JPH10323189A - Oligonucleotide for detection or strain identification of acid-fast bacterium and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligonucleotide containing a part or the whole of a specific base sequence, capable of detecting an acid-fast bacterium such as a human-type tubercule bacillus or identifying the strain of the acid-fast bacterium in a quick, sure and simple manner, etc., and useful as a primer for nucleic acid multiplication, etc. SOLUTION: This new oligonucleotide contains a part or the whole of nucleic acids sequences expressed by formulae I to IV [A is adenine; C is cytosine; G is guanine; T is thymine; T at an arbitrary position may be substituted with uracil (U)], etc., or a part or the whole of complementary sequences of the above sequences and is useful as a probe, etc., for detecting or identifying strain of acid-fast bacteria such as a human-type tubercule bacillus (Mycobacterium tuberculosis) in a quick, sure and simple manner on clinical diagnosis. These oligonucleotides are obtained by finding the fact that a base sequence specific to an acid-fast bacterium exists on 16S liposomal RNA of the acid-fast bacterium and finding a nucleic acid sequence suitable for detecting or identifying strain of acid-fast bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for rapidly, reliably and easily detecting or identifying bacterial species of the genus Mycobacterium including Mycobacterium tuberculosis in clinical diagnosis. The present invention relates to an oligonucleotide, a method for detecting or identifying an acid-fast bacterium utilizing the oligonucleotide, and a reagent thereof.

[0002]

2. Description of the Related Art Mycobacteria are Gram-positive bacilli having acid-fast properties, and there are many pathogenic bacteria that cause serious lesions, such as Mycobacterium tuberculosis and Mycobacterium leprae. In general, acid-fast bacteria grow slowly and require a considerable amount of time to culture.

[0003] Among them, M. tuberculosis is a pathogen causing tuberculosis in humans, and its examination is extremely important clinically. Pulmonary tuberculosis predominates in the pathogenesis caused by M. tuberculosis, but causes tuberculous lesions in almost all organs,
Causes tuberculous pleurisy, tuberculous meningitis, caries, intestinal tuberculosis, renal tuberculosis, joint tuberculosis, etc. The source of infection is the patient, which is transmitted through the respiratory tract by droplet infection or the like.

Conventionally, tuberculosis bacteria have been tested by a culture method. In general, M. tuberculosis is isolated and cultured on Ogawa medium,
Bacterial species are identified based on the properties of the medium (growth rate / temperature, colony shape, pigment production, etc.), niacin test, nitrate reduction test, heat-resistant catalase test, Tween 80 hydrolysis test, and the like. However, Mycobacterium tuberculosis grows slowly, requiring about 3 to 4 weeks for isolation culture alone, and about 2 to 3 weeks for various subsequent tests. For this reason, the detection or identification test of M. tuberculosis takes about one month or more, despite its clinical importance and urgency, and has been a great constraint on flexible diagnosis and medication. Methods have been developed to detect proteins produced by M. tuberculosis by antigen-antibody reaction.However, it has not yet been possible to identify sufficient strains, and there is a problem with sensitivity. What was needed remained the same.

[0005] Mycobacterium tuberculos
is), other than Mycobacterium avium,
Mycobacterium intracellulare
iumintracellulare), Mycobacterium kansasii, Mycobacterium kansasii
Mycobacterium scrofulaceum, Mycobacterium fortuitum (Mycobacterium scrofulaceum)
Atypical mycobacteria such as fortuitum) are known to be pathogenic to humans. Symptoms are milder and less infectious than tuberculosis, but they are resistant to antituberculosis drugs and there are no effective drugs.
These, like M. tuberculosis, affect human lungs, lymph nodes, skin, and the like, and are particularly frequent in pulmonary infections.

Generally, pulmonary tuberculosis is extremely difficult to distinguish from clinical symptoms and histopathological findings, and must be identified by identifying the bacteria. Pathogenicity is Mycobacterium kansasii> Mycobacterium avium, Mycobacterium intracellulare (Mycobacterium intracellulare)
e)> The other three are the largest, and the top three strains are particularly important.

Conventionally, these atypical mycobacteria have been isolated and cultured on Ogawa's medium in the same manner as Mycobacterium tuberculosis, and their properties (growth rate / temperature, colony shape, pigment production, etc.)
Bacterial species are identified from the results of the niacin test, nitrate reduction test, heat-resistant catalase test, Tween 80 hydrolysis test, light emission test, and the like. However, these also generally grow slowly and often require about one month or more to identify the bacterial species.

[0008] Identification of Mycobacterium tuberculosis and atypical mycobacteria is extremely important clinically. Mycobacterium tuberculosis has a serious medical condition, but treatment with an antituberculous agent such as streptomycin, rifampicin, ethambutol, or isonicotinic hydrazide is effective, so that treatment must be started early. On the other hand, atypical mycobacteria are generally resistant to these drugs. The identification of the species greatly determines the course of subsequent treatment. However, as mentioned above, these bacteria grow slowly and require several weeks to identify the species. In addition, Mycobacterium smegma
tis) and similar non-pathogenic bacteria may be detected.
This is why a rapid and reliable detection and species identification method is desired.

