JPH10191982A - Bacterium-detecting oligonucleotide probe belonging to genus legionella and detection of the same bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oligonucleotide probe for detecting a genus Legionella bacterium, capable of specifically detecting various species of genus Legionella bacteria with high accuracy by one kind of probe in a short time and capable of easily preparing, and to specifically detect various species of genus Legionella bacteria with high accuracy in a short time by using the probe. SOLUTION: A bacterium belonging to genus Legionella is detected by performing hybridization by bringing a label oligonucleotide probe obtained by labeling an oligonucleotide probe having a base sequence of 5'AGC AAA CGC GAT AAG TTG AC3' with a radioactive element, an enzyme, a fluorescent substance or a chemical substance into contact with a bacterium belonging to genus Legionella or its DNA, and using the label as an indicator.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to Legionella (Legionella )
onella ), an oligonucleotide probe and a labeled oligonucleotide probe for detecting, identifying or quantifying a bacterium belonging to the genus (hereinafter, sometimes referred to as a Legionella bacterium), a reagent for detecting a Legionella bacterium containing the labeled oligonucleotide probe, and This is a method for detecting Legionella bacteria using these.

[0002]

2. Description of the Related Art In the detection of Legionella from the environment, Legionella is usually quantified by low pH treatment followed by culturing in a Legionella selective medium such as WYOα. However, no common and specific morphology and biochemical properties of Legionella have been reported, so that after colony formation, auxotrophy, gram staining, morphological observation, reactivity with antibodies, etc. are tested The results of some of these identification tests have led to the determination of Legionella.

[0003] In other words, in the method by cultivation, it takes 5 to 10 days for colonies to exhibit the unique color, shape, and other characteristics, and since the colonies are not completely selective media, some of the resulting colonies include Legionella. Some bacteria form colonies similar to those described above, and it is essential to identify the auxotrophy and Gram stain by collecting the colonies. For this reason, there is a problem that detection of Legionella bacteria requires a long period of about two weeks. In addition, the growth conditions in the environment where Legionella grew are greatly different from the growth conditions using the selective medium, and there is also a problem that depending on the physiological state of Legionella, there may be a case where colonies are not formed on the medium.

[0004] As another means for detecting and quantifying Legionella, there is a method using an antibody. However, no antibody capable of detecting a wide range of species of Legionella has been reported. In addition, the method using an antibody has a problem that it takes time and effort to obtain the antibody.

Incidentally, Japanese Patent Application Laid-Open No. 61-88900 discloses a DNA
A method for detecting Legionella using a probe is described. However, since this method uses a fragment mixture of genomic DNA obtained after a certain separation operation by partially decomposing genomic DNA with a restriction enzyme, it is difficult to repeat exactly the same fragment mixture. There is a problem that there is a difference in sex. In addition, there is a problem that it cannot be used for so-called in situ hybridization. Furthermore, when a genomic DNA fragment mixture is used as a probe, the portion to be detected is not clear, and it is difficult to make the obtained results academically meaningful.

Japanese Patent Publication No. 5-78319 discloses a nucleic acid probe.
Describes how to detect, identify or quantify organisms using
Examples include 23 species of Legionella,
TypicallyL. pnueumophila(PHA1),L. micdadei(HEBA),L.
 bozemanii(WIGA),L. dumoffi(TEX-KL),L. garmanii
(LS-13),L. jordanis(BL540),L. longbeachae,L. MS
H9,L. oakridgenis(Oak Rage 10),L. lansing2,
L. SC-32C-C8,L. WA-316,L. WO-44-3C (L. feeleii),
L. phoenix 1,L. WA-270A,L. PF-209C-C2,L.SC 65C3
(ORW),L. jamestown 26G1-E2,L. MSH-4,L. lansing 
3,L. SC18-C9,L. SC63-C7, L-81-716 (L. wadsworthii)
Describes an exemplary probe that can specifically detect
Have been. However, this nucleic acid probe is also a mixture.
Therefore, there is the same problem as described above. The base of the probe
The length or sequence is not clear,
It is not clear whether they are aware, and there is a problem with the detection accuracy.

