JPH10179196A - Oligonulceotide probe for detecting bacterium belonging to legionella and detection of bacterium belonging to degionella - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligouncleotide probe for detecting bacteria belonging to Legionella capable of specifically detecting many kinds of bacterial belonging to Legionella in a high accuracy in a short time and readily being prepared, and to provide a method for detecting the bacterial belonging to Legionella by which many kinds of the bacterial belonging to Legionella is specifically detected in a high accuracy at a short time by using the oligouncleotide probe. SOLUTION: Bacteria belonging to Legionella is determined by bringing a labeled oligonucleotide probe obtained by labeling an oligonucleotide probe having the base sequence of 5'GGT GAT GGT ACT ACT ACT GC3' or 5'GAA CAC AAA GCA GTT GC3' by a radioisotope, an enzyme, a fluorescent material or a chemical material, into contact with the bacteria belonging to Legionella or the DNA thereof to perform hybridization thereof and thereafter detecting the bacteria belonging to the Legionella by 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(Akuri ク リ 10),L. lansing2,L. SC-
32C-C8,L. WA-316,L.WO-44-3C (L. feeleii),L. phoe
nix 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)
Illustrative probes that can be detected differentially are described
ing. However, this nucleic acid probe is also a mixture,
There are similar problems as described above. In addition, the base length of the probe
The sequence is not clear, which part of the rRNA
Is unclear, and there is a problem in 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, the known 3
The problem is that only about 55% of the eight 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]

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 and with high precision, and to easily prepare Legionella spp. An object of the present invention is to provide an oligonucleotide probe for use in detecting Legionella bacteria in a short period of time and to specifically detect a large number of species of Legionella bacteria with high accuracy.

[0012]

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 a deoxyribonucleotide or a ribonucleotide having a partial sequence of a gene encoding a common antigen of bacteria belonging to the genus Legionella , and having a base sequence of 5'GGT GAT GGT ACT ACT ACT GC3 ' Or an oligonucleotide probe for detecting a bacterium belonging to the genus Legionella, comprising 5′GAA GGT CAC AAA GCA GTT GC3 ′. (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) Hybridization is performed by contacting the labeled oligonucleotide probe described in (2) or the reagent described in (3) with a sample containing a bacterium belonging to the genus Legionella or its DNA, and performing detection using the label as an index. A method for detecting bacteria belonging to the genus Legionella.

In the present invention, 5'GGT GAT GGT ACT ACT AC
Oligonucleotide probe having a base sequence of TGC3 '(hereinafter referred to as first probe) and 5'GAA GGT CAC
AAAGCA GTT GC3 oligonucleotide probes having the nucleotide sequence of '(hereinafter, referred to as a second probe) is, Legion
The nucleotide sequence of a gene encoding a known common antigen of ella pneumophila (58-kDa common antigen (htpB) gene),
Comparing the nucleotide sequence of the gene encoding a known common antigen Legionella micdadei, the nucleotide sequence of the heat shock genes E. coli and P. aeruginosa (Hsp60), Leg
It is obtained by selecting a nucleotide sequence that is common to the nucleotide sequence of the gene encoding the common antigen of ionella pneumophila and Legionella micdadei and that differs from the nucleotide sequence of Hsp60 of E. coli and P. aeruginosa. It is.

[0014] PAUL S. HOFFMAN et al.
Temperature-Dependent Expression in Escherichia c
oli of a Legionella pneumophila Gene Coding for a
geunus-Common 60-Kilodalton Antigen, PAUL S. HOFFM
AN, CHARLES A, BUTLER, ANDFREDERCK D. QUINN, INFEC
TION AND IMMUNITY, June 1989, p. 1731-1739), a Legionella pneumophila 60 kDa Legionella common antigen protein (later reported by JACQUELYN S. SAMPSON et al. As 58 kDa (Nucleotide Sequence of htpB, the Legionella)
pneumophila Gene Encoding the 58-Kilodalton (kda) C
ommon Antigen, Formarly Designated the 60kDa Commo
n Antigen, JACQUELYN S. SAMPSON, STEVEN P. OCOMMO
R, BRIAN P. HOLLOWAY, BONNIE B. PLIKAYTIS, GEORGE
M. CARLONE, AND LEONARD W. MAYER, INFECTION AND IMM
UNITY, Sept, 1990, p. 3154-3157] proved to be a heat shock protein, and reported that this protein showed homology to the E. coli heat shock protein GroEL. In addition, an antigen protein of similar molecular weight is Legionel
Also reported in la micdadei (Cloning and express
ion of the Legionella micdadei common antigen in E
scherichia coli , JM BANGSBORG, MT COLLINS, N.
HФIBY AND P. HINDERSSON, APMIS 97: 14-22, 198
9). From these facts, the protein of about 60 kDa and the heat shock protein (Hsp60) in the protein called the common antigen
Is a functional protein of the same type, and it is presumed that the gene is conserved across genera, and the genes were compared as described above.

