JPH11332596A - Detection of pseudomonas aeruginosa and kit for detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligonucleotide having a specific base sequence and useful for detecting Pseudomonas aeruginosa which is one of causative bacteria of opportunistic infection derived from a 16SrRNA gene of the Pseudomonas aeruginosa. SOLUTION: This oligonucleotide is a new one having a base sequence represented by the formula and corresponding to a sense strand of the base Nos. 138-156 of a 16SrRNA gene of Pseudomonas aeruginosa and is called Bacillus pyocyaneus and a Gram-negative bacillus widely present in environment such as mainly water or soil and is used for a detection method, etc., with a high specificity for the Pseudomonas aeruginosa which is one of causative bacteria of opportunistic infection. The oligonucleotide is obtained by procuring the 16SrRNA sequences of bacteria of the genus Pseudomonas from gene databases, carrying out detailed comparison, finding out a sequence specific for the Pseudomonas aeruginosa and chemically synthesizing the oligonucleotide according to a phoshoramidite method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Pseudomonas
The present invention relates to a method for detecting Aeruginosa (hereinafter referred to as Pseudomonas aeruginosa), a kit for detection, and an oligonucleotide used for the kit.

[0002]

2. Description of the Related Art Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a gram-negative bacillus widely present in the environment such as water and soil, and is known as one of the causative bacteria of opportunistic infections. It is also an important fungus in quality control during the production of foods, medicines, cosmetics, and the like. Usually Pseudomonas
The detection and identification of aeruginosa are performed by a combination of selective medium and examination of physiological and biochemical properties by a culture method. However, in principle, the culture method requires at least one day or more for bacteria to grow to a detectable level, and lacks rapidity. The physiological and biochemical properties of Pseudomonas aeruginosa are similar to other Pseudomonas bacteria except for pyocyanin productivity, and it is difficult to make accurate determinations especially for mutants that have lost pyocyanin productivity. The PCR method is
A gene sequence sandwiched between oligonucleotides of about 20 to 30 bp having a sequence complementary to a specific target gene sequence (hereinafter referred to as primers)
A method of amplifying and synthesizing using A synthase, a method that is routinely used in the field of gene analysis,
It is useful as a means for rapidly and highly sensitively detecting and identifying genes. Further, if a gene specific to a certain microorganism is targeted, it can be used for highly sensitive detection and simple identification of the microorganism, and a detection kit using this principle is also commercially available.

[0003] Pseudomonas ae reported so far
Ruginosa detection primers are roughly classified into the following two types. Targeting a gene that is specifically present in Pseudomonas aeruginosa Targeting a gene that exists in all bacteria but has a slightly different sequence is an example of a Macin targeting the aldD gene.
Tosh, K targeting ExotoxinA gene
De V targeting oprL gene by Han et al.
os and others. These are problems in that it cannot be verified that the target gene specifically exists in Pseudomonas aeruginosa. An example of Kur
And Tyler et al. Have reported the gene sequence of the intermediate region connecting 16S rRNA and 23S rRNA and the gene of 16S rRNA. Regarding the former, the target gene is Pseudomona
The problem is that it is not possible to verify the specific existence of s aeruginosa.

[0004] Regarding the latter, 16S rRNA sequences of various fungi have been analyzed by many researchers around the world, and the results are readily available via the Internet, and it seems that there is no problem in verification. Until now, O '
Callaghan et al. (J. Clin. Pathol. 47, 222-226, 1994),
Kingsford et al. (Veterinary Microbiology 47 61-70, 199
5), Karpati et al. (Molecular and Cellular Probes 10,
397-408, 1996] reported a primer for detecting Pseudomonas aeruginosa. However, as a result of a homology search by BLAST search of NCBI of the present inventors, it was found that these primers had a problem in specificity.

[0005] First, the primer of O'Callaghan et al. Is forward primer: 5'-GTTACCAACAGAATAAGC-3 'reverse primer: 5'-AGCTCAGTAGCTTTTGGA-3', but P.aeruginosa
X06684, P. aeruginosa M34133, P. aeruginosa Z76651, as well as P. stutzeri, P. citronellolis, P. oleovorans, P.
alcaligenes, Flavobacterium lutescens, P. nitroredu
Cens, P.resinovorans, P.fragi, and P.stutzeri also showed homology. On the other hand, the only one having homology to the reverse primer is P. aeruginosa X06684 (see FIG. 3).
Bacteria other than ruginosa X06684 may not be detected (see Table 1).