Recently, a method for detecting pathogens with high sensitivity by artificially amplifying DNA or RNA (for example, PCR; see JP-A-60-281) and detecting the amplified nucleic acid has been developed. Have been. These technologies have also been applied to the detection of acid-fast bacilli such as Mycobacterium tuberculosis, including several DNA probes such as “DNA probe“ Chugai-MTD ”(Chugai Pharmaceutical) and“ Amplicor Mycobacterium ”(Nihon Roche). Such a reagent kit has been developed. With these,
Mycobacterium tuberculosis can be detected from clinical samples such as sputum without performing operations such as culture. Conventionally about 5
These techniques, in which the detection of M. tuberculosis, which took ６6 weeks, is completed within a minimum of about 1 day, have been revolutionary.

[0010] However, these gene diagnosis methods also have problems to be further improved. First of all, these kits are operated in almost all procedures in almost all the steps. Therefore, it takes a lot of trouble, and the operation is likely to fail and the result is likely to vary.

[0011]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel oligonucleotide for directly, conveniently, rapidly and reliably detecting or identifying a mycobacterium, and using the oligonucleotide. An object of the present invention is to provide a method for detecting an acid-fast bacterium or a method for identifying a species of the bacterium, and a reagent therefor.

[0012]

Means for Solving the Problems The present inventors have developed 16S rRNA (ribosomal R) of mycobacteria and other organisms.
As a result of various studies on the NA) gene, a nucleic acid sequence suitable for detection of mycobacteria or identification of the species was obtained.
The detection of the bacterium using it was established, and the present invention was completed.

That is, the present invention provides a sequence listing and SEQ ID NO: 1
To 11 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position may be substituted with uracil (U)). Oligonucleotides characterized in that they contain part or all or part or all of their complementary sequences.

The present invention also relates to the nucleic acid sequences described in the Sequence Listing and SEQ ID NOs: 1 to 3 (where A is adenine, C is cytosine,
G represents guanine, and T represents thymine. Further, T at any position may be substituted with uracil (U). ) Or an oligonucleotide containing a part or all of their complementary sequence.

Furthermore, the present invention provides a sequence listing and SEQ ID NOs: 4-1.
1 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position may be substituted with uracil (U)) Oligonucleotides containing a part or the whole, or a part or the whole of their complementary sequence, and a probe for detecting a nucleic acid, characterized by having a label.

The present invention relates to the nucleic acid sequences described in the Sequence Listing and SEQ ID NOs: 1 to 3 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is uracil ( U), or an oligonucleotide containing a part or all of a complementary sequence thereof, or a nucleic acid amplification primer. This is a reagent kit for detecting or identifying the mycobacteria.

The present invention relates to the nucleic acid sequences described in the Sequence Listing and SEQ ID NOs: 4 to 11 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position represents uracil ( Or an oligonucleotide containing a part or all of a complementary sequence thereof, and a probe for detecting a nucleic acid, characterized by having a label. A reagent kit for detecting an acid-fast bacterium or identifying a bacterial species.

The present invention relates to the nucleic acid sequences described in the Sequence Listing and SEQ ID NOS: 1 to 3 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is uracil ( Or primers for nucleic acid amplification, which are oligonucleotides containing a part or all of their complementary sequences, and primers for sequence amplification and SEQ ID NOs: 4 to 11. Nucleic acid sequence (provided that
A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. Further, T at any position may be substituted with uracil (U). ), Or an oligonucleotide containing a part or all of a complementary sequence thereof, comprising a probe for detecting a nucleic acid having a label, characterized in that it comprises a nucleic acid detection probe having a label. It is an identification reagent kit.

According to the present invention, there is provided a method for detecting or identifying an acid-fast bacterium, wherein the nucleic acid amplified by using the nucleic acid amplification primer is detected or identified by using the nucleic acid-detecting probe. This is a method for identifying bacterial species.

[0020] The present invention relates to a method for converting RNA in a sample into at least two types of primers for nucleic acid amplification,
And in the presence of ribonucleic acid (RNA), a reverse transcriptase, R
Method for amplifying RNA having a sequence complementary to RNA in a sample by allowing NaseH, DNA polymerase and RNA polymerase to act thereon, and detecting the acid-fast bacterium or identifying the species using the nucleic acid detection probe Wherein the at least two kinds of nucleic acid amplification primers are nucleic acid sequences described in Sequence Listing and SEQ ID NOs: 1 to 3 (where A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. T at any position may be substituted with uracil (U)) or a part or all of their complementary sequence, wherein at least one of the oligonucleotides The species oligonucleotide is an oligonucleotide having a T7 promoter at the 5 ′ end, and the nucleic acid detection probe is 11. The nucleic acid sequence according to 11, wherein A
Represents adenine, C represents cytosine, G represents guanine, and T represents thymine. Further, T at any position may be substituted with uracil (U). ), Or a method for detecting an acid-fast bacterium, wherein the oligonucleotide is a labeled oligonucleotide containing a part or all of the complementary sequence thereof, or a labeled oligonucleotide. It is.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION One embodiment of the present invention relates to a forward primer (F
1 primer) and a reverse primer (R1 primer) having a sequence complementary to the sequence described in SEQ ID NO: 3
And a primer for amplifying acid-fast bacterium nucleic acids.

Another embodiment of the present invention provides a T7 at the 5 'end.
Oligonucleotide (SEQ ID NO: 1) to which a promoter sequence (AATTCTAATA CGACTCACTA TAGGGAG) is bound
2) A primer for amplifying mycobacteria.

One embodiment of the present invention relates to an acid-fast bacterium comprising a probe having the sequence shown in SEQ ID NO: 4 (M1 probe) and a probe having the sequence shown in SEQ ID NO: 5 (M2 probe). This is a detection probe.