[0007] Japanese Patent Publication No. 1-503356 describes a method for detecting Legionella using a nucleic acid probe having a specific sequence. In Examples, 21 types (serotypes) were obtained using three types of mixed probes. Except Legionella spp.). Table 1 shows the Legionella species targeted.

[0008]

[Table 1]

However, in the above detection method, it is necessary to prepare and use three types of probes.
The problem is that only about 55% of the species Legionella (1994) have been tested and it is unclear whether the remaining 45% can be detected.

[0010] Japanese Patent Publication No. 5-507206 also discloses a method for detecting Legionella species using a probe having a specific sequence. However, this probe is for detecting a sequence amplified by PCR using a primer specific to Legionella, and has a problem that the Legionella specificity of the probe is not clear.

[0011] Also, in situ identification of Legion
ellaceae using 16S rRNA-targetedoligonucleotide pr
obes and confocal laser scanning microscopy, Werne
r Manz, Rudolf Amann et.al. Microbiology (1995), 1
41, 29-39 report the evaluation results of two types of probes as probes for detecting the Legionella family. One is 226-243 of 16SrRNA (16SrRN of E. coli)
The other is a probe targeting A numbering), and the other is a probe targeting 705-722.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to detect a large number of species of Legionella spp. In a short time with a single type of probe, and to easily prepare them. An object of the present invention is to provide a possible oligonucleotide probe for detecting Legionella bacteria, and to use the oligonucleotide probe to specifically detect a large number of species of Legionella bacteria with high accuracy in a short time.

[0013]

SUMMARY OF THE INVENTION The present invention provides an oligonucleotide probe and a labeled oligonucleotide probe for detecting the following Legionella bacteria, a reagent for detecting the Legionella bacteria containing the labeled oligonucleotide probe, and a Legionella using the same. This is a method for detecting genus bacteria. (1) A probe consisting of deoxyribonucleotides or ribonucleotides and having a sequence complementary to the variable region of 16S rRNA of a bacterium belonging to the genus Legionella , having a base sequence of 5'AGC AAA CGC GAT AAG TTG AC3 ' An oligonucleotide probe for detecting bacteria belonging to the genus Legionella. (2) A labeled oligonucleotide probe for detecting bacteria belonging to the genus Legionella, wherein the oligonucleotide probe according to (1) is labeled with a radioactive element, an enzyme, a fluorescent substance, or a chemical substance. (3) A reagent for detecting a bacterium belonging to the genus Legionella, comprising the labeled oligonucleotide probe according to (2). (4) Bacteria belonging to the genus Legionella, their DNA or rRNA
A bacterium belonging to the genus Legionella, which is obtained by contacting the labeled oligonucleotide probe according to (2) or the reagent according to (3) with a specimen containing Detection method.

The probe of the present invention is a known Legionella 16Sr
It is obtained by selecting from the sequences complementary to the variable region of RNA a portion that is highly conserved in Legionella and that differs from other genera. That is, the sequence of the probe of the present invention is Legionella pneumophila (Legionella pneumophila) .
onella pneumophila ) and other Legionella 16S rRNA
Corresponds to the nucleotide sequence 871-890 (numbering of 16S rRNA of Escherichia coli). Sequences of the probes of the present invention therefore, Legionella pneumophila (Legionella p
neumophila ) and the complementary sequence of the base sequence 871-890 of DNA encoding 16S rRNA of other Legionella (numbering of 16S rRNA of Escherichia coli) or the base sequence 871-8 of 16S rRNA
It targets 90 (numbering of E. coli 16S rRNA) and is sufficient to recognize these sequences.

[0015] Escherichia coli, Ps. Aeruginosa, Legi
Table 2 shows a partial nucleotide sequence of the 16S rRNA sequence of onella pneumophilia and other Legionella .