The primer of Kingsford et al. Is forward primer: 5'-GCGCGCGTAAGTGGTTCAGC-3 'reverse primer: 5'-CCTTAGAGTGCCCACCCGAG-3'. Only (see FIG. 4),
On the other hand, for the reverse primer, P. aeruginosa X06
684, P. aeruginosa M34133, P. aeruginosa Z76651, P. mendocina, Azomonas macrocytogenes, Azobacte
r paspali and Azobater chroococcum show homology, and in the case of a combination of these primers, P. aeruginosa
Bacteria other than X06684 may not be detected (see Table 2).

The primer of Karpati et al. Is forward primer: 5'-GTGCCTGCAGCCGCGGTAAT-3 'reverse primer: 5'-TGCGCCACTAAGATCTCAAG-3', but the forward primer is universal, and P. aeruginosa X0668
4, P. aeruginosa M34133, P. aeruginosa Z76651,
P.stutzeri, P.putida, P.oleovorans, P.mendocina,
Since P. alcaligenes also shows homology, in this combination, Pseudomonas aerug
inosa cannot be distinguished (see Table 2).

[0008] As described above, the primers of O'Callaghan et al. And Kingsford et al. Correspond to only one of the 16S rRNA gene sequences of Pseudomonas aeruginosa whose three primers are presently known, and Pseudomonas aerugi
You may not be able to detect the entire nosa. The primers of Karpati et al. Are pseudomonas aeruginosa
monas stutzeri, Pseudomonas putida, Psudomonas alc
aligenes etc. are also detected, and the specificity is low, which is a problem.

[0009]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to enable the detection of highly specific Pseudomonas aeruginosa.

[0010]

Means for Solving the Problems The present inventors have proposed Pseudomo
By comparing the gene sequences of closely related species of Pseudomonas aeuginosa and Pseudomonas aeruginosa,
A combination of primer sequences that can detect only ruginosa was found. That is, the present invention provides an oligonucleotide having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The present invention also provides a method for detecting Pseudomonas aeruginosa, wherein at least one of the oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 is used as a probe. Pseudomonas aeru characterized in that at least one, and preferably both, of the oligonucleotides having the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are used as gene amplification reaction primers.
Provides a method for detecting ginosa. Pseudomonas further comprising at least one of the oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
Provide a kit for detection of aeruginosa.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Almost complete 16S rRNA of Pseudomonas aeruginosa currently registered in a database
The sequence is Pseudomonas ae registered by Moore et al.
ruginosa LMG1242T strain (ACCESSION No. Z76651), Tosch
Pseudomonas aeruginosa DSM500 registered by ka et al.
71 strains (ACCESSION No. X06684), Pseudomonas aeruginosa ATCC 25330 strains registered by Woese et al. (ACCESSION
No. M34133). We have found that Pseudom
16S rRNA sequences of other bacteria belonging to the genus Pseudomonas, which are related to onas aeruginosa, were obtained from various gene databases and compared in detail. As a result, Pseudomonas ae
It was found that the sequences described in SEQ ID NOS: 1 and 2 were conserved in only a small number of other species of bacteria other than ruginosa.

That is, as shown in FIG.
aeruginosa X06684, Pseudomonas aeruginosa M34133,
16SrRN of three strains of Pseudomonas aeruginosa Z76651
In the A sequence, base numbers 138 to 156 (SEQ ID NO: 1)
However, SEQ ID NO: 1 is conserved among other Pseudomonas bacteria, because Pseudomonas fluorescen
s and Pseudomonas tolaasii only (see Table 1). On the other hand, as shown in FIG. 2, Pseudomonas aeruginosa X0668
4, Pseudomonas aeruginosa M34133, Pseudomonas aeru
A sequence complementary to SEQ ID NO: 2 is found at base positions 1152-1133 of the 16S rRNA sequences of the three strains of ginosa Z76651. Other strains in which this sequence is conserved include Pseudomonas mendocina and Azomonas macrocytogene.
s, Azobacter paspali, and Azobater chroococcum (see Table 1).

The oligonucleotide of the present invention can be prepared by any suitable method. For example, the whole or a part of the sequence may be chemically synthesized according to the method described in Examples below. For example, it can be chemically synthesized using an automatic DNA synthesizer. Or, Pseudomonas
It is also possible to cut out directly from the aeruginosa gene using a restriction enzyme or the like.
It is also possible to clone the plasmid into a plasmid such as li, collect the plasmid after propagation of the bacteria, cut out the plasmid, and use it.