[0015] Legionella pneumophila and Legionella
Table 2 shows a partial nucleotide sequence of the gene encoding the common antigen of micdadei and a partial nucleotide sequence of the heat shock gene (Hsp60) of E. coli and P. aeruginosa .

[0016]

[Table 2]

The nucleotide sequence of the first probe of the present invention (the nucleotide sequence of SEQ ID NO: 1) is a gene encoding a common antigen of L. pneumophila (58-kDa common antigen (htpB) gene).
Alternatively , it corresponds to the nucleotide sequence at positions 253 to 272 of the gene encoding the common antigen of Legionella micdadei . The nucleotide sequence of the second probe of the present invention (the nucleotide sequence of SEQ ID NO: 2) is a gene encoding a common antigen of L. pneumophila (58-kDa common antigen (htpB) gene) or Legion.
301 of the gene encoding the common antigen of ella micdadei
This corresponds to the base sequence at positions 320 to 320. Therefore, the sequence of the first probe of the present invention is a gene encoding a common antigen of L. pneumophila (58-kDa common antigen (htpB) gen
e) Alternatively , targeting the complementary sequence at positions 253 to 272 of the gene encoding the common antigen of Legionella micdadei and a nucleotide sequence similar thereto, the sequence of the second probe of the present invention is as follows: Targets complementary sequences and similar nucleotide sequences.

As described above, the first and second probes of the present invention have a clear nucleotide sequence and a length of 20 nucleotides.
Since it is about a base, it can be easily synthesized by chemical synthesis from nucleotides or deoxynucleotides. Therefore, the same probe can be easily obtained any number of times, and the specificity does not differ depending on the lot. Further, since the first and second probes of the present invention have a relatively short number of bases, they can be used for in situ hybridization. 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 first probe or the second probe with a labeling substance such as a radioactive element, an enzyme, a fluorescent substance or a chemical substance. . As such a labeling substance, conventionally used labeling substances can be used, and specific examples thereof include radioactive elements such as ³² P; fluorescent substances such as FITC and rhodamine; haptens such as dicoxigenin; alkaline phosphatase;
Enzymes such as peroxidase; biochemical substances 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-described labeled oligonucleotide probe, and may be composed of only the labeled oligonucleotide probe, or may be one 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 the reagent containing the same is hybridized with a Legionella bacterium or a specimen containing the DNA,
Thereafter, detection is performed using the labeling substance as an index, for example, by detecting using radioactivity, color development, fluorescence, luminescence, etc. as an index, the Legionella bacterium hybridized with the probe can be visually or microscopically specifically and precisely. Can easily detect, identify or quantify.

Table 3 shows some of the species of Legionella species that can be detected, identified or quantified with the probes of the present invention.

[0023]

[Table 3]

Notes to Table 3 * 1 First probe of the present invention * 2 Second probe of the present invention ○: detectable ×: not detectable

As shown in Table 3, when the probe of the present invention in combination with the first and second two probes, L. e
All Legionella bacterial species except rythra can be detected. In the present invention, when testing the reactivity between Legionella and the probe, 35 species of Legionella (including L. pneumo
phila subspecies). As a result, Table 3
It was possible to detect 11 more Legionella species in addition to the Legionella species shown in (1) (see Table 4 below). These results indicated that more species and types of Legionella can be detected by the probe of the present invention.

The first probe of the present invention, CA / GL253
Can detect 15 Legionella species in Table 3, and there are 6 Legionella species that cannot be detected. But Table 3
In addition to the above species, at least 9 Legionella species can be detected (see Examples and Table 4), and a total of 24 or more Legionella species (excluding subspecies) can be detected. Thus, the first probe of the present invention can detect more species of Legionella than the conventional probe.

The second probe of the present invention, CA / GL301, can detect 10 Legionellas in Table 3.
Furthermore, in addition to the Legionella species of Table 3, at least seven Legionella species (excluding subspecies) can be detected (see Examples and Table 4). Thus, the second probe of the present invention can detect more species of Legionella than the conventional probe.