[Table 2]

Since the nucleotide sequence of the probe of the present invention is clear as described above and the length of the nucleotide sequence is about 20 nucleotides, it can be easily synthesized from nucleotides or deoxynucleotides by chemical synthesis. For this reason, the same probe can be easily obtained any number of times, and the specificity does not differ between lots. Further, since the probe of the present invention has a relatively short number of bases, it can be used for in situ hybridization, and there are many types of Legionella bacteria that can be detected from one type of probe. From these points, it is excellent as a detection reagent.

The labeled oligonucleotide probe for detecting bacteria belonging to the genus Legionella of the present invention is a probe obtained by labeling the probe with a labeling substance such as a radioactive element, an enzyme, a fluorescent substance or a chemical substance. As such a labeling substance, a conventionally used labeling substance can be used, and specific examples thereof include radioactive elements such as ³² P; fluorescent substances such as FITC and rhodamine; haptens such as dicoxygenin; alkaline phosphatase, peroxidase and the like. Enzymes;
Biochemicals such as biotin; These labeling substances can be introduced into the probe by a known method.

The reagent for detecting bacteria belonging to the genus Legionella of the present invention contains the above-mentioned labeled oligonucleotide probe, and may be composed of only the labeled oligonucleotide probe, or may be dissolved in a buffer or the like. A kit may be used so that hybridization with Legionella bacteria can be easily carried out.

The labeled oligonucleotide probe of the present invention or a reagent containing the same is a Legionella bacterium or a bacterium belonging to the genus Legionella.
A Legionella bacterium to which the probe has hybridized by performing hybridization with a sample containing DNA and then detecting with a labeling substance as an index, for example, by detecting with radioactivity, color development, fluorescence, luminescence, etc. as an index Can be easily detected, identified or quantified with high precision, visually or by a microscope.

The probe of the present invention can be detected, identified or quantified by a single species, and there are many species of Legionella bacteria, and at least 34 species of Legionella (see Table 3 below).
Can be detected. Thus, the probe of the present invention can detect more kinds of Legionella with one kind of probe than the conventional probe.

In the detection method of the present invention, the labeled oligonucleotide probe or reagent is brought into contact with a bacterium belonging to the genus Legionella or a specimen containing the DNA thereof, and hybridization is carried out. It is a method of detecting. Hybridization can be performed by a method similar to the conventional method. For example,
In the case of bacteria, filter the DNA of Legionella bacteria by infiltrating the cells into a filter such as a nylon filter, lysing on the filter, and then irradiating with ultraviolet light or raising the temperature to 80 ° C or higher. Fix on top. This filter is usually immersed in a hybridization solution for 1 hour or more, and further immersed in a hybridization solution to which a labeled oligonucleotide has been added, and reacted for 2 to 24 hours. It is preferable to select a temperature at the time of hybridization that shows sufficient specificity and detection sensitivity, and usually the Tm value of the probe minus 5 to 10 ° C is used as a standard. The temperature at which sufficient specificity and detection sensitivity are obtained can be determined in advance by experiments. Wash the probe and the filter after the reaction with buffer,
Non-specifically bound probes are removed. Washing is preferably performed two to three times with a new buffer solution. Washing is preferably performed at a temperature that maintains sufficient sensitivity, and is usually set lower than the hybridization temperature. The temperature at which sufficient sensitivity is maintained can be determined in advance by experiments.

Detection after hybridization can be performed by a known method depending on the type of the labeling substance. For example, when labeled with a radioactive element, it can be detected by measuring radioactivity by a known method. When labeled with an enzyme, it can be detected by measuring the enzyme activity by a known method. When labeled with a fluorescent substance, it can be detected by measuring the amount of light by a known method. In the case of labeling with an antigen or an antibody, antigen-antibody reaction can be performed using an antibody or an antigen that specifically reacts with the labeled antigen or antibody, and the reaction product can be measured by a known method.