One embodiment of the present invention relates to a M. tuberculosis bacterium comprising a probe having the sequence of SEQ ID NO: 6 (T1 probe) and a probe having the sequence of SEQ ID NO: 10 (T2 probe). This is a probe for identifying bacterial species.

One embodiment of the present invention relates to a Mycobacterium avium strain comprising a probe having the sequence of SEQ ID NO: 7 (A1 probe) and a probe having the sequence of SEQ ID NO: 11 (A2 probe). This is a probe for identifying bacterial species.

One embodiment of the present invention is a probe for identifying Mycobacterium intracellulare species comprising a probe (I1 probe) having the sequence shown in SEQ ID NO: 8 in the Sequence Listing.

One embodiment of the present invention is a probe for identifying Mycobacterium kansasii species comprising a probe (K1 probe) having the sequence shown in SEQ ID NO: 9 in the Sequence Listing.

As one means for amplifying a nucleic acid using the oligonucleotide of the present invention, NASBA (Nucleic acid s
equense-based amplification) nucleic acid amplification technology (Nature; 350, 91, 1991), and a DNA probe automatic measurement system (Japan Clinical Laboratory Automation) that automates this method and the sandwich hybridization method in a microplate. An RNA detection system that combines a combination of academic journals; 20, 728 pages, 1995) has already been developed.

The NASBA method uses a combination of a forward primer having the same sequence as the target RNA and a reverse primer having a sequence complementary to the target RNA and having a T7 promoter added to the 5 'end. Transcriptase, RNaseH, DNA polymerase and T7RN
This is a technique for amplifying RNA having a nucleic acid sequence between a forward primer and a reverse primer by allowing A polymerase to act. The nucleic acid to be amplified by the method comprises a target R
RNA having a sequence complementary to NA. The introduction of the NASBA method, which can amplify RNA in a single-step enzymatic reaction, greatly simplifies the nucleic acid amplification step in operation, and the introduction of a DNA probe automatic measurement system makes the detection step after amplification almost impossible. Be automated. In the present invention, it is preferable to use the above-mentioned RNA detection system for the detection or identification of the acid-fast bacterium.

RNA is a transcript of gene DNA,
The number present in bacteria is much higher than that of DNA. Among them, attention has been paid to 16S rRNA, which is particularly present in a large number and whose sequence is well conserved within or between bacterial species. The oligonucleotide of the present invention, as described below, It is a sequence having excellent properties selectively from the inside.

The oligonucleotides of the present invention have been determined so far by 16S rRNA (ribosomal RNA) gene sequences of mycobacterium (Mycobacterium) bacteria and other organisms (J. Bacteriol .; 172, 116, 1989). Year, Int.J.
Syst. Bacteriol .; 40, 217, 1990, Int.J.Syst.B
acteriol .; 40, 323, 1990, Nucleic Acids Re
s .; 17, 7843, 1989, Nucleic Acids Res .; 18
Volume 5, p. 5558, 1990, etc.), the results of repeated theoretical and experimental verifications of the homology of the gene sequences among the various species resulted in the finding of Mycobacterium tuberc
ulosis) in the 16S rRNA gene, a portion corresponding to a region of about 250 base pairs from the 1st to the 250th, that is, a portion corresponding to the sequence shown in FIG.

FIG. 1 shows 16SrRN of various mycobacteria.
FIG. 2 is a diagram showing A gene sequences in parallel. Only the entire sequence of M. tuberculosis in the first line is shown. For the following bacteria, only bases different from those of M. tuberculosis are displayed, and the same bases are indicated by dots. In other words, a portion corresponding to the 250 base pair region of M. tuberculosis (250 base pairs in M. tuberculosis, but may have a different length in other species) is a mycobacterium species It consists of a highly conserved part that is conserved between the two (hereinafter referred to as the acid-fast bacterium common region), and a part whose sequence differs greatly depending on the species within the acid-fast bacterium (hereinafter referred to as the species-specific region). .

In the present invention, the above-mentioned mycobacterial common region is used as an amplification primer, and R
NA amplification is performed, and a plurality of bacterial species are theoretically amplified by a single amplification, and then detection and bacterial species identification are performed using a detection probe. However, a primer for nucleic acid amplification of any sequence cannot be used for the NASBA method. NAS
Primers for nucleic acid amplification in the BA method were theoretically designed and selected after confirming their performance experimentally. In this manner, among the plurality of candidates, particularly, the amplification primer of the present invention includes a forward primer having a sequence described in Sequence Listing and SEQ ID NO: 1 (hereinafter, referred to as F1 primer, also shown in FIG. 1), A reverse primer (hereinafter referred to as R1 primer, also shown in FIG. 1) having a complementary sequence to the sequence described in SEQ ID NO: 3 is preferable.

These sequences of the present invention can exhibit the expected performance even in nucleic acid amplification techniques other than the NASBA method, for example, PCR (see JP-A-60-281). However, the present invention is not limited to amplification.

The R2 primer used in the NASBA method includes a promoter sequence, for example, a T7 promoter sequence (AATTCTAATA CGACTCACTA TAGGGA) due to the necessity of the NASBA method.
G) must be linked to the 5 'end, the entire sequence of one example of which is set forth in the Sequence Listing SEQ ID NO: 12.