In the detection method of the present invention, the labeled oligonucleotide probe or the 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, when a bacterium is targeted, the cells are impregnated into a filter such as a nylon filter, lysed on the filter, and then irradiated with ultraviolet light or a high temperature of 80 ° C. or higher to obtain DNA of the genus Legionella. Is fixed on the filter. 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. The filter which has been reacted with the probe is washed with a buffer to remove the non-specifically bound probe.
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 according to 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.

Thus, 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.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION

Examples and Comparative Examples 1) Preparation of Oligonucleotide Probe The first probe and the second probe of the present invention were 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.

Each of the above probes was used for DIG oligoprobe tailed
Labeling was performed with dicoxygenin using a label kit (Boehringer Mannheim). The hybridization solution (0.75M) was added to the oligonucleotide probe labeled with dicoxigenin so that the concentration of the oligonucleotide probe became 0.3 pmol / ml.
NaCl, 75mM sodium citrate, 1% (w / v) Blockingreagen
t, 0.1% (w / v) N-lauroylsarkosine, 0.02% (w / v) Sodium
dodesyl sulfate (SDS), 0.1 mg / l Poly (a)] and used for 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 spp., 35 species shown in Tables 3 and 4 (of which two were L. pneumophila subspecies) were obtained, and after DNA extraction, dot blot was performed. . In addition, as a comparative example, E. colacal (ATCC 2335), E.
coli (ATCC 25922), K. preunhial (ATCC 13883), P. vul
garis (ATCC 13315), P. aeruginosa (ATCC 27853), S. t
yphimurium (ATCC 14028), S. marcesseus (ATCC 8100),
Using S. aureus (ATCC 12600), use D
After extracting NA, it was 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. DNA
Extraction of Current Protocols in Molecular Biology. 2.
4.1. Preparation was performed according to Genomic DNA form Bacteria.

The absorbance of the extracted DNA at A260 nm was measured, and each DNA was adjusted to have the same concentration. Then, 100 ng of the DNA was dot-blotted on a nylon filter. After air drying, the filters were sequentially placed on filter paper moistened with each of the following solutions for 5 minutes to denature the DNA.
(1) 0.5N NaOH, 0.5M NaCl, (2) 1.5M NaCl, 0.5MT
ris-HCl pH7.4, (3) 0.3M NaCl, 30mM sodium citora
te pH 7.0. Thereafter, the mixture was heated at 120 ° C. for 30 minutes to fix the DNA on 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
citrate, 1% (w / v) Blockingreagent, 0.1% (w / v) N-lauro
ylsarkosine, 0.02% (w / v) sodium dodesyl sulfate (SDS), 0.1 mg / l Poly (a)] was added thereto, and air bubbles were removed to seal. The reaction was carried out at 68 ° C. for 1 hour or more with gentle shaking in a hybridization oven. The prehybridization solution was discarded, and the 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 removing bubbles and sealing, gently shake in a hybridization oven
The reaction was performed at 50 ° C for 2 to 3 hours.

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 4 shows the results.

[0040]

[Table 4]

[0041]

[Table 5]

[0042]

[Table 6]

Note 4 in Table 4 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 in Table 4, it can be seen that when two types of mixed probes were used, strong signals were obtained from all Legionella except L. erythra among the Legionella subjected to the test. Among the bacteria other than the provided Legionella, K. preun
A strong signal was generated for ohial , but little signal was obtained from others. From this, it was confirmed that the probe of the present invention was able to detect most Legionella species, and had almost Legionella specificity.

[0045]

The oligonucleotide probe of the present invention forms a base pair specifically complementary to a gene encoding a common antigen of Legionella bacteria of many species. Therefore, it can be suitably used as a probe for specifically detecting a large number of species of Legionella bacteria with high accuracy in a short time. In addition, since the base is short, it can be easily prepared.

In the labeled oligonucleotide probe of the present invention, since the above-mentioned oligonucleotide probe is labeled with a labeling substance, the Legionella bacterium can be detected in a short period of time by hybridization to specifically and highly accurately detect many species of Legionella bacterium. can do.

Since the reagent of the present invention contains the above-described labeled oligonucleotide probe, it is possible to specifically detect a large number of species of Legionella bacteria with high accuracy by hybridization in a short time.

