JPH06253845A - Method for detecting streptococcus aureus and its reagent kit - Google Patents

Abstract

PURPOSE:To obtain a direct, simple, rapid and sure method for detecting Staphylococcus aureus and a new oligonucleotide and a reagent for detection used therefor. CONSTITUTION:This oligonucleotide is characterized by hybridizing with a nucleic acid-sequence common to respective coagulase types present in a coagulase gene. Furthermore, a labeled oligonucleotide is prepared by labeling the oligonucleotide. This method for detecting Staphylococcus aureus and this reagent kit for detection use the oligonucleotide or the labeled oligonucleotide as a probe or a primer.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oligonucleotide which hybridizes with a nucleic acid sequence common to each coagulase type present in a coagulase gene, and to rapidly suppress Staphylococcus aureus in a sample using the oligonucleotide,
The present invention relates to a reliable and simple method for detection and a reagent kit for the detection.

[0002]

2. Description of the Related Art Staphylococcus aureu
s) is a typical Pseudomonas aeruginosa that causes a disease collectively called skin and soft tissue infection. However, severe infections such as empyema and Staphylococcus aureus-like skin syndrome may occur, and the production of enterotoxin may cause food poisoning, which causes various symptoms. Also, in cases where the so-called surgical invasion is strong, such as in patients with severe burns or immediately after surgery, or when the immune function is extremely reduced due to leukemia, cancer, etc., severe symptoms occur in the deep part of the body. Sometimes. Especially in these patients, rapid and reliable identification of the causative bacteria is required. More recently, MRSA
The emergence of Staphylococcus aureus, which is called (methicillin-resistant Staphylococcus aureus) and which is resistant to all β-lactam drugs including methicillin, has been a problem. In patients with immunodeficiency, severe burns, premature babies, etc., if only the usual administration of antibiotics is resorted to without knowing that the causative bacterium is MRSA, a serious condition may occur. Therefore, MRSA
The rapid detection of is very important. The sequence of the methicillin resistance gene (mecA gene) in MRSA has already been determined (FEBS Lett.; 221: 167, 1987, Gene; 9.
4 vol. 137 p. 1990), it takes at least 24 hours to judge,
Moreover, instead of the drug susceptibility test, which was difficult to obtain stable results, the sequence of this gene was used to
Gene amplification by polymerase or the like (PCR; JP-A-60-2
(See Japanese Patent No. 81) or a DNA hybridization method, etc., it has become possible to make a reliable determination of methicillin resistance within 2 to 3 hours. However, the methicillin resistance gene was found in the Staphylococcus epiderm
Staphylo other than Staphylococcus aureus (idis)
may also have the genus coccus). However, pathogenicity is often a problem for S. aureus, and in addition to determination of methicillin resistance, it is necessary to distinguish S. aureus from other staphylococci.

Coagulase plays an important role in the pathogenicity of Staphylococcus aureus, and a coagulase-based plasma coagulation test is currently carried out to determine Staphylococcus aureus. There is a slide method (clamping factor test) as a simple method, which is mixed with rabbit plasma on a slide glass and judged in 3 minutes. I can't hope so much. In general, rabbit plasma was inoculated with fresh cultures in vitro and 3
Let stand for 3 hours at 7 ° C and check for solidification. However, even in this case, it is not possible to make a clear judgment in many cases, and in the case of a negative result, it must be left for another 18 hours and judged according to the degree of coagulation. After all, it takes 20 hours or more after the staphylococcus has been isolated to make a sufficient judgment, and it requires a certain degree of skill and sufficient experience.

There are eight types of coagulase due to antigenicity, and it is considered that the genes are mutated even with the same type, so that there are more types at the gene level. Three types of coagulase gene sequences have been determined to date (J. Biochem .; 102: 1177, 1987, Nucleic
Acids Res .; 17 pp. 8871 1989, Mol. Microbiol .; 4 vol. 39
However, even if the nucleic acid probe is prepared by using the restriction enzyme fragments of these genes as they are, it cannot be expected to react with all coagulase genes.

[0005]

An object of the present invention is to provide a direct, convenient, rapid, and reliable method for detecting Staphylococcus aureus, a novel oligonucleotide used therefor, and a reagent for detecting the same.

[0006]

Means for Solving the Problems As a result of various studies on the coagulase gene, the present inventors obtained a nucleic acid sequence suitable for the detection of Staphylococcus aureus, established a detection method using the same, and The invention was completed. That is, the present invention is an oligonucleotide characterized in that it hybridizes with a nucleic acid sequence common to each coagulase type present in the coagulase gene.