[0036] RNAs commonly amplified by the acid-fast bacterium by the nucleic acid amplification primer of the present invention need to be identified according to the type of bacteria. In the present invention, the following probes utilizing a species-specific region contained in the amplification region are preferable. For example, for identification of M. tuberculosis species,
A probe having the sequence of SEQ ID NO: 6 (hereinafter referred to as T1 probe, also shown in FIG. 1) and a probe having the sequence of SEQ ID NO: 10 (hereinafter referred to as T2 probe,
FIG. 1), a probe having the sequence described in Sequence Listing and SEQ ID NO: 7 (hereinafter referred to as A1 probe, also shown in FIG. 1) and a sequence listing for identification of M. avium species.
A probe having the sequence described in SEQ ID NO: 11 (hereinafter referred to as A2 probe, also shown in FIG. 1) is exemplified, and has a sequence described in SEQ ID NO: 8 for identification of Mycobacterium intracellulare species. Probe (hereinafter I1
For example, a probe having the sequence described in Sequence Listing and SEQ ID NO: 9 (hereinafter referred to as K1 probe, also shown in FIG. 1) for identification of Mycobacterium kansasii species Is exemplified.

A, a probe for identifying Mycobacterium avium species
Since the two probes have a sequence common to many mycobacteria other than Mycobacterium tuberculosis and Mycobacterium bovis, they are also used for their detection. T1 probe, T2
Probes, A1 probes, I1 probes, K1 probes and the like have high bacterial species specificity and can be used alone for dot blot hybridization and the like.

On the other hand, a probe having the sequence shown in SEQ ID NO: 4 (hereinafter referred to as M1 probe, also shown in FIG. 1) and a sequence A probe having the sequence of SEQ ID NO: 5 (hereinafter referred to as M2 probe, also shown in FIG. 1) is exemplified.

A method for detecting an acid-fast bacterium or identifying a species using these nucleic acid amplification primers and nucleic acid detection probes will be described below. First, RNA is taken out from a clinical specimen considered to contain unknown mycobacteria, and for example, by combining the F1 primer and the R1 primer,
Amplify by NASBA method. The amplified RNA product is collected and measured by an automatic DNA probe measurement system incorporating the above nucleic acid detection probe, thereby identifying various bacterial species.

The DNA probe automatic measurement system used in the present invention employs a sandwich hybridization method from the viewpoint of adapting to automation and ensuring more strict bacterial species specificity, and two types of probes are used for one type of measurement. It is preferred to use For example, if the T1 probe and the T2 probe are used, M. tuberculosis is detected, if the A1 probe and the A2 probe are used, M. avium is detected, and if the I1 probe and the A2 probe are used, the mycobacterium is detected. Um intracellulare is detected and K
One probe and A2 probe Mycobacterium kansasii are detected.

If probes corresponding to other strains are prepared based on the sequence shown in FIG. 1, more strains can be identified.
It becomes possible with a single nucleic acid amplification. However, when detecting Mycobacterium tuberculosis, mycobacterium bovis is also detected, and related bacteria species cannot be identified.However, their affinity is well known to doctors and the like, and the frequency of appearance is rare. There is little impact.

When it is desired to detect only the most important M. tuberculosis bacillus with high sensitivity, rather than simultaneously detecting or identifying the species of mycobacteria, 16S rRNA of the mycobacteria is widely and commonly amplified. The method can be sensitively disadvantaged by the presence of RNA from other mycobacteria in the sample. In the present invention, in such a case, a primer having a sequence described in Sequence Listing and SEQ ID NO: 2 specific to the sequence of M. tuberculosis (hereinafter referred to as F2 primer, shown in FIG. 1)
Use If RNA is amplified by combining the F2 primer and the R1 primer, more sensitive detection becomes possible. After amplification, the T1 probe and the T2 probe may be used for detection as described above.

The detection according to the present invention is to detect the entire mycobacteria and various mycobacteria in various clinical materials such as sputum and blood by detecting the sequence of the 16S rRNA gene of each microbe. is there. Determining the species of the isolated and cultured cells is also included. The sample in the present invention includes, in addition to the above-mentioned various clinical materials and cultured cells, nucleic acids isolated and purified therefrom.

The oligonucleotide used in the nucleic acid probe of the present invention does not need to use the entire sequence described in Sequence Listings 4 to 11 or its complementary sequence. Tm value) and the like, and an appropriate region may be used with an appropriate length. However, when it is desired to provide the probe with sufficient specificity, the length is desirably at least 10 bases, more desirably at least about 20 bases.

The oligonucleotide used for the nucleic acid amplification primer in the present invention may also be prepared from the sequence shown in Sequence Listings 1 to 3 and its complementary sequence using a sequence of an appropriate length depending on amplification conditions and the like. Also, some substitution may be made in the oligonucleotide depending on the conditions used, the purpose, and the like.

Since the oligonucleotide of the present invention can be prepared by chemical synthesis, it is possible to obtain an oligonucleotide of constant quality easily, in large quantities and at low cost, as compared with cloned oligonucleotides or polynucleotides. The oligonucleotide of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, a thymidine residue (T) is read as a uridine residue (U). In addition, the DNA may be a DNA containing a uridine residue by performing synthesis by changing T at an arbitrary position to U at the time of synthesis. Similarly, RNA containing a thymidine residue in which U at any position is changed to T may be used. The oligonucleotide may have a point mutation such as deletion, insertion or substitution, or a modified nucleotide.

