 20 bases (primer 3) represented by the sequence of SEQ ID NO: 3 in the sequence listing and the oligonucleotide of 20 bases (primer 4) represented by the sequence of SEQ ID NO: 4 in the sequencing listing were each 380A- DNA
It was synthesized under the condition of trityl-one using a synthesizer (Applied Biosystems). The column was cut with a 27% aqueous ammonia solution and heated at 55 ° C. overnight. 2 ml of the obtained solution is diluted with 1 ml of ion-exchanged water, and the diluted solution is diluted with OPC (Oligonuclideide purif).
application column cartridge (Applied Biosystems) at a rate of 1-2 drops / sec. The eluate was washed three times with 5 ml of diluted ammonia aqueous solution, twice with 5 ml of ion-exchanged water, twice with 5 ml of 2% TFA solution, and further twice with 5 ml of ion-exchanged water. To obtain a purified oligonucleotide adsorbed Nukure <br/> Oh Chi de eluted 3 times with 20% acetonitrile solution 1 ml. The following Examples 2 and 4 were carried out using the primers 1 to 4 thus obtained.

Example 2: Amplification of various microorganism DNAs and bases
Determination of Sequence (1) DNA was extracted with phenol / chloroform from the cultured cells of the 19 acid-fast bacterium standard strains. Subsequently, Table 1 was placed in a 500 μl Eppendorf tube.
Was prepared, and DNA was amplified using the primers 1 to 4 prepared in Example 1. In Table 1, the Ta q DNA polymerase, Ampl
iTaq DNA polymerase (Cetus) was used. Also, M. chelone
Regarding i), primers 1 and 2 shown in Table 1 were changed to primers 3 and 4.

[0022]

[Table 1]

Next, the reaction solution was treated with mineral oil (Sigma).
a Cal M-3516) with 100 μl. The obtained sample was used as a DNA Thermal Cycler (P
erkin-Elmer Cetus).
(I) 1 minute at 94 ° C, (ii) 2 minutes at 65 ° C and (ii)
i) 25 cycles of a three-step amplification at 72 ° C. for 2 minutes
Repeated times. In the last cycle of the 25th,
Step (iii) was performed for 5 minutes. Each 10 μl of the reaction solution obtained by the DNA amplification operation was subjected to electrophoresis on a 2% agarose gel containing ethidium bromide (0.5 μg / ml). The results are shown in FIG. In FIG. 1, “TB complex” at the top indicates a group of Mycobacterium tuberculosis, “I” is type I according to Runyon's classification,
“II” is a type II according to the Runyon classification, and “III”
"Is a type III according to Runyon's classification, and" IV "
Is a fast-growing bacterium according to the Runyon classification, and "ot
"hers" is a bacterial species that does not belong to the above classification, the middle row shows the type of microorganism, and the numerical value on the right side (236b
p) means the size of base pairs. As is clear from FIG. 1, DNA was amplified in all 18 kinds of antibacterial acids except Mycobacterium cheloni and 236 bp
A band appeared. As a result of using Mycobacterium chelonii with primers 3 and 4, a band appeared at 225 bp, and it was confirmed that the band was amplified.

(2) The nucleotide sequence is determined by the method of Sanger et al. (Proc. Natl. Acad. Sci. US
A74: 5463-5467, 1977). That is, an EcoRI site (GATGATTC) was placed at the 5 ′ end of the upstream primer of the dnaJ gene primer.
And a HindIII site (GAT
AAGCTT), PCR was performed, and the amplified D
NA was confirmed on a 2% agarose gel. Extracted with chloroform for the purpose of removing oil, precipitated with ethanol,
The mixture was allowed to stand at 30 ° C. for 30 minutes, then centrifuged, washed with water, dried and digested with two restriction enzymes, EcoRI and HindIII. Run the entire reaction mass on a 2% agarose gel,
The gel containing the R amplification product was cut out, and the DNA was purified by silica beads after dissolving the gel with an aqueous sodium iodide solution (DNAPrep; Diatron). On the other hand, M13mp18, a sequence vector, was digested with EcoRI and HindIII at 37 ° C. for 1 hour as described above. PCR amplification product digested with restriction enzymes
Mix with p18 at a ratio of 1: 4 and add 16 ° C. with T4 ligase.
For 1 hour and transformed into E. coli by the calcium chloride method.
Selected by PTG. After selecting white plaques, the cells were cultured with shaking at 37 ° C. for 5 hours. Purifying the single-stranded DNA in the supernatant,
The nucleotide sequence was determined by the dideoxy method. Table 2 shows the results.
It is shown in Table 8.

