JP6661963B2 - How to detect staphylococci - Google Patents

Description

The present invention relates to a method for detecting staphylococci and a composition therefor.

Staphylococci are Gram-positive cocci belonging to the genus Staphylococcus (hereinafter, when a microorganism name is represented by a genus and its subclass (species, etc.), the genus name is abbreviated as S.). . In particular, Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) have pathogenicity to humans and are frequently isolated clinically (Non-Patent Document 1).

Further, S.I. aureus and S. aureus. It is known that epidermidis exhibits drug resistance by having a drug resistance gene such as a methicillin resistance gene (mecA) (Patent Document 1). Therefore, there is a need for a method that can rapidly detect and classify these bacteria for the purpose of selecting a therapeutic agent, preventing the spread of infection, preventing food poisoning, and the like.

JP 2013-048620 Table 2013-535951 JP 2001-518283 Special table 2003-511015

Genome biology and evolution, 7 (5), 1313-1328

A method for detecting a plurality of types of staphylococci by nucleic acid identification is known as a known technology. However, in the conventional technology, it is necessary to prepare a plurality of primer sets in order to classify the species (Patent Documents 2 and 4). ), The operation is complicated and the cost is high.

There is also a method of detecting a plurality of types of staphylococci using universal primers, but it has been difficult to identify staphylococcal species by electrophoresis (Patent Documents 3 and 4). In order to identify the bacterial species by this method, it was necessary to further add a detection probe to the reaction system.

An object of the present invention is to provide a method for easily detecting a plurality of types of staphylococci by distinguishing one from another by using one primer set and a composition therefor.

The present inventors have conducted intensive studies and found that multiple types of staphylococci can be easily detected and distinguished from each other by nucleic acid identification using one primer set having a specific base sequence. Reached. That is, the outline of the present invention is as follows.

[Item 1]
A method for distinguishing and detecting plural types of staphylococci in a sample, comprising the following steps (1) and (2):
(1) Step of performing nucleic acid amplification using one primer set consisting of a forward primer represented by the following (A) and a reverse primer represented by the following (B) to obtain an amplification product (A) From the following (A1) to (A1) A4) an oligonucleotide represented by any one of the nucleotide sequences (A1) a nucleotide sequence represented by SEQ ID NO: 1 (A2) a nucleotide sequence represented by SEQ ID NO: 2 (A3) 6 nucleotides at the 3'-terminal end of SEQ ID NO: 1 (A4) any of (B1) to (B4) below the sequence (B) that partially contains the sequence of the 6 bases at the 3 ′ end of SEQ ID NO: 2 The oligonucleotide (B1) represented by the nucleotide sequence of (B1) The nucleotide sequence represented by SEQ ID NO: 3 (B2) The nucleotide sequence represented by SEQ ID NO: 4 (B3) Include in some Column (B4) the most 3 'end of the 6 nucleotide sequence of SEQ ID NO: 4, the sequence comprising a part (2) (1) detecting the amplification product obtained in step [section 2]
Item 2. The method according to Item 1, wherein the amplified products are distinguished from each other by a difference in length in the step (2) and detected.
[Item 3]
Item 1. The method according to Item 1, wherein in step (2), the amplification products are detected separately from each other based on a difference in base sequence.
[Item 4]
Multiple species of staphylococci are S. aureus. aureus and S. aureus. Item 4. The method according to any one of Items 1 to 3, which is two kinds of staphylococci of Epidermidis.
[Item 5]
Multiple species of staphylococci are S. aureus. aureus, S.A. epidermidis and S. Item 5. The method according to any one of Items 1 to 4, which is three kinds of S. captis.
[Item 6]
Item 6. The method according to any one of Items 1 to 5, wherein one primer set is a forward primer represented by (A2) or (A4) and a reverse primer represented by (B2) or (B4).
[Item 7]
A primer set comprising a forward primer represented by the following (A) and a reverse primer represented by the following (B).
(A) an oligonucleotide represented by any one of the following nucleotide sequences (A1) to (A4): (A1) a nucleotide sequence represented by SEQ ID NO: 1 (A2) a nucleotide sequence represented by SEQ ID NO: 2 (A3) SEQ ID NO: Sequence (A4) containing the sequence of the most 3′-terminal 6 bases of No. 1 as a part thereof (A4) Following sequence (B) including the sequence of the most 3′-terminal 6 nucleotides of SEQ ID NO: 2 as a part thereof Oligonucleotide (B1) represented by any one of base sequences (B1) to (B4) (B1) base sequence represented by SEQ ID NO: 3 (B2) base sequence represented by SEQ ID NO: 4 (B3) most 3 ′ of SEQ ID NO: 3 Sequence containing a terminal 6-base sequence as a part thereof (B4) Sequence containing the most 3 ′ terminal 6-base sequence of SEQ ID NO: 4 as a part [Item 8]
Item 9. A composition for detecting a plurality of types of staphylococci in a sample, wherein the composition comprises the primer set according to item 7.
[Item 9]
Item 9. A kit for detecting a plurality of types of staphylococci in a sample by distinguishing each other, comprising the primer set according to item 7.