It is preferable that a label or a substance capable of binding to the label is bound to the nucleic acid detection probe of the present invention. As the label, a known label such as a radioactive substance, an enzyme, a fluorescent substance, or a luminescent substance can be used. Examples of the substance capable of binding to the label include biotin. Labeling can be performed at the end of the oligonucleotide,
It may be in the middle of the sequence. Furthermore, labeling to a sugar, phosphate group or base moiety may be used. For example, enzyme-labeling can be performed by substituting a nucleotide having a linker arm as a member of an oligonucleotide of a nucleic acid sequence (Nucleic Acids Res .; 14, 6115, 19).
1986). As an example, uridine having a linker arm at the 5-position can be chemically synthesized from deoxyuridine by a synthesis method disclosed in Japanese Patent Application Laid-Open No. 60-500717 and introduced into the above oligonucleotide.

The oligonucleotide of the present invention can be bound to a solid support and used as a capture probe. In this case, a sandwich assay (see JP-A-58-40099) may be carried out using a combination of a capture probe and a labeled probe, or a method of labeling and capturing a target nucleic acid (see JP-A-62-200).
-265999). There is also a method in which an oligonucleotide is labeled with biotin, and after hybridization, captured with an avidin-bound carrier.

[0049]

The present invention will be described below with reference to examples.

Example 1 Confirmation of Performance Using Cultured Strains (1) Synthesis of NASBA Primer for Detection of Mycobacterium Bacteria Using the ABI DNA synthesizer type 392, the sequence listing / sequence number 1 was obtained by the phosphoramidite method. Oligonucleotide having the sequence shown (F1 primer), oligonucleotide having the sequence shown in SEQ ID NO: 2 (F2 primer) and SEQ ID NO: 12
An oligonucleotide (R1 primer) having the sequence shown in (1) was synthesized. The procedure followed the ABI manual, and the deprotection of various oligonucleotides was carried out at 55 ° C. overnight with aqueous ammonia. Purification is FPLC manufactured by Pharmacia
At an anion exchange column.

(2) Synthesis of Oligonucleotide Having Linker Arm An oligonucleotide (M1 probe) having the sequence shown in SEQ ID NO: 4 was prepared by the phosphoramidite method using an ABI DNA synthesizer Model 392. Oligonucleotide (M2 probe) having the sequence shown in Sequence Listing / SEQ ID NO: 5, Oligonucleotide (T1 probe) having the sequence shown in Sequence Listing / SEQ ID NO: 6, Oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 7 Nucleotides (A1 probe), oligonucleotides having the sequence shown in SEQ ID NO: 8 (I1 probe),
Oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 9 (K1 probe), oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 10 (T2 probe), having the sequence shown in Sequence Listing / SEQ ID NO: 11 Oligonucleotides (A2 probe) were synthesized. At this time, uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine according to the synthesis method disclosed in Japanese Patent Publication No. 60-500717 was introduced into the oligonucleotide. The uridine can replace any T in the oligonucleotide, but here the 5 ′ T
Were substituted at the 5 'end and those without T at the 5' end were added to the 5 'end. The synthesized linker oligonucleotide was subjected to deprotection treatment with ammonia water at 50 ° C. overnight, and then purified by Pharmacia's FPLC using an anion exchange column.

(3) Labeling of Linker Oligonucleotide with Enzyme / Alkaline Phosphatase Of the linker oligonucleotide obtained in the above (2),
For M2, T2, A2, alkaline phosphatase was bound via its linker arm. The procedure was performed according to the literature (Nucleic Acids Res .; 14: 6115, 1986). Linker oligonucleotide 1.5 A ₂₆₀
Was dissolved in 12.5 μl of 0.2 M NaHCO ₃ , and 25 μl of 10 mg / ml disuccinimidyl suberate (DSS) was added thereto.
The reaction was performed at room temperature for 2 minutes. The reaction solution was 1 mM CH ₃ COONa (pH
Gel filtration was performed on a Sephadex G-25 (Pharmacia) column (1 cm φ x 30 cm) equilibrated in 5.0) to remove excess DSS. The linker oligonucleotide in which the terminal amino group was activated was further added to a twice molar amount of alkaline phosphatase (Boehringer Mannheim, (100 mM NaHCO3)
₃ , 3M NaCl) at room temperature for 16 hours to obtain an alkaline phosphatase-labeled nucleic acid probe. The obtained labeled probe was purified by Pharmacia FPLC using an anion exchange column. The fractions containing the labeled probe were collected and concentrated by ultrafiltration using Centricon 30K (Amicon).

(4) Binding of linker oligonucleotide to microtiter plate Of the linker oligonucleotide obtained in (2) above,
For M1, T1, A1, I1, and K1, binding to the inner surface of the microtiter plate was performed via the linker arm. The linker oligonucleotide was added to a solution of 50 mM borate buffer (pH 10) and 100 mM MgCl ₂ at 0.05 pmol /
After diluting the mixture to a microtiter plate (MicroLite2, Dynatech), 100 µl each was dispensed, and allowed to stand at room temperature for about 15 hours to allow the linker oligonucleotide of probe 2 to reach the inner surface of the microtiter plate. Was bound. After that, 0.1pmol dNTP
Substitution was performed with 0.5% PVP.5xSSC, and blocking for suppressing nonspecific reaction was performed at room temperature for about 2 hours. Finally 1xSS
Washed with C and dried.