The oligonucleotide thus prepared can be used as a primer and
It can also be used as a probe for detecting domonas aeruginosa. However, it is preferable to use it as a primer because the specificity of detection of Pseudomonas aeruginosa increases. Such an oligonucleotide or a partial base sequence that can be used as a primer or a probe may have a site capable of binding to a label or a solid phase carrier, if necessary. Here, in the case of performing detection using a primer, a site where a site capable of binding to a label or a solid phase carrier may be introduced may be any site which does not hinder the extension reaction of the primer. Terminals are most preferred. In the case of performing detection using a probe, the position at which a site capable of binding to a label or a solid phase carrier may be introduced may be a 3′-terminal or 5′-terminal hydroxyl group portion, or a base portion or a phosphate diester portion. Although it is conceivable, it is desirable to consider the base sequence and length of the probe so as not to hinder hybridization.

As the label, either a radioactive substance or a non-radioactive substance may be used. Examples of the non-radioactive label include those that can be directly labeled, such as fluorescent substances such as fluorescein derivatives, rhodamine and its derivatives, chemiluminescent substances, and delayed fluorescent substances. It is also possible to indirectly detect a label using a substance that specifically binds to the label. Examples of such a label include biotin and hapten. For biotin, avidin or streptavidin is used, and for hapten, an antibody that specifically binds to avidin or streptavidin is used. As the hapten, a compound having a 2,4-dinitrophenyl group, digoxigenin, or the like can be used. All of these labels may be used alone or in combination of two or more as necessary.
It can be introduced into a probe or primer.

When it is necessary to specifically bind a specific fragment of a nucleic acid to a solid phase carrier such as sandwich hybridization, the site capable of binding to the solid phase carrier may be any site that can selectively bind to the carrier. Anything is fine. For example, biotin, fluorescein, compounds having a 2,4-dinitrophenyl group, and haptens such as digoxigenin can be mentioned, and any of these can be introduced into a probe or a primer alone or in combination of two or more as needed.

As the solid phase carrier, a solid phase material such as polystyrene balls, agarose beads, polyacryl beads, latex, microtiter wells, etc. capable of capturing the binding site introduced into the primer, for example, streptoa Pididine, antibodies and the like are introduced. For example, to capture a PCR product from a biotin-introduced primer, a carrier having streptavidin bound to a solid phase, and to capture an elongation reaction product from a primer into which fluorescein or the like has been introduced, use an antibody against each. Is used.

Furthermore, by making the solid phase carrier into fine particles, the target nucleic acid can be easily determined by the presence or absence of aggregation or precipitation. In addition, one of the primers has a site capable of binding to the solid phase carrier, and the other primer has a label introduced therein. There is also a method of washing away with a suitable solvent, and the target nucleic acid is immobilized on the solid support in a form having a label, and is specifically detected.

Next, a method for detecting Pseudomonas aeruginosa using the oligonucleotide of the present invention as a primer will be described. According to the present invention, the oligonucleotide represented by SEQ ID NO: 1 is used as an upstream forward primer, and the oligonucleotide represented by SEQ ID NO: 2 is used as a downstream reverse primer, preferably by PCR.
(Polymerase Chain Reaction)
onas aeruginosa can be detected.

Tables 1 and 2 show the bacteria that can be detected using the primer of the present invention and the conventional primer. In Tables 1 and 2, Pseudomonas is abbreviated as P. It can be seen that the primer of SEQ ID NO: 1 has higher specificity for the three strains of Pseudomonas aeruginosa than the conventional forward primer.

[0021]

[Table 1]

[0022]

[Table 2]

Accordingly, the oligonucleotide represented by SEQ ID NO: 1 was used as a forward primer, and this was added to a downstream known primer such as Kingsford et al.
Even if the reverse primer of rpati et al. is combined, Pseud
omonas aeruginosa can be specifically detected.
Alternatively, the oligonucleotide represented by SEQ ID NO: 2 may be used as a downstream reverse primer, which may be combined with a known upstream primer such as a forward primer of O'Callaghan et al.

Here, the basic form of the two primers may be one in which the two primers are not modified at all, or as described above, a label detectable on at least one of the two primers. Or, one in which a part capable of binding to a solid phase carrier is introduced, one in which a label is introduced in one of two kinds of primers, and one in which a site capable of binding to a solid phase carrier is introduced in the other, and both kinds of primers And those into which a moiety capable of binding to a solid phase carrier has been introduced.

Various methods have been devised for the enzymes and reaction conditions used for the gene amplification reaction. The oligonucleotides of the present invention have a sufficient length and base sequence to be used in various PCR methods. It can be used to reliably and easily detect Pseudomonas aeruginosa.