Tables 2 to 8 show that M. tuberculosis (M.tu
A (adenine residue), T (thymine residue), G (guanine residue) and G (guanine residue)
(Cytosine residue), and the amino acid sequence deduced from the nucleotide sequence is represented by one letter code (IUPAC-IUB)
Committee on Biochemical
Nomenclature Recommendation
on). Also, in the base sequences of other acid-fast bacteria,
These bases are shown only when they differ from the nucleotide sequence of M. tuberculosis, and "-" is described in the same case. In addition, in the amino acid sequences of other acid-fast bacteria, those amino acids are shown only when they differ from the amino acid sequences of M. tuberculosis, and the descriptions are omitted in the same case. Note that "*" means missing.

As is clear from Tables 2 to 8, M. tuberculosis and M. bovis BCG (M. bovi)
BCG), 141 of the dnaJ gene of M. bovis, M. africanum and M. microti.
The 4th to 1609th nucleotide sequences were completely identical. Mycobacterium avium (M.avium), known as aviumu complex and cannot be distinguished by biochemical identification
And Mycobacterium intracellulare (M.intracel
lulare) showed a high homology of 94% identity at the nucleotide level. Mycobacterium avium and Mycobacterium intracellulare are similar to Mycobacterium tuberculosis at the nucleotide level in the former at 88%, at the latter at 86%, and at the amino acid level, the former at 97% and the latter at 94%, respectively. Had. In addition, Mycobacterium avium is mycobacterium paratuberculosis (M. paratuberculos)
is) shows extremely high homology of 99% at the nucleotide level, and is completely identical at the amino acid level. Furthermore,
M. kansasi (M. kansasi) is thought to have similar pathogenicity and susceptibility to antituberculosis drugs.
The homology between i) and M. tuberculosis was as low as 74% at the nucleotide level and 72% at the amino acid level.

Example 3 Preparation of Probe From the results of Example 2, sites specific to each acid-fast bacterium were selected, and the following 15 types of probes 1 to 15 were synthesized in the same manner as in Example 1. Probe 1: Specific to M. tuberculosis: Oligonucleotide consisting of 20 bases described in SEQ ID NO: 5 in the sequence listing Probe 2: Mycobacterium kansasii
Specific to ii): Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 6 in the sequence listing 3: Specific to Mycobacterium avium (M.avium): Description in the sequence of SEQ ID NO: 7 in the sequence listing Oligonucleotide probe consisting of 20 bases 4: Specific to M. intracellulare: Oligonucleotide probe consisting of 20 bases described in SEQ ID NO: 8 in the sequence listing 5: Mycobacterium marinaum (M.marinu
m) specific: oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 9 in the sequence listing Probe 6: M. gastri
Specific to: Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 10 in the sequence listing Probe 7: Mycobacterium scrofuraceum (M.
Specific to S. scrofulaceum): Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 11 in the sequence listing Probe 8: M. szulga
Specific to i): Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 12 in the sequence listing 9: Mycobacterium gordonaire (M.gord)
onae): An oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 13 in the sequence listing. Probe 10: M. xenopi
Specific to: Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 14 in the Sequence Listing 11: M. simiae
Specific to e): Oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 15 in the sequence listing 12: Specific to M. fortuitum: Sequence of SEQ ID NO: 16 in the sequence listing Oligonucleotide probe 13 consisting of 20 bases described in M.chel
specific to onei): oligonucleotide probe consisting of 20 bases described in the sequence of SEQ ID NO: 17 in the sequence listing Probe 14: Mycobacterium hemofilm (M.he
Mophilum) specific: oligonucleotide probes 15 consisting of 20 bases as set forth in the sequence of SEQ ID NO: 18 in Sequence Listing: specific for Mycobacterium parametric tuberculosis (M.paratuberculosis): sequence of SEQ ID NO: 19 in the Sequence Listing The oligonucleotide consisting of 20 bases according to the above

The 20-mer oligonucleotide was synthesized and purified using an OPC cartridge. 10 pmol of the purified oligonucleotide, λ- ³² PATP and polynucleotide kinase were mixed in a buffer, reacted at 37 ° C. for 30 minutes, and a ³² P label was added to the 5 ′ end. Unreacted isotopes were separated by a spin column and used for the following dot hybridization.