(5) Preparation of Samples 29 samples of cultured bacterial samples already identified as the bacterial species listed in FIG. 2 were prepared, collected, washed, and subjected to the method of R. Boom et al.
urnal of Clinical Microbiology 28 3 495
Years). This technique is a method in which nucleic acid such as RNA is bound to silica particles and purified and isolated by repeating washing.

(6) Sample RNA by NASBA method
Amplification of NAS using RNA solution prepared in (5) above
According to the BA method (Nature; 350, 91, 1991) 16SrRN
RNA was replicated and amplified from A. F1 primer as the forward primer and R as the reverse primer
One primer was used. The amplification method and its conditions
Basically, according to Nature; 350, 91, 1991. First,
5 μl of the sample RNA was mixed with 10 μl of a solution containing a composition other than the enzyme (including the primer), and incubated at 65 ° C. for 5 minutes to denature the RNA. Thereafter, 5 μl of the enzyme mixture was added and reacted at 41 ° C. for 90 minutes.
The SBA process has been completed.

(7) Support in microtiter plate
Switch hybridization The NASBA reaction solution amplified in the above (6) is increased by 10 times.
Dilute, denature in 0.3 N NaOH, 20 μl for each test
With 200 mM citrate phosphate buffer (pH 6.0)
Raaf AG (Nippon Seika Co., Ltd.), 750 mM NaCl, 0.1%
 NaN_Three100 μl of the above solution, and prepared in (4) above.
Microtiter plate with capture probe
I put it in. Liquid paraffin is layered to prevent evaporation,
Shake at 50 ° C. for 1 hour. Thus, sample D
The probe with immobilized NA allows for specific microphone
Captured on a titer plate. Next, in (3) above
Alkaline phosphatase labeled detection pro
5xSSC (pH7.0) containing 0.1%
Scruff AG, 0.5% PVP, 10mM MgCl_Two, 1mM ZnCl _Two,
0.1% NaN_ThreeWith 100 μl of the same solution and also prevent evaporation
Liquid paraffin, and shake at 50 ° C for 1 hour.
I let you. This allows the captured sample DNA to
Specific binding of potassium phosphatase-labeled probe
did.

Thereafter, the above probe solution was added to 2 × SSC (pH 7.
0), Substitution with 0.1% Scruff AG, incubation at 50 ° C. for 10 minutes, further substitution with 1 × SSC, and washing. After draining the washing solution, Lumiph, a luminescent substrate for alkaline phosphatase
Inject 100 μl of os 480 (Lumigen) and incubate at 37 ° C for 15
After warming, the luminescence was measured in a dark room using a photon counter (Hamamatsu Photonics). All of these steps are
It is performed automatically by the A-probe automatic measurement system (Journal of the Japan Society of Clinical Laboratory Automation; 20: 728, 1995), and the required time is about 2 hours.

This time, (1) capture probe: M1-detection probe: M2 (common detection of Mycobacterium),
(2) Capture probe: T1-detection probe: T2 (detection of M. tuberculosis), (3) Capture probe: A
1-detection probe: A2 (detection of M. avium), (4) capture probe: I1-detection probe: A2
(M.intrace Intracellulare
llulare) detection), (5) Capture probe: K1-detection probe: A2 (M. kansasii (M. kansasii)
i) Detection) were detected by five types of sandwich hybridization methods.

(8) Results The results are shown in Table 1. The numerical value is the luminescence (cps, count / se
cond), tentatively positive if 5000 cps or more and negative if less. In (1), all were positive, and all the mycobacteria tested this time were detected. (2)
Only M. tuberculosis was positive.
In (3), only M. avium became positive. In (4), only Mycobacterium intracellulare (M. intracellulare) was positive. (5)
Mycobacterium kansasii (M. kansasii) and the related bacterium Mycobacterium scrofuraseum (M. scroful
aceum), Mycobacterium gastri (M. gastri), and Mycobacterium simiae (M. simiae). Naturally, negative results were obtained when distilled water was used instead of RNA.

[0060]

[Table 1]

From the above, it was possible to rapidly detect (within one day) or identify the species of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium kansasii. It has been shown that this is possible.

Example 2 Confirmation of Performance Using Clinical Specimens (Sputum) (Part 1) (1) Preparation of Samples The seven clinical specimens listed in Table 2 were collected and washed, and were completely identical to those in Example 1 (5). Similarly, the method of R. Boom et al. (Journal
of Clinical Microbiology 28: 3, 495 (1990)).

(2) Sample RNA by NASBA Method
Using the RNA solution of the above (1), the NASBA method was used in the same manner as in Example 1 (6) (Nature; 350: 91).
(1991) RNA was replicated and amplified from 16S rRNA. The primer used was also F1-R1 as in Example 1 (6).

(3) Sandwich Hybridization in Microtiter Plate The NASBA reaction solution of the above (2) was diluted 10-fold and detected by various sandwich hybridizations as in Example 1 (7). This time, (1) Capture probe: T1
-Detection probe: T2 (M. tuberculosis)
Detection), (2) capture probe: A1-detection probe: A2
(Detection of Mycobacterium avium (M.avium)), (3) Capture probe:
I1-detection probe: A2 (detection of M. intracellulare), (4) capture probe: K1-detection probe: A2 (detection of M. kansasii) Was detected by the sandwich hybridization method.