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

[0049]

[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 name: Legionella pneumophila sequence GGTGATGGTA CTACTACTGC 20

SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism: Legionella pneumophila sequence GAAGGTCACA AAGCAGTTGC 20

SEQ ID NO: 3 Sequence length: 50 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Legionella pneumophila sequence CTGGTGATGG TACTACTACT GCAACAGTAT TGGCTCGTTC TATTCTTGTT 50

SEQ ID NO: 4 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Legionella micdadei sequence CAGGTGATGG TACTACTACT GCTACGGTAT TGGCACGTGC CATTGTCGTT 50

SEQ ID NO: 5 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli sequence CTGCAGGCGA CGGTACCACC ACTGCAACCG TACTGGCTCA GGCTATCATC 50

[0054] SEQ ID NO: 6 SEQ Length: 50 sequence types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism:. Ps aeruginosa sequence CTGCCGGTGA CGGCACCACC ACCGCGACCG TCCTGGCCCA GGCCATCGTC 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 pneumophila sequence GAAGGTCACA AAGCAGTTGC TGCTGGTATG AATCCAATGG ATCTCAAACG 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 micdadei sequence GAAGGCCACA AAGCAGTTGC AGCAGGCATG AACCCAATGG ATTTGAAACG 50

SEQ ID NO: 9 Sequence length: 50 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli sequence ACTGAAGGTC TGAAAGCTGT TGCTGCGGGC ATGAACCCGA TGGACCTGAA 50

[0058] SEQ ID NO: 10 SEQ Length: 50 sequence types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism:. Ps aeruginosa sequence AACGAAGGCC TGAAGGCCGT TGCCGCCGGC ATGAACCCGA TGGACCTGAA 50

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] February 4, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction contents]

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(Akuri ク リ 10),L. lansing2,L. SC-
32C-C8,L. WA-316,L.WO-44-3C (L. feeleii),L. phoe
nix 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)
Illustrative probes that can be detected differentially are described
ing. However, this nucleic acid probe is also a mixture,
There are similar problems as described above. In addition, the base length of the probe
The sequence is not clear, which part of the rRNA
Is unclear, and there is a problem in detection accuracy.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction contents]

[0008]

[Table 1]

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction contents]

As described above, the first and second probes of the present invention have a clear nucleotide sequence and a length of 20 nucleotides.
Since it is about a base, it can be easily synthesized by chemical synthesis from nucleotides or deoxynucleotides. Therefore, it is possible to obtain the same probe easily any number of times, is not that is different specificity by lot. Further, since the first and second probes of the present invention have a relatively short number of bases, they can be used for in situ hybridization. From these points, it is excellent as a detection reagent.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

[0023]

[Table 3]

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction contents]

2) Preparation of specimens Among the reported Legionella spp., 35 species shown in Tables 3 and 4 (of which two were L. pneumophila subspecies) were obtained, and after DNA extraction, dot blot was performed. . As a comparative example, Enterobacter cloacae (ATCC
2335 5 ), Escherichia coli (ATCC 25922), Klebsiella p
neumoniae (ATCC 13883), Proteus vulgaris (ATCC 1331
5), Pseudomonas aeruginosa (ATCC 27853), Salmonella
typhimurium (ATCC 14028), Serratia marcescens (ATCC
8100), Staphylococcus aureus (ATCC 12600),
DNA was extracted in the same manner as in Legionella, and then subjected to dot blotting.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction contents]

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. The anti-self digoxigenin alkaline phosphatase-conjugated antibody was added 1/10000 amount of the liquid volume, was reacted with 20 minutes gentle shaking. Take out the filter and put 100m in a new container
M 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.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction contents]

[0042]

[Table 6]

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0044

[Correction method] Change

[Correction contents]

From the results in Table 4, it can be seen that when two types of mixed probes were used, strong signals were obtained from all Legionella except L. erythra among the Legionella subjected to the test. Among the bacteria other than the provided Legionella, K. pneum
A strong signal was produced for oniae , but little signal was obtained from others. From this, it was confirmed that the probe of the present invention was able to detect most Legionella species, and had almost Legionella specificity.

Claims (4)

[Claims] Claims 1. A sequence having a partial sequence of a gene encoding a common antigen of a bacterium belonging to the genus Legionella ,
An oligo for detecting a bacterium belonging to the genus Legionella, comprising a probe consisting of deoxyribonucleotide or ribonucleotide, having a base sequence of 5′GGT GAT GGT ACT ACT ACT GC3 ′ or 5′GAA GGT CAC AAA GCA GTT GC3 ′. Nucleotide probe. 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 bacterium belonging to the genus Legionella or its D
Claims: 1. A bacterial strain belonging to the genus Legionella, which is obtained by contacting a labeled oligonucleotide probe according to claim 2 or a reagent according to claim 3 with a sample containing NA and performing hybridization using the label as an index. Detection method.