Example 4: Hybridization (Pro
Specificity of tuberculosis) Among the standard strains described in the above list 1, two types of M. tuberculosis (M. tuberculosis) and M. bovis (M. bovis) and 14 types of non- PCR was performed on the standard mycobacterial bacterium strain in the same manner as in Example 2. Separately, M. tuberculosi
s), M. kansasii,
Mycobacterium scrofulaceum
m), Mycobacterium intracellulare (M.intra
cellulare), Mycobacterium simiae (M.simia)
e) DNA of clinical isolates of M. avium and M. avium was extracted with phenol / chloroform,
Amplified by PCR in the same manner as described above. Each PC
100 μl of an alkali denaturing solution was added to 2 μl of the R amplification product,
After allowing to stand for 5 minutes, suction was performed with a dot hybridization device. The nylon membrane was neutralized, air-dried, and fixed with a UV lamp. The labeled probe prepared in Example 3 (10 ⁻⁶ CPM / ml) was added, and hybridization was performed at 62 ° C. for 2 hours in a hybridization buffer containing trimethylammonium chloride. After washing three times with 6 × SSC, it was air-dried and subjected to autoradiography. The results are shown in FIGS. 5 shows the position of each PCR amplification product in FIGS. 2 to 4, FIG. 7 shows the position of each PCR amplification product in FIG. 6, and the symbols in FIGS. 5 and 7 are as follows. A: Mycobacterium tuberculosis (M. tuberculosis; NIHJ1633) B: Control (blank) C: Mycobacterium bovis (M. bovis: M. bovi)
s; NIHJ 1607) D: Mycobacterium kansasii (ATCC)
12478) E: M.marinum; NIHJ
1620) F: M. simiae; NIHJ 162
7) G: M. scrofula
ceum; NIHJ 1626) H: M. szulgai; NCTC
10831) I: M. gordonae; NI
HJ 1617) J: Mycobacterium intracellulare
ellulare; ATCC 13950) K: Mycobacterium avium: M.av
ium; NIHJ 1605) L: M. xenopi; NIHJ 1638
M: M. gastri; NIHJ 161
6) N: Mycobacterium fortuitum
m; NIHJ 1615) O: M. chelonei; NIHJ
1611) P: M. hemophilum;
KK 49-01) Q: Mycobacterium paratuberculosis (M.para)
tuberculosis; ATCC 19698) 1-6: Mycobacterium tuberculosis (clinical isolate) 7-11: Mycobacterium kansasii (clinical isolate) 12: Mycobacterium scrofuraseum (clinical isolate) 13-17: Mycobacterium Um intracellulare (clinical isolate) 18: Mycobacterium simiae (clinical isolate) 19-23: Mycobacterium avium (clinical isolate)

FIG. 2 shows the results obtained using the probe 1 (specific for M. tuberculosis) prepared in Example 3. It was confirmed that the M. tuberculosis specifically reacted with the M. tuberculosis standard strain, M. bovis M. tuberculosis standard strain, and M. tuberculosis clinical isolates. FIG. 3 shows the results obtained using the probe 2 (specific for Mycobacterium kansasii) prepared in Example 3. It was confirmed that it specifically reacted with the standard strain and clinical isolate of Mycobacterium kansasii. FIG. 4 shows the results obtained using the probe 4 (specific for Mycobacterium intracellulare) prepared in Example 3. It was confirmed that it specifically reacted with the standard strain of Mycobacterium intracellulare and the clinical isolate. FIG. 6 shows the results obtained using the probe 3 (specific for Mycobacterium avium) prepared in Example 3. It did not react with Mycobacterium intracellulare having a homology of 94%, and it was confirmed that M. avium could be specifically recognized.