(4) Results The results are shown in Table 2. The numerical value is the luminescence (cps, count / se
cond), tentatively positive if 5000 cps or more and negative if less. Sputum 1, sputum 2, and sputum 3 were positive in (1), and the presence of M. tuberculosis was detected. Sputum 4
Sputum 5 was positive in (3) and the presence of Mycobacterium intracellulare was detected. Sputum 6 / Sputum 7
Was negative for all detection systems.

From the results of the subsequent culture and strain identification test, S. tuberculosis was detected in sputum 1, sputum 2, and sputum 3, and Mycobacterium intracellulare was detected in sputum 4 and sputum 5. Completely in agreement with the results of the inventive method. Sputum 6 and sputum 7 were positive in the liquid medium (Bactec System, manufactured by Becton Dickinson), but were negative in the Ogawa medium, and thus the bacterial species was not identified.

[0067]

[Table 2]

From the above, it was shown that the method of the present invention is also effective for actual clinical specimens. The sensitivity was considered to be higher than the smear staining method and equivalent to the culture method using the Ogawa medium.

Example 3 Performance Confirmation Using Clinical Specimens (Sputum) (Part 2) (1) Sample Preparation For the 12 clinical specimens listed in FIG.
In exactly the same manner as in Example 1 (5), the method of R. Boom et al. (Journa
RNA was extracted according to l. Clinical Microbiology 28: 3, 495 (1990).

(2) Sample RNA by NASBA Method
Using the RNA solution prepared in the above (1), the NASBA method was used in the same manner as in Example 1 (6) (Nature; 350
(P. 91, 1991) RNA was replicated and amplified from 16S rRNA. The primers used were the same as in Examples 1 and 2 (A)
F1-R1 and (B) F2-R1 specific to M. tuberculosis
There are two ways.

(3) Sandwich Hybridization in Microtiter Plate The NASBA reaction solution amplified in the above (2) is diluted 10-fold and detected by various kinds of sandwich hybridization as in Example 1 (7). . This time, capture probe: T1-detection probe: T2 (M. tuberculosis (M.tuber
culosis) was detected only by the sandwich hybridization method.

(4) Results The results are shown in Table 3. The numerical value is the luminescence (cps, count / se
cond), tentatively positive if 5000 cps or more and negative if less. (A) shows 8 sputum, 12 sputum, 1 sputum
4. Positive in sputum 15 and the presence of M. tuberculosis was detected. (B) In sputum 8, sputum 12, sputum 14, sputum 1
In addition to 5, sputum 13 and sputum 18 were also positive.

Compared with the result of Amplicor mycobacterium (Roche Japan), which is a similar gene diagnostic kit, the present invention (A) may have a slight lack of sensitivity, but similar results are obtained in (B). Was done. In addition, a clearer judgment can be made as compared with the amplicor. Furthermore, the detection of the present invention is automated, and is superior to Amplicon in operability. Other prior art techniques have not yielded satisfactory results, and the results for the Ogawa medium have not been obtained at this stage in time. In conclusion, if you want to obtain higher sensitivity only for Mycobacterium tuberculosis, (B) F2
It was shown that it is better to use the primer of -R1.

[0074]

[Table 3]

[0075]

Industrial Applicability According to the present invention, it is possible to directly, simply, rapidly and surely detect and identify bacteria of the genus Mycobacterium.
In addition, an oligonucleotide having a part or the whole of the sequence of the present invention or its complementary sequence is suitable as a primer for nucleic acid amplification such as the NASBA method, and also as a detection probe after an amplification reaction. Utilizing the oligonucleotide of the present invention, by these methods, conventionally, compared to tuberculosis bacterium cultivation and subsequent biochemical tests that took several weeks, the time can be significantly shortened, simple, And a reproducible and reliable result is obtained. Moreover, since it can be directly detected from clinical materials such as sputum and blood, its clinical significance is great.
In addition, the method according to the present invention is completed in a shorter time than conventional gene diagnosis methods, requires no skill, and is simple.

[0076]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Location: 1..20 Method for determining characteristics : S Other characteristics: Mycobacterium genus 16Sr
It has a sequence complementary to the sequence of the RNA gene. Sequence GGCGTGCTTA ACACATGCAA 20

SEQ ID NO: 2 Sequence length: 21 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..21 Method determined: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of the 16S rRNA gene. Sequence GGAAAGGTCT CTTCGGAGATA 21

SEQ ID NO: 3 Sequence length: 20 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..20 Determined method: S Other characteristics: Mycobacterium bacterium 16Sr
It has a sequence complementary to the sequence of the RNA gene. Sequence CTACCCGTCG TCGCCTTGGT 20

SEQ ID NO: 4 Sequence length: 22 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..22 Determined method: S Other characteristics: Mycobacterium bacterium 16Sr
It has a sequence complementary to the sequence of the RNA gene. Sequence AGTGGCGAAC GGGTGAGTAA CA 22

SEQ ID NO: 5 Sequence length: 22 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..22 Determined method: S Other characteristics: Mycobacterium bacterium 16Sr
It has a sequence complementary to the sequence of the RNA gene. Sequence TAAGCCTGGG AAACTGGGTC TA 22

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..20 Method determined: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of the 16S rRNA gene. Sequence TAGGACCACG GGATGCATGT 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..20 Method determined: S Other features: Mycobacterium avium 16S
It has a sequence complementary to the sequence of the rRNA gene. Sequence TAGGACCTCA AGACGCATGT 20