Hereinafter, the detection method of the present invention will be described according to a general PCR method. That is, the method is carried out by adding the primers and the DNA polymerase to the sample, repeating denaturation treatment to single-stranded DNA, annealing and extension reaction several tens of times, and detecting the amplified gene.

The sample used in the detection method of the present invention is not particularly limited, and various samples in which it is desired to determine the presence or absence of Pseudomonas aeruginosa in fields such as research, medical care, environment, and quality control are used. Inspection of PCR from these materials
It is necessary to extract the nucleic acid component of Pseudomonas aeruginosa, which is a sample of Pseudomonas aeruginosa, but since Pseudomonas aeruginosa is a gram-negative bacterium with low cell wall strength, the number of bacteria contained in the sample can be determined without special extraction operation. If it is large, it can be detected by the present detection method. In addition, more efficient DNA extraction can be achieved by a method using physical operations such as heat treatment, pressure treatment, and grinding, a method using an alkali or a surfactant, and a method using a protease such as proteinase K. And high sensitivity inspection can be expected. In addition, there are many copies of 16S
By transcribing rRNA to DNA using reverse transcriptase, the sensitivity can be further improved.

A part of these nucleic acid extraction solutions can be directly subjected to PCR, but the sensitivity of PCR can be increased by adding nucleic acid purification and concentration operations. As a method for purification or concentration, a method using an organic solvent such as phenol as a protein denaturant, or a method using other specific protein denaturants [Beutler et al., Bi
oTechniques 9, P. 166 (1990)], a method using a carrier such as latex particles having a capture and concentration probe immobilized thereon (JP-A-63-117262), and the like.
Other commercially available DNA extraction kits and the like may be used.

As the polymerase used for PCR, it is preferable to use a heat-resistant DNA polymerase, for example, Taq DNA polymerase. More preferably 3 '→
By using a thermostable DNA polymerase having 5 'exonuclease activity, an improvement in detection sensitivity can be expected. As preferable PCR conditions, for example, when a heat-resistant DNA polymerase is used and the temperature cycle is performed in three steps of denaturation, annealing, and extension, the temperature is 90 ° C. to 96 ° C. for 1 second to 60 seconds, and 50 ° C. to 70 ° C., respectively.
For 1 second to 120 seconds, and 65 to 75 ° C. for 1 second to 120 seconds. When the reaction is carried out on a scale using a 0.2 mL tube which is usually used, the reaction is performed at 94 ° C. for 30 seconds
The conditions of 55 ° C. for 60 seconds and 72 ° C. for 60 seconds are preferred. When annealing and elongation are performed in two steps in one step, the steps are performed at 90 to 96 ° C. for 1 to 60 seconds and 65 to 65 ° C., respectively.
It is preferably performed at 72 ° C. for 1 to 120 seconds. When performing the reaction on a scale using a 0.2 mL tube, the conditions of 94 ° C. for 30 seconds and 68 ° C. for 60 seconds are preferred.

PCR using primers of the present invention
DNA amplification is determined by a method in which the size of the generated amplified DNA is confirmed by electrophoresis, a method in which PCR is further performed on the amplified product, or a method in which the amplified product is confirmed using an internal probe and the sensitivity is simultaneously improved. , A method of confirming PCR amplification in real time by fluorescently labeling the internal probe, and using a labeled primer for identification.
All commonly used methods such as a method of performing CR and capturing and identifying a labeled amplified DNA on a solid phase, and an in situ PCR method can be used.

As a method for capturing and identifying the amplified DNA, a method using a solid phase in which a substance specifically binding to a label is immobilized in advance (Japanese Patent Laid-Open No. 1-252300),
There is a method in which a capture probe having a sequence complementary to the sequence of the target amplified DNA is immobilized on a solid phase in advance, and specific binding is performed by hybridization. For such a solid phase, a microtiter well is used. Alternatively, beads or the like can be used, but it is advantageous to use microtiter wells for processing multiple samples at once.

When a clinical specimen is used as a direct sample for examination, even if Pseudomonas aeruginosa is present in the sample, the number of Pseudomonas aeruginosa is small, and DN
It is conceivable that a considerable number of temperature cycles for A amplification need to be repeated. In such cases, even with the use of carefully studied primers, the danger of non-specific amplified DNA cannot be completely eliminated, and the capture of amplified DNA requires a high level with the capture probe. It is desirable to use hybridization. The method of Kawai et al. For the immobilization of capture probes on microtiter wells [Analytic
al Biochemistry 209. P.63-69] can be used.