Further, the present invention provides a method for detecting a coregrass gene in a sample with a labeled probe or a nucleic acid primer, wherein the labeled probe or the nucleic acid primer hybridizes with a nucleic acid sequence common between the coagulase types present in the coagulase gene. A method for detecting Staphylococcus aureus characterized by containing an oligonucleotide.

Further, the present invention is a labeled probe containing an oligonucleotide which hybridizes with a nucleic acid sequence common between the coagulase types present in the coagulase gene.

The present invention is also a nucleic acid primer containing an oligonucleotide that hybridizes with a nucleic acid sequence common to each coagulase type present in the coagulase gene.

The present invention is a reagent kit for detecting Staphylococcus aureus, which comprises a labeled probe containing an oligonucleotide that hybridizes with a nucleic acid sequence common between coagulase types existing in a coagulase gene.

The present invention is a reagent kit for detecting Staphylococcus aureus, which comprises a nucleic acid primer containing an oligonucleotide that hybridizes with a nucleic acid sequence common to each coagulase type present in the coagulase gene.

As described above, there are eight types of coagulase due to their antigenicity and the like, and it is considered that the genes are mutated even with the same type, so there are more types at the gene level. Three types of coagulase gene sequences have been determined so far (J. Biochem .; 102: 1177, 1987).
Nucleic Acids Res .; 17 pp. 8871 1989 Mol. Micr
obiol .; Vol. 4, p. 393, 1990), it is not possible to react with all coagulase genes even if a nucleic acid probe is simply prepared by directly using the restriction enzyme fragments of these genes.

As a result of repeated experimental verification with reference to the coagulase gene sequence determined up to now, the present inventors have found that the 81 base pair repeating sequence and its surroundings are well conserved among the coagulase genes. I found a thing. Furthermore, the fact that all coagulase genes can be detected by detecting this repetitive sequence, and the use of a part or all of the nucleic acid sequence of SEQ ID NO: 1 or its complementary sequence as a probe or primer It was confirmed that the sequence could be detected.

The nucleic acid sequence shown in Sequence Listing / SEQ ID NO: 1 is
It is a putative consensus sequence of 81 base pair repeats within the coagulase gene that were experimentally confirmed by the present inventors. That is, by using a part or all of the nucleic acid sequence shown in Sequence Listing or SEQ ID NO: 1 or its complementary sequence as a probe or primer, the coagulase gene can be detected regardless of the difference in coagulase type. is there. This is considered to be due to the fact that this repetitive sequence is well conserved among coagulase genes and that the effect of mutation is small because the target sequence is repetitive.

The oligonucleotide of the present invention has a nucleic acid sequence common to each coagulase type present in the coagulase gene, preferably 81 unique to the coagulase gene.
It is an oligonucleotide that hybridizes with a repetitive sequence of base pairs, and more preferably in Sequence Listing, SEQ ID NO: 1.
(Where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position may be substituted with uracil (U)) or its complement. An oligonucleotide that is part or all of a sequence.

The oligonucleotide used in the nucleic acid probe of the present invention does not necessarily have to use the entire region of the sequence shown in Sequence Listing / SEQ ID NO: 1 or its complementary sequence, and the dissociation temperature may be adjusted according to the nucleic acid hybridization conditions to be used. (Tm value) may be calculated, and an appropriate area may be used with an appropriate length. However, if the probe is desired to have sufficient specificity, the length is preferably 10 bases or more, more preferably 20 bases or more. The oligonucleotide used for the nucleic acid primer in the present invention may also be prepared from the sequence shown in Sequence Listing / SEQ ID NO: 1 and its complementary sequence using a sequence having an appropriate length. In addition, since the sequence shown in Sequence Listing / SEQ ID NO: 1 is a putative consensus sequence, depending on the conditions of use, purpose, etc.,
It can easily be expected that some substitutions may be made in the oligonucleotide.

Since the oligonucleotide of the present invention can be prepared by chemical synthesis, it is possible to obtain an oligonucleotide of constant quality easily, in large quantities and at a low cost, as compared with a cloned oligonucleotide or polynucleotide. The oligonucleotide of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, it goes without saying that the thymidine residue (T) is read as the uridine residue (U). Alternatively, a DNA containing a uridine residue may be used by synthesizing by changing T at any position to U at the time of synthesis. Similarly, T at any position U
It may be RNA containing a thymidine residue changed to. The oligonucleotide may have point mutations such as deletion, insertion or substitution, and modified nucleotides.