According to the present invention, by using one primer set, a plurality of types of staphylococci (for example, S. aureus and S. epidermidis, and S. captis, one of the genus of coagulase-negative staphylococci, can be used as the two species). In addition, the present invention has provided a method for easily detecting the three added species separately from each other, and a composition therefor. Use of the method and the composition can greatly contribute to selection of a therapeutic agent for staphylococcal infection, prevention of spread of infection, prevention of food poisoning, and the like.

S. aureus, S.A. epidermidis, S.P. FIG. 2 is a diagram in which genomic DNA sequences of S. capitis are aligned. The design of the forward primer was performed from this region. S. aureus, S.A. epidermidis, S.P. FIG. 2 is a diagram in which genomic DNA sequences of S. capitis are aligned. The design of the reverse primer was performed from this region. FIG. 7 is a diagram showing the results of Example 1. From left, marker, S.M. aureus, S.A. epidermidis, S.P. capitis, E .; faecalis, a marker. FIG. 9 is a diagram showing the results of electrophoresis of Example 2. The left and right edges are markers. 5 shows the results of a sequence analysis of a band of about 280 bp obtained by electrophoresis in Example 2. This base sequence is S. aureus and high homology. 5 shows the results of a sequence analysis of a band of about 400 bp obtained by electrophoresis in Example 2. This base sequence is S. Epidermidis showed high homology. 5 shows the results of sequence analysis of a band of about 180 bp obtained by electrophoresis in Example 2. This base sequence is S. capitis and high homology.

One embodiment of the present invention is a method for distinguishing and detecting a plurality of types of staphylococci in a sample, comprising the following steps (1) and (2).
(1) Step of performing nucleic acid amplification using one primer set consisting of a forward primer represented by the following (A) and a reverse primer represented by the following (B) to obtain an amplification product (A) From the following (A1) to (A1) A4) an oligonucleotide represented by any one of the nucleotide sequences (A1) a nucleotide sequence represented by SEQ ID NO: 1 (A2) a nucleotide sequence represented by SEQ ID NO: 2 (A3) 6 nucleotides at the 3'-terminal end of SEQ ID NO: 1 (A4) any of (B1) to (B4) below the sequence (B) that partially contains the sequence of the 6 bases at the 3 ′ end of SEQ ID NO: 2 The oligonucleotide (B1) represented by the nucleotide sequence of (B1) The nucleotide sequence represented by SEQ ID NO: 3 (B2) The nucleotide sequence represented by SEQ ID NO: 4 (B3) Include in some Column (B4) the most 3 'end of the 6 nucleotide sequence of SEQ ID NO: 4, detecting the amplification product obtained in the step sequence (2) (1) includes in its part

[About detection target]
[Staphylococcus]
Staphylococci to be detected in the present invention are not particularly limited as long as they are Gram-positive cocci belonging to the genus Staphylococcus. For example, S. aureus, S. epidermidis, and one of the coagulase-negative staphylococci, S. aureus. captis and the like. S. captis is a staphylococcus that is highly distributed on the human scalp and sometimes causes endocarditis and meningitis.

According to the method of the present invention, a plurality of types of staphylococci can be detected separately from each other, and the combination thereof is not particularly limited. For example, at least aureus and S. aureus. From a sample containing two types of staphylococci epidermidis, both types can be detected separately from each other. In addition, at least two types of S.p. From a sample containing three types of staphylococci to which capitis is added, the three types can be detected separately from each other. In addition, S.I. aureus, S.A. epidermidis and S. It is possible to determine which staphylococci of the three species are contained from a sample containing at least one of the group consisting of C. captis.

[About sample]
As a measurement sample that can be used in the present invention, not only a biological sample or food but also a prepared nucleic acid or the like can be used, but is not limited thereto.

The biological sample is not particularly limited, but includes blood, blood culture solution, pus, cerebrospinal fluid, pleural effusion, pharyngeal swab, nasal swab, sputum, tissue section, skin, vomit, feces, separated culture colony, catheter washing solution, etc. Is mentioned. When a biological sample is used as a measurement sample, pretreatment such as dilution or suspension may be performed according to each biological sample.

Foods include, but are not limited to, water, alcoholic beverages, soft drinks, processed foods, vegetables, livestock products, marine products, eggs, dairy products, raw meat, raw fish, side dishes, and the like. When a food is used as a measurement sample, not only a part or all of the food but also a food whose surface is wiped can be used, but the present invention is not limited thereto. In addition, pretreatment such as fine crushing depending on the sample or suspension in purified water may be performed.

Further, a material obtained by wiping a cooking utensil or a door knob or a liquid obtained by cleaning the material can also be used as a sample.

The prepared nucleic