In the case where PCR is performed using a primer with an identifiable label and a labeled amplified DNA is detected, a general method may be used according to the labeling substance to be used. For example, if the labeling substance is a radioisotope, the activity may be measured as it is. For example, if biotin is used, an avidin-enzyme conjugate may be used. And detection may be performed by optical or fluorescent means.

[0034] The present invention also relates to Pseu
Provided is a detection kit for performing the domonas aeruginosa detection method. The detection kit of the present invention is a kit containing at least one of the oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and further containing reagents necessary for PCR. The reagents included in the detection kit include DNA polymerase, primers, buffer (concentration buffer if necessary), dATP, dCTP,
Each solution of dTTP and dGTP, if necessary, a control primer and the like. Preferably, these components may be dispensed into a PCR tube for each use of one sample in a PCR tube, or tableted. .

For example, the composition of the final reaction solution is 0.1 to 5
mM Tris-HCl, pH 7.5-8.5,0-0.
5 mM MgCl ₂ , 1-500 mM KCl, 1-5
00 μM dATP, 1 to 500 μM dCTP, 1
500 μM dGTP, 1-500 μM dTTP,
0.1 to 1000 μM primer, 0.1 to 2 U / 5
A kit set to be 0 μl Taq DNA polymerase can be exemplified, but the concentration of each component may be any range as long as a normal PCR reaction is possible, and is not limited thereto. Other components can be added as long as they do not interfere with the PCR reaction.

The oligonucleotide of the present invention can be used as a probe for detection of Pseudomonas aeruginosa, but the detection method used as a primer is more effective than Pseudomonas aeruginosa.
Monas aeruginosa is preferred because it increases the specificity of detection. In general, detection methods using a probe include dot hybridization, southern hybridization, and in situ hybridization, and any of these methods can use the above-described labeled oligonucleotide to perform hybridization as usual.

In recent years, in order to increase the sensitivity of hybridization, rRN has been used in many copies per cell.
Although a method using A as a detection target has been developed, the oligonucleotide in the present invention is an oligonucleotide containing a region encoding 16S rRNA, and can be used for rRNA detection by a similar method. Also, i
In the n-situ hybridization, detection with a good S / N ratio is generally possible by using rRNA as a detection target, but the oligonucleotide of the present invention can be subjected to detection in the same manner as described above. In addition, methods based on sandwich hybridization have been developed for simplification of the operation, and Pseudomonas aeruginosa can be detected using the oligonucleotide of the present invention also by these methods.

[0038]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Selection of primers) (l) Preparation of specimen DNA was extracted from cultures of the following strains by a heat treatment method.
Prepared. Pseudomonas aeruginosa IFO3899 Pseudomonas aeruginosa IFO12689 Pseudomonas aeruginosa IFO13275 Pseudomonas aeruginosa IFO13736 Pseudomonas alcaligenes IFO14 159 Pseudomonas fluorescens IFO14160 Pseudomonas putida IFO14164secosamonFOSA164FOC

(2) Synthesis and Labeling of Primers Oligonucleotides as primers were synthesized by a phosphoramidite method using a DNA synthesizer manufactured by Applied Biosystems. Amino link at the 5 'terminal base
Amino groups were introduced using II (trade name: Applied Biosystems), and then purified by gel filtration using Sephadex G-50.

(3) PCR 20 pmol / μl was added to a 0.2 ml reaction solution tube.
2.5 μl each of the above primers, 1 μl each of the above samples, 25 μl of Premix Taq (Takara), and 19 μl of sterile distilled water, were added to a PCR reaction tank TP-3200 (Ta
kara), and 25 cycles of reaction were carried out at 94 ° C., 30 seconds, 68 ° C., 60 seconds. After the reaction, 10 μl was taken from the reaction solution, and subjected to agarose gel electrophoresis and ethidium bromide staining to detect an amplified nucleic acid chain. As a result, one clear band was confirmed at a length of about 1000 base pairs, which is expected from only four strains of Pseudomonas aeruginosa.