The present invention also provides a nucleic acid sequence common to each coagulase type in the coagulase gene, preferably a sequence listing
The nucleic acid sequence of SEQ ID NO: 1 (where A is adenine, C
Represents cytosine, G represents guanine, and T represents thymine. Also,
T at any position may be replaced with uracil (U). ) Or a complementary sequence thereof, which is a labeled probe obtained by labeling an oligonucleotide having a part or all of its detection region as a detection region, or a nucleic acid primer obtained by directly or labeling the oligonucleotide.

Known labels such as radioactive substances, enzymes, fluorescent substances and luminescent substances can be used as the labeling substance. The label may be attached to the oligonucleotide either by end labeling or in the middle of the sequence. A label may be attached to the sugar, the phosphate group, and the base moiety. For example, an enzyme can be labeled by substituting a nucleotide having a linker arm as a member of an oligonucleotide (Nuclei
c Acids Research; 14: 6115, 1986). As an example, uridine having a linker arm at the 5-position is disclosed in JP-A-60-5.
It can also be chemically synthesized from deoxyuridine by the synthetic method disclosed in 00717 and introduced into the above-mentioned oligonucleotide.

The present invention is a method for detecting Staphylococcus aureus by detecting the coregrass gene in a sample,
In particular, it is characterized by detecting a common nucleic acid sequence in each coagulase type in the coagulase gene. Desirably, it is a method of detecting Staphylococcus aureus by detecting an 81 base pair repeating sequence in the coagulase gene. Specifically, an oligonucleotide that hybridizes with a nucleic acid sequence common to each coagulase type in the coagulase gene, preferably the nucleic acid sequence shown in Sequence Listing / SEQ ID NO: 1 (where A is adenine, C is cytosine, and G is Guanine, T represents thymine, and T at any position may be substituted with uracil (U)) or a part or all of its complementary sequence is used as a probe or primer. This is a method for detecting Staphylococcus aureus.

The oligonucleotide of the present invention can be bound to a solid phase carrier and used as a capture probe. In this case, a sandwich assay (see Japanese Patent Laid-Open No. 58-40099) may be carried out using a combination of a capture probe and a labeled probe, or a method of labeling and capturing a target nucleic acid (Japanese Patent Laid-Open No. 62-62).
-265999). There is also a method in which an oligonucleotide is labeled with biotin, and after hybridization, it is captured with an avidin-binding carrier.

When the oligonucleotide of the present invention is used as a primer, it is necessary to amplify only the sequence specific to the coagulase gene by performing nucleic acid amplification (PCR; see JP-A-60-281) by DNA polymerase or the like. You can In PCR, the coagulase gene can be easily detected by incorporating a radiolabeled nucleotide during the reaction or by fractionating the amplification product by electrophoresis and detecting a specific band. Further, the amplification product can be directly detected by using the above-mentioned labeled oligonucleotide as a primer.

Further, the reagent kit for detecting Staphylococcus aureus of the present invention contains a reaction carrier, a detection reagent and the like in addition to the above-mentioned oligonucleotide for detecting Staphylococcus aureus or the probe labeled with the oligonucleotide.

The reagent kit for detecting Staphylococcus aureus of the present invention contains, in addition to the nucleic acid primer for detecting Staphylococcus aureus, a DNA synthase, a reaction solution, etc., and a reagent for detecting an amplification product in some cases. .

The rapid method for detecting Staphylococcus aureus in the present invention is to detect Staphylococcus aureus in various clinical samples such as sputum and blood by detecting the coagulase gene. It goes without saying that it also includes determining whether or not the isolated and cultured bacterial cells are Staphylococcus aureus. Further, the sample in the present invention includes the above-mentioned various clinical samples and cultured bacterial cells, as well as nucleic acids isolated and purified from these. It may also be an amplified nucleic acid.

[0026]

EXAMPLES Next, by showing examples of the present invention,
The effect of the present invention will be further clarified. Example 1 (1) Synthesis of Staphylococcus aureus detection oligonucleotide having linker arm Using a DNA synthesizer type 392 manufactured by ABI, the sequence listing by the phosphoamidite method was performed.
An oligonucleotide (28 bases) having a sequence complementary to the 29th to 56th sequences among the sequences shown in SEQ ID NO: 1 was synthesized. At this time, special table Sho 60-500717
Uridine having a linker arm at the 5-position, which was chemically synthesized from deoxyuridine, was introduced into the above-mentioned oligonucleotide by the synthetic method described in the publication. This uridine can replace any T in the oligonucleotide, but in this example, the T at the 5'end was replaced. The sequence of the prepared oligonucleotide is shown below.