As described above, by using the probe of the present invention, a wide range of Legionella species of the genus Legionella can be specifically detected by hybridization. Therefore, a plurality of operations for identifying Legionella colonies, which have been conventionally performed, can be omitted, and detection can be easily performed. That is, in the conventional culture method, each colony must be picked and used for identification,
By performing colony hybridization using the probe of the present invention, for example, it is possible to determine at a time whether all colonies growing on a medium plate are Legionella. In principle, Legionella that cannot be cultured cannot be detected by culture, but Legionella species that cannot be cultured by the detection method of the present invention can also be detected. It is also possible to clarify how Legionella is distributed in biofilms and tissues in the environment. Further, since the probe is not a genomic DNA fragment mixture, a probe having the same performance can be easily obtained.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION

Examples and Comparative Examples 1) Preparation of Oligonucleotide Probe The probe of the present invention was synthesized by a known solid phase phosphoramidite method. The synthesized probe was purified by high performance liquid chromatography. After purification, it was freeze-dried and stored. The subsequent operation was performed by dissolving in deionized water.

The above probe was used for DIG oligoprobe tailed la
Labeling was performed with dicoxygenin using a bel kit (Boehringer Mannheim). The oligonucleotide probe labeled with dicoxygenin was added to the hybridization solution (0.75 M NaOH) to a concentration of 0.3 pmol / ml.
Cl, 75mM sodium citrate, 1% (w / v) Blockingreagent,
0.1% (w / v) N-lauroylsarkosine, 0.02% (w / v) Sodium dod
esyl sulfate (SDS), 0.1mg / l Poly (a))
It was used in the hybridization described below. Note that dicoxigenin is a type of hapten and is a compound that acts as an antigen.

2) Preparation of specimens Among the reported Legionella species, 35 species shown in Table 3
(Of which two areL. pneumophilaGet the subspecies)
After DNA extraction, dot blot was performed. Compare
For example,Enterobacter cloacae(ATCC 23355),Esch
erichia coli(ATCC 25922),Klebsiella pneumoniae(AT
CC 13883),Proteus vulgaris(ATCC 13315),Pseudomon
as aeruginosa(ATCC 27853),Salmonella typhimurium
(ATCC 14028),Serratia marcescens(ATCC 8100),Stap
hylococcus aureus(ATCC 12600) with Legionella
Similarly, DNA was extracted and subjected to dot blot.

That is, Legionella was treated with BCYEα agar medium (Buffered charcoal-yeast extractagar supplemented).
with α-ketoglutarate, composition; yeast extract 10.0 g, activated carbon powder 1.5 g, ACES buffer 10.
0 g, agar 15.0 g, L-cysteine monohydrochloride 0.4 g, soluble iron pyrophosphate 0.25 g, α-ketoglutarate monopotassium 1.0
g medium containing pH 6.9 ± 0.05) and scraped from the plate with a platinum loop. For DNA extraction, see Current Protocols in Molecular Biology. 2.4.
1. Performed according to the Preparation of Genomic DNA form Bacteria.

The absorbance of the extracted DNA at A ₂₆₀ nm was measured, and each DNA was adjusted to have the same concentration.
Dot blot was performed on a nylon filter in 100 ng increments.
After air drying, the filters were sequentially placed on the filter paper moistened with each of the following solutions for 5 minutes to denature the DNA. (1) 0.5
N NaOH, 0.5M NaCl, (2) 1.5M NaCl, 0.5M Tris-HCl pH
7.4, (3) 0.3 M NaCl, 30 mM sodium citorate pH 7.0.
Thereafter, the mixture was heated at 120 ° C. for 30 minutes to fix the DNA to the filter, and used for the following hybridization.