Hereinafter, embodiments included in the present invention will be described. (1) An oligonucleotide having the nucleotide sequence of SEQ ID NO: 1. (2) an oligonucleotide having the base sequence of SEQ ID NO: 2; (3) A primer comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1. (4) A primer containing an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2. (5) A probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1. (6) A probe comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2. (7) A primer having the nucleotide sequence of SEQ ID NO: 1 or 2 into which a site capable of binding to a label and / or a solid support is introduced. (8) A probe having the nucleotide sequence of SEQ ID NO: 1 or 2 into which a site capable of binding to a label and / or a solid support is introduced. (9) A primer comprising a combination of two types of oligonucleotides, wherein at least one of the oligonucleotides is selected from oligonucleotides having the nucleotide sequence of SEQ ID NO: 1 or 2, and each oligonucleotide is labeled. And / or a primer to which a site capable of binding to a solid support may be introduced. (10) A primer having the base sequence of SEQ ID NO: 1 or 2, wherein a part of the base sequence of the oligonucleotide is deleted or substituted or added with another base or base sequence. (11) The site capable of binding to a label or a solid support is
A primer or probe having the nucleotide sequence of SEQ ID NO: 1 or 2 introduced into the 5 'end of the oligonucleotide. (12) A method for detecting Pseudomonas aeruginosa, wherein at least one kind of oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2 is used as a probe. (13) Pseudomonas aeru characterized in that at least one kind of oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 or 2 is used as a gene amplification reaction primer.
Ginosa detection method. (14) A method for detecting Pseudomonas aeruginosa, wherein both an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 are used as primers for gene amplification. (15) A sample for which the presence or absence of Pseudomonas aeruginosa is to be detected is prepared, if necessary, the cells in the sample are crushed, and a primer having the nucleotide sequence of SEQ ID NO: 1 or 2 is added to the sample to extend the primer. And performing a detection operation on the obtained extension reaction product, wherein Pseudomonas aeruginosa is detected. (16) A sample whose presence or absence of Pseudomonas aeruginosa is to be detected is prepared, if necessary, the cells in the sample are crushed, and a primer having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2 are added to the sample. A method for detecting Pseudomonas aeruginosa, comprising: performing an extension reaction of a primer by adding both of the primers having the formula (1); and performing a detection operation on the obtained extension reaction product. (17) The method for detecting Pseudomonas aeruginosa according to (15) or (16), wherein the extension reaction of the primer is PCR.

[0043]

As described above, the oligonucleotide of the present invention is a 16S rRNA of Pseudomonas aeruginosa.
These oligonucleotides have a sequence corresponding to the sense strand from base numbers 138 to 156 of the gene (SEQ ID NO: 1) and a sequence corresponding to the antisense strand from base numbers 1133 to 1152 (SEQ ID NO: 2). Pseudomonas aeruginosa can be specifically detected by using at least one of the above as a probe, or by using at least one of the above oligonucleotides, preferably a combination of both, as a primer.

[0044]

[Sequence list] SEQ ID NO: 1 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA Sequence characteristics: Other characteristics: 16S rRNA gene of Pseudomonas aeruginosa Has a sequence complementary to a part of the sequence. Sequence: TAGTGGGGGA TAACGTCCG 19

SEQ ID NO: 2 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Other characteristics: One of 16S rRNA gene of Pseudomonas aeruginosa It has a sequence complementary to the sequence of the part. Sequence: CTTAGAGTGC CCACCCGAGG 20

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an explanatory diagram showing the sequence of base numbers 131 to 160 of the 16S rRNA gene of Pseudomonas aeruginosa.

FIG. 2 is an explanatory diagram showing a sequence from base numbers 1131 to 1160 of a 16S rRNA gene of Pseudomonas aeruginosa.

FIG. 3 is an explanatory diagram showing a sequence from base numbers 631 to 660 of the 16S rRNA gene of Pseudomonas aeruginosa.

FIG. 4 is an explanatory diagram showing the sequence of base numbers 571 to 600 of the 16S rRNA gene of Pseudomonas aeruginosa.

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁶ Identification code FI (C12N 15/09 ZNA C12R 1: 385) (72) Inventor Kensuke Tanaka 1-3-7 Honjo, Sumida-ku, Tokyo Lion Inside the corporation

Claims (5)

[Claims] 1. An oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 TAGTGGGGGA TAACGTCCG. 2. An oligonucleotide having the base sequence of SEQ ID NO: 2 CTTAGAGTGC CCACCCGAGG. 3. A method for detecting Pseudomonas aeruginosa, wherein at least one of the oligonucleotides according to claim 1 or 2 is used as a probe. 4. A method for detecting Pseudomonas aeruginosa, comprising using at least one of the oligonucleotides according to claim 1 as a primer for a gene amplification reaction. A kit for detecting Pseudomonas aeruginosa, comprising at least one of the oligonucleotides according to claim 1 or 2.