5'-UGTGTTGTTA CGTTATATGC ATTTGTTT-3 '(U represents uridine having a linker arm at the 5-position) The synthesized linker oligonucleotide was deprotected with aqueous ammonia at 50 ° C. overnight, and then Pharmacia Co. It was purified by FPLC manufactured by using an anion exchange column.

(2) Labeling of Linker Oligonucleotide with Alkaline Phosphatase Alkaline phosphatase was labeled with the above-mentioned linker oligonucleotide and its linker arm (Nucleic
Acids Research; 14: 6115, 1986). Linker oligonucleotide 1.5 A ₂₆₀ to 0.2 M NaHC
It was dissolved in 12.5 μl of O _3, 25 μl of 10 mg / ml disuccinimidyl suberate (DSS) was added thereto, and reacted at room temperature for 2 minutes. The reaction mixture was subjected to gel filtration with a Sephadex G-25 column (1 cm φx30 cm) equilibrated with 1 mM CH ₃ COONa (pH 5.0) to remove excess DSS. Alkaline phosphatase-labeled nucleic acid probe was prepared by reacting a linker oligonucleotide with activated terminal amino group with twice the molar amount of alkaline phosphatase (dissolved in 100 mM NaHCO ₃ , 3M NaCl) at room temperature for 16 hours. Got The obtained labeled probe was purified by Pharmacia FPLC using an anion exchange column. Fractions containing the labeled probe were collected and concentrated by an ultrafiltration method using Centricon 30K (Amicon).

(3) Hybridization with bacterial cell coating 66 bacterial strains including clinical isolates (of which Staphylococcus aureus (Staph)
A colony of ylococcus aureus (23 strain)) was scraped off with a toothpick or the like and smeared on a small amount on a nylon membrane, and lysed under alkaline conditions to immobilize nucleic acid on the nylon membrane. After neutralizing the membrane, the membrane was transferred to a hybridization bag (BRL) and the hybridization buffer containing the alkaline phosphatase-labeled nucleic acid probe solution (5xSSC, 0.5
% Bovine serum albumin, 0.5% polyvinylpyrrolidone,
1% Sodium dodecyl sulfate) was added and the mixture was sealed with a policyr, and hybridization was carried out at 50 ° C. for 15 minutes. Remove the membrane from the plastic bag and wash solution 1 (1xSSC,
Washing with 1% sodium dodecyl sulfate) at 50 ° C. for 10 minutes with shaking. Further, washing with washing solution 2 (1xSSC, 0.5% Triton X-100) was carried out at room temperature for 10 minutes with shaking. Finally, cleaning solution 3 (1xSS
C) was washed with shaking at room temperature for 5 minutes. Transfer the membrane to a new hybridization bag and add the substrate solution (0.1M Tris-H
Cl, 0.1M NaCl, 0.1M MgCl ₂ , 0.3 mg / ml nitroblue tetrazolium, 0.3 mg / ml bromchloroindoferryl phosphate pH 7.5) was added, and the mixture was sealed with a policyr and incubated at 37 ° C. for 1 hour.

(4) Results Spots of purple dye generated by alkaline phosphatase were visually determined. Those with confirmed spots were regarded as positive. As a result, all 23 strains of Staphylococcus aureus were determined to be positive, while strains of other strains were determined to be negative. Among Staphylococcus aureus, one strain each had a coagulase type determined to be type II, type III, type V, type VI, and type VII was included, but for other types of Staphylococcus aureus, coagulase type was included. Is not known. In addition to the clinical isolates, two of them were deposited at Osaka University (IFO No. 3060, IFO No. 3).
761) included.