3) Hybridization Hybridization is performed using DIG oligoprobe tailed labe
It was performed based on lkit Standard 5 xSSC procedure.
The procedure is shown below. Put the filter DNA was fixed in a plastic bag, a hybridization solution [0.75 M NaCl in 100 cm ² per 20 ml, 75 mM sodium ci
trate, 1% (w / v) Blockingreagent, 0.1% (w / v) N-lauroyl
sarkosine, 0.02% (w / v) Sodium dodesyl sulfate (S
DS), 0.1 mg / l Poly (a)] and sealed without bubbles. The reaction was carried out at 68 ° C. for 1 hour or more with gentle shaking in a hybridization oven. The pre-hybridization solution was discarded, and a new hybridization solution containing the oligonucleotide probe prepared in the above 1) at a concentration of 0.3 pmol / ml was placed in a plastic bag so as to be 2.5 ml per 100 cm ² . After sealing by removing bubbles, the mixture was reacted at 50 ° C. for 2 to 3 hours with gentle shaking in a hybridization oven.

Thereafter, the filter was taken out of the plastic bag, and 0.3 M NaCl, 30 mM sodium citrate pH 7.0,
The 0.1% SDS solution in a container such as Tupperware 100 cm ² per 50ml
Then, the filter was immersed, washed by shaking, and the weakly bound probe was removed. After 5 minutes, the solution was replaced and washed for another 5 minutes. Then, 15 mM NaCl, 1.5 mM sodium citra
te pH 7.0, 0.1% SDS solution in a container such as tapper 100cm ²
The filter was immersed, shaken and washed to remove weakly bound probes. After 15 minutes, the solution was replaced and washed for another 15 minutes.

Filter the filter with 160 ml of 100 mM Ma per 100 ml
leic acid, 150 mM NaCl pH7.5 solution. Next, the filter was immersed in a solution obtained by adding 1% blocking reagent to the above solution, and reacted with gentle shaking for 20 minutes. An anti-dicoxygenin alkaline phosphatase-conjugated antibody was added in an amount of 1 / 10,000 of the above volume, and reacted with gentle shaking for 20 minutes. Remove the filter and put 100mM in a new container
Maleic acid, 150 mM NaCl pH7.5, 0.3% tween20 was added, and the filter was immersed and washed for 10 minutes while gently shaking. Changed to a new solution and repeated. The solution was replaced with a new solution, and the process was repeated.

100 mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM
The filter was put in the MgCl ₂ solution and left for 2 minutes or more.
CDP-Star (Boehringer Mannheim) with the above solution
/ 100, the filter was immersed and left for 5 minutes.
Excess liquid from the filter was drained, put in a plastic bag, and reacted at 37 ° C. for 10 minutes. Then X-ray film (ECL
(Hyperfilm, Amersham) for 30 minutes,
Developed. Since the portion where the probe reacted appeared as a black signal, the generated signal was visually observed to determine the reactivity in four stages. Table 3 shows the results.

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

[Table 5]

Note 3 in Table 3 Nos. 1 to 36 were used in Examples, Comparative examples of 101 to 108 bacteria Evaluation criteria ++: strong signal +: signal ±: signal is slightly confirmed-: reaction not confirmed * 1 DNA could not be extracted and hybridization was not performed

From the results shown in Table 3, it can be seen that using the probe of the present invention, strong signals were obtained from all Legionella except L. islaelensis among the Legionella subjected to the test. Almost no signal was obtained from bacteria other than Legionella. From this, the probe of the present invention can detect most Legionella species,
It was confirmed that there is Legionella specificity.

[0038]

Industrial Applicability The oligonucleotide probe of the present invention has a specific nucleotide sequence, a sequence at a specific position of 16S rRNA of many species of Legionella bacteria and a complementary sequence of a DNA encoding 16S rRNA, and a specific sequence. To form a base pair complementary to Therefore, it can be suitably used as a probe for specifically detecting a large number of species of Legionella bacteria with one type of probe in a short time and with high accuracy. In addition, since the base is short, it can be easily prepared.