[0031]

According to the present invention, the entire nucleotide sequence of the DNA encoding the dnaJ protein of 19 microorganisms belonging to the genus Mycobacteria is determined, and various probes that specifically recognize each bacterial species are prepared based on the nucleotide sequences. By using these probes, it has become possible to specifically detect and separate microorganisms belonging to the genus Mycobacteria with high sensitivity and quickly.

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

[Table 5]

[0036]

[Table 6]

[0037]

[Table 7]

[0038]

[Table 8]

[0032]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single-stranded Sequence: GGGTGACGCG GCATGGCCCA

SEQ ID NO: 2 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: CGGGTTTCGT CGTACTCCTT

SEQ ID NO: 3 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Sequence: AGAGGGGGTG ACGCGACATG

SEQ ID NO: 4 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: TCGTCGTACT CCTTGCGCTT

SEQ ID NO: 5 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: AAGGGAATGG GTCGAAAAAG

SEQ ID NO: 6 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: AATCAAGCGA GTGGCTCGAA

SEQ ID NO: 7 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: GCTGCCGGCG AACGATTCAA

SEQ ID NO: 8 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: GCCGCCGGTG AACGATTCAA

SEQ ID NO: 9 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: TATCCGAGGC CAAAGAGGTG

SEQ ID NO: 10 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: GTCTCCTCTG ACGCCACCGC

SEQ ID NO: 11 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: AGCGGTCTCC GAGGCCTACT

SEQ ID NO: 12 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: GGCTTACCGC AAGCTGGCTT

SEQ ID NO: 13 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: CTCCGATGCC GACGCCAAGC

SEQ ID NO: 14 Sequence length: 20 Sequence type: number of nucleic acid chains: single-stranded Sequence: CAACGATCCG CGCGCTGCAG

SEQ ID NO: 15 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single-stranded Sequence: AGCGCGTGAT CTGCACCCGG

SEQ ID NO: 16 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single-stranded Sequence: CGATGCGGGA GCGGCGGAGC

SEQ ID NO: 17 Sequence length: 20 Sequence type: Number of nucleic acid chains: Single-stranded Sequence: ACCGTTACAA GGCCGTCAGC

SEQ ID NO: 18 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Sequence: CCTACATCCC GACGCGAACC

SEQ ID NO: 19 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Sequence: CGATTCAAGG CGGTGTCCGA

[Brief description of the drawings]

FIG. 1 is a photograph instead of a drawing, showing the results of electrophoresis of DNA amplified by a PCR method.

FIG. 2 is a photograph, instead of a drawing, showing the result of performing dot hybridization using a probe specific to M. tuberculosis.

FIG. 3 is a photograph, instead of a drawing, showing the result of performing dot hybridization using a probe specific to Mycobacterium kansasii.

FIG. 4 is a photograph instead of a drawing showing the result of performing dot hybridization using a probe specific to Mycobacterium intracellulare.

FIG. 5 is an explanatory diagram showing the position of each PCR amplification product in FIGS. 2 to 4;

FIG. 6 is a photograph, instead of a drawing, showing the result of performing dot hybridization using a probe specific to M. avium.

FIG. 7 is an explanatory diagram showing the position of each PCR amplification product in FIG.

────────────────────────────────────────────────── (5) References JP-A-4-36199 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/00-15 / 10 BIOSIS (DIALOG) GenBank / EMBL / DDBJ / GeneSeq

Claims (3)

(57) [Claims] 1. A method for detecting a microorganism belonging to the genus Mycobacteria, which comprises using an oligonucleotide probe consisting of any one of the base sequences represented by SEQ ID NOS: 5 to 19. Wherein the oligonucleotide probe consisting of either one of the nucleotide sequences of the base sequence represented by SEQ ID NO: 5-19. 3. A nucleotide containing the nucleotide sequence represented by SEQ ID NO: 3.
No. 1 oligonucleotide primer and SEQ ID NO: 4
Second oligonucleotide ply containing the base sequence to be
2. The detection method according to claim 1, wherein
Primer set for delivery method .