The breakdown of the negative strains is as follows: Bacillus subtilis (1 strain) Citrobacter freundii (1 strain) Clostridium sticklandii (1 strain) Enterobacter・ Aerogenes 3 strains Enterobacter cloacae 1 strain Enterococcus avium 1 strain Enterococcus durans 1 strain Enterococcus faecalicus 5 strains Enterococcus faecalicus faecium 1 strain Escherichia coli 5 strains Klebsiella oxytoca 1 strain Klebsiella pneumoniae 2 strains Pseudomonas aeruginosa Pseudomonas aeruginosa 3 strains Staphylococcus e pidermidis 8 strains Staphylococcus haemolyticus 1 strain Phthaphylococcus highus strain Staphylococcus 1・ Staphylococcus saprophyticus 2 strains Staphylococcus xylosus 1 strain Streptococcus agalactae 1 strain S. pyogenes 1 strains Strains Vibrio parahaemolyticus (Vibrio parahaemolyticus) 1 strain is 43 strains.

Particularly, Staphylococcus epidermidis, which belongs to the same genus Staphylococcus as Staphylococcus aureus, Staphylococcus haemolyticus (Staphylococcus)
haemolyticus Staphylococcus hyicu
s) Staphylococcu
s saprophyticus) Staphylococcus xylo
It is important that no reaction was seen with sus). Also,
The fact that no reaction was observed with various Pseudomonas aeruginosa other than Staphylococcus aureus indicates that this nucleic acid probe has extremely high specificity for S. aureus and is very effective in practice. Furthermore, it was also confirmed that this nucleic acid probe did not react with human placental DNA.

From the above results, it was revealed that the determination method of Staphylococcus aureus according to the present invention can obtain exactly the same results as the determination by the conventional method of directly detecting the coagulase activity with good reproducibility. Moreover, there was no ambiguous result that was difficult to judge like the conventional method. Moreover, in the method for determining Staphylococcus aureus according to the present invention, the determination was completed about 2 hours after the colony was isolated.

Example 2 (1) Synthesis of PCR primer for detecting Staphylococcus aureus Using the DNA synthesizer type 392 manufactured by ABI, by the phosphoamidite method, from the first of the sequences shown in SEQ ID NO: 1 Oligonucleotides having up to the 15th sequence (15 bases, hereinafter referred to as primer F) and 61 of the sequences shown in SEQ ID NO: 1 in the sequence listing
An oligonucleotide (20 bases, hereinafter referred to as primer R) having a sequence complementary to the sequence from the 80th sequence to the 80th sequence was synthesized. The method follows the ABI manual, and various oligonucleotides are deprotected with ammonia water at 55 ° C.
It was carried out overnight. Purification was carried out by FPLC manufactured by Pharmacia on an anion exchange column. The sequences of these oligonucleotides are shown below.

Primer F: 5'-GCTCGCCCAA CACAA-3 'Primer R: 5'-CCGTATGATA CTTGACCATT-3'

(2) Preparation of Samples 15 clinical isolates of Staphylococcus (Staph aureus (Staph)
ylococcus aureus 9 strains, Staphylococ
cus epidermidis 6 strains) was scraped off with a toothpick or the like, suspended in 100 μl of pure water, and heated at 94 ° C. for 10 minutes.
5 μl of this suspension was added to 50 μl of the PCR reaction solution.

(3) PCR The reaction solution composition was as follows: Primer F and Primer R 1 μM each
0 mM Tris-HCl (pH 8.9), 1.5 mM MgCl ₂ , 80 mM KCl, 500 μg
/ ml BSA, 0.1% sodium cholate, 0.1% TritonX-100,
0.2 mM dATP, dCTP, dGTP, dTTP and Tth D, respectively
NA polymerase (Toyobo) 40 units / ml. The reaction conditions are as follows. Thermal denaturation: 94 ° C., 0.5 minutes Annealing: 50 ° C., 2 minutes Polymerization reaction: 75 ° C., 1.5 minutes 30 times. These operations are Perkin Elmer /
It was performed using a DNA thermal cycler manufactured by Cetus (Perkin-Elmer / Cetus).

(4) Detection 5 μl of the reaction solution was electrophoresed on 1.5% agarose gel and stained with ethidium bromide, and then fluorescence from ultraviolet rays was detected. The electrical conditions for electrophoresis are constant voltage of 100 V and time of 30.
I went for a minute. The operating method and other conditions are described in Mole of Maniatis et al.
The method described in cular Cloning (1982) was followed. In addition to the reaction solution, a molecular weight marker was also electrophoresed at the same time, and the length of the detected DNA fragment was calculated by comparing the relative mobilities. Since the detection region is a repeating sequence of 81 base pairs, in the combination of primer F and primer R, amplification products of various lengths are used as a ladder of a fluorescent band that increases every 81 base pairs with a minimum of 80 base pairs. was detected. The number of fluorescent bands depended on the number of repetitive sequences in the coagulase gene possessed by the strain. A fluorescent band did not appear at all in the strain having no coagulase gene.