In the labeled oligonucleotide probe of the present invention, since the above oligonucleotide probe is labeled with a labeling substance, the Legionella bacterium can be hybridized in a short time so that many species of Legionella bacterium can be specifically treated with one kind of probe. Can be detected with high accuracy.

Since the reagent of the present invention contains the above-mentioned labeled oligonucleotide probe, Legionella bacteria can be detected by hybridization in a short period of time with high accuracy and with high accuracy by using one kind of probe to detect many species of Legionella bacteria. be able to.

In the detection method of the present invention, the labeled oligonucleotide probe or reagent is brought into contact with a Legionella bacterium or its DNA to perform hybridization, and then the detection is performed using the label as an index. Can detect a large number of species of Legionella bacteria in a short time with a single type of probe and with high precision.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Legionella pneumophila sequence AGCAAACGCG ATAAGTTGAC 20

SEQ ID NO: 2 Sequence length: 48 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli sequence TTGAGGCGTG GCTTCCGGAG CTAACGCGTT AAGTCGACCG CCTGGGGA 48

[0044] SEQ ID NO: 3 Length of sequence: 50 SEQ types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism:. Ps aeruginosa sequence CCTTGAGATC TTAGTGGCGC ACGTAACGCG ATAAGTCGAC CGCCTGGGGA 50

SEQ ID NO: 4 Sequence length: 50 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella pneumophila sequence ATGAAAATAA TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTGGGGA 50

SEQ ID NO: 5 Sequence length: 50 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella pneumophila sequence ATGAAAATAA TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTRGGGA 50

SEQ ID NO: 6 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella pneumophila sequence ATGAAAATAA TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTGGGGA 50

SEQ ID NO: 7 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella sp. Sequence ATGAATATAA TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTGGGGA 50

SEQ ID NO: 8 Sequence length: 50 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella erythra sequence ATGAATGAGA TTAGTGGCGC AGCAAACGCG ATWAGTTGAC CGCCTNGGGA 50

SEQ ID NO: 9 Sequence length: 50 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella micdadei sequence ATGAATGAGG TTAGTGACGA AGCAAACGCG ATAAGTTGAC CGCCTNGGGA 50

SEQ ID NO: 10 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella spiritensis sequence ATAAATNAGG TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTNGGGA 50

SEQ ID NO: 11 Sequence length: 50 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella hackeliae sequence ATAAATGAGA TTAGTGGCGC AGCAAACGCG ATAAGTTGAC CGCCTNGGGA 50

SEQ ID NO: 12 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella bozemanii sequence GTGAAAATCA TTAGTGGCGC AGCAAACGCG ATWAGTTGAC CGCCTNGGGA 50

SEQ ID NO: 13 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella longbeachae sequence ATGAATNTRT TTAGTGGCGC AGCAAACGCG ATAAGTWGAC CGCCTNGGGA 50

Claims (4)

[Claims] 1. A probe consisting of deoxyribonucleotides or ribonucleotides and having a sequence complementary to the variable region of 16S rRNA of a bacterium belonging to the genus Legionella , wherein the nucleotide sequence is 5 'AGC AAA CGC GAT AAG TTG
An oligonucleotide probe for detecting a bacterium belonging to the genus Legionella, comprising AC3 '. 2. A labeled oligonucleotide probe for detecting bacteria belonging to the genus Legionella, wherein the oligonucleotide probe according to claim 1 is labeled with a radioactive element, an enzyme, a fluorescent substance or a chemical substance. 3. A reagent for detecting bacteria belonging to the genus Legionella, comprising the labeled oligonucleotide probe according to claim 2. 4. A hybridization method comprising contacting a sample containing a bacterium belonging to the genus Legionella, its DNA or rRNA with the labeled oligonucleotide probe according to claim 2 or the reagent according to claim 3, and performing hybridization. A method for detecting a bacterium belonging to the genus Legionella, wherein the bacterium belongs to the genus Legionella.