SEQ ID NO: 8 Sequence length: 20 Sequence type: Number of nucleic acid chains: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Features Determined method: S Other characteristics: It has a sequence complementary to the sequence of Mycobacterium intracellulare 16S rRNA gene. Sequence TAGGACCTTT AGGCGCATGT 20

SEQ ID NO: 9 Sequence length: 20 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..20 Method determined: S Other characteristics: Mycobacterium kansasii (Mycobact
erium kansasii) has a sequence complementary to the sequence of the 16S rRNA gene. Sequence TAGGACCACT TGGCGCATGC 20

SEQ ID NO: 10 Sequence length: 18 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..18 Features Method determined: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of the 16S rRNA gene. Sequence GCGCTTTAGC GGTGTGGG 18

SEQ ID NO: 11 Sequence length: 20 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence features Location: 1..20 Features Method determined: S Other features: Mycobacterium avium 16S
It has a sequence complementary to the sequence of the rRNA gene. Array AAAGCTTTTG CGGTGTGGGA 20

SEQ ID NO: 12 Sequence length: 47 Sequence type: Number of nucleic acid strands: Both forms Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Location: 1..47 Method determined: S Other characteristics: Mycobacterium tuberculosi
s) It has a sequence complementary to the sequence of the 16S rRNA gene. Sequence AATTCTAATA CGACTCACTA TAGGGAGCTA CCCGTCGTCG CCTTGGT 47

[Brief description of the drawings]

FIG. 1 is a diagram in which 16S rRNA gene sequences (approximately 1 to 250 bases) of various mycobacteria are described in parallel.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 325) (C12Q 1/68 C12R 1:33) (72) Inventor Katsuya Daimon 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Inside Research Institute Co., Ltd.

Claims (15)

[Claims] 1. A nucleic acid sequence described in the Sequence Listing and SEQ ID NOS: 1 to 11 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position represents uracil (U ), Or a part or all of their complementary sequences. 2. The nucleic acid sequence described in the Sequence Listing and SEQ ID NOs. 1 to 3 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position is uracil (U ), Or an oligonucleotide containing a part or all of their complementary sequence, or a part or all of their complementary sequences. 3. A nucleic acid sequence described in SEQ ID NOs: 4 to 11 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is uracil (U ), Or an oligonucleotide containing a part or all of a complementary sequence thereof, and having a label, characterized in that it has a label. 4. A forward primer (F1 primer) having a sequence described in Sequence Listing / SEQ ID NO: 1;
A primer for nucleic acid amplification comprising a reverse primer (R1 primer) having a sequence complementary to the sequence of SEQ ID NO: 3. 5. A T7 promoter sequence (AATT) at the 5 ′ end.
A primer for nucleic acid amplification, which is an oligonucleotide (SEQ ID NO: 12) bound to CTAATA CGACTCACTATAGGGAG). 6. A probe for detecting an acid-fast bacterium nucleic acid comprising a probe (M1 probe) having the sequence of SEQ ID NO: 4 and a probe (M2 probe) having the sequence of SEQ ID NO: 5. 7. A probe for identifying Mycobacterium tuberculosis strains comprising a probe having the sequence of SEQ ID NO: 6 (T1 probe) and a probe having the sequence of SEQ ID NO: 10 (T2 probe). 8. A probe for identifying Mycobacterium avium species comprising a probe (A1 probe) having the sequence of SEQ ID NO: 7 and a probe (A2 probe) having the sequence of SEQ ID NO: 11. 9. A probe for identifying Mycobacterium intracellulare species comprising a probe (I1 probe) having the sequence of SEQ ID NO: 8. 10. A probe for identifying a Mycobacterium kansasii strain comprising a probe (K1 probe) having the sequence shown in the sequence listing and SEQ ID NO: 9. 11. A reagent kit for detecting an acid-fast bacterium or identifying a bacterial species, comprising the nucleic acid amplification primer according to claim 2. 12. A reagent kit for detecting an acid-fast bacterium or identifying a species thereof, comprising the nucleic acid detection probe according to claim 3. 13. A reagent kit for detecting an acid-fast bacterium or identifying a species thereof, comprising the nucleic acid amplification primer according to claim 2 and the nucleic acid detection probe according to claim 3. 14. A method for detecting or identifying an acid-fast bacterium by using the nucleic acid detection probe according to claim 3 for a nucleic acid amplified using the nucleic acid amplification primer according to claim 2. A method for detecting or identifying a mycobacterium belonging to the genus Mycobacterium. 15. An RNA in a sample is treated with reverse transcriptase, RNa in the presence of at least two nucleic acid amplification primers, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Method of amplifying RNA having a sequence complementary to RNA in a sample by reacting seH, DNA polymerase and RNA polymerase, and detecting the acid-fast bacterium or identifying the species using a probe for detecting the RNA with the nucleic acid Wherein the at least two kinds of nucleic acid amplification primers are nucleic acid sequences described in SEQ ID NOs: 1 to 3 (where A represents adenine, C represents cytosine, G represents guanine, and T represents thymine.
Further, T at any position may be substituted with uracil (U). Or a part or all of the complementary sequence thereof, wherein at least one oligonucleotide of the oligonucleotide has an T7 promoter at the 5 ′ end, The nucleic acid detection probe is a nucleic acid sequence described in SEQ ID NOs: 4 to 11 (where A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and T at any position is uracil ( Or an oligonucleotide containing a part or all of the complementary sequence thereof, wherein the oligonucleotide is a labeled oligonucleotide. A method for detecting or identifying a bacterial species of a genus Bacteria.