(5) Results After agarose gel electrophoresis and ethidium bromide staining, those in which the ladder of the above fluorescence band was confirmed by ultraviolet rays were regarded as positive. As a result, a fluorescent band was confirmed in all 9 strains of Staphylococcus aureus and could be determined as positive, whereas a fluorescent band was not seen in any of the 6 strains of Staphylococcus epidermis, and it could be determined as negative. From the above, it was proved that by using the oligonucleotide according to the present invention for PCR, it is possible to easily obtain exactly the same result as the conventional method. Moreover, there was no vague result that was difficult to judge like the conventional method. Further, since detection by nucleic acid amplification such as PCR can be expected to have high sensitivity, it can be directly detected from clinical materials before isolation of bacteria.

[0040]

EFFECT OF THE INVENTION The orgonucleotide of the present invention, which hybridizes with the nucleic acid sequence common to each coagulase type present in the coagulase gene, enables direct, convenient, rapid and reliable detection of Staphylococcus aureus. . In particular, an oligonucleotide having a part or all of the nucleic acid sequence shown in SEQ ID NO: 1 of the Sequence Listing of the present invention or its complementary sequence is used as a primer for an amplification reaction, a probe for direct detection, or a combination thereof. Can also be used, the time can be shortened as compared with the conventional plasma coagulation test, and simple and reproducible and reliable results can be obtained. Moreover, it becomes possible to detect Staphylococcus aureus directly from clinical samples such as sputum and blood by nucleic acid amplification using the oligonucleotide.

[0041]

[Sequence list]

SEQ ID NO: 1 Sequence length: 81 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..81 Method for determining features : S Other features: Has a sequence complementary to that of S. aureus. Sequence GCTCGCCCAA CACAAAACAA GCCAAGCGAA ACAAATGCAT ATAACGTAAC AACACATGCA 60 AATGGTCAAG TATCATACGG A 81

SEQ ID NO: 2 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..28 Features Method determined: S Other characteristics: having a sequence complementary to that of S. aureus. Sequence UGTGTTGTTA CATTATATGC ATTTGTTT 28

SEQ ID NO: 3 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..15 Method determined: S Other characteristics: having a sequence complementary to that of S. aureus. Sequence GCTCGCCCAA CACAA 15

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..20 Features Method determined: S Other characteristics: having a sequence complementary to that of S. aureus. Sequence CCGTATGATA CTTGACCATT 20

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display part // (C12Q 1/14 C12R 1: 445) (72) Inventor Norimitsu Otsuka Nishiri-shi, Hyogo Prefecture 3-10-4 Nakamachi (72) Inventor Keiko Yamashita 3-43-811 Uedacho, Nishinomiya City, Hyogo Prefecture

Claims (9)

[Claims] 1. An oligonucleotide, which hybridizes with a nucleic acid sequence common to each coagulase type present in a coagulase gene. 2. The oligonucleotide according to claim 1, wherein the nucleic acid sequence common to each coagulase type present in the coagulase gene is a repeating sequence of 81 base pairs. 3. An oligonucleotide that hybridizes with a nucleic acid sequence common to each coagulase type present in the coagulase gene has the nucleic acid sequence of SEQ ID NO: 1 (where A is adenine, C is cytosine, and G is guanine, T is thymine, and T at any position may be substituted with uracil (U)) or a part or all of its complementary sequence.
The described oligonucleotide. 4. A method for detecting a coagulase gene in a sample with a labeled probe or a nucleic acid primer, wherein the labeled probe or the nucleic acid primer hybridizes with a nucleic acid sequence common between the coagulase types present in the coagulase gene. A method for detecting Staphylococcus aureus, which comprises: 5. The Staphylococcus aureus according to claim 4, wherein the oligonucleotide that hybridizes with a nucleic acid sequence common between the coagulase types present in the coagulase gene is the oligonucleotide according to claims 2 to 3. Detection method. 6. A labeled probe containing the oligonucleotide according to claim 1. 7. A nucleic acid primer containing the oligonucleotide according to claim 1. 8. A reagent kit for detecting Staphylococcus aureus, comprising the labeled probe according to claim 6. 9. A reagent kit for detecting Staphylococcus aureus, which comprises the nucleic acid primer according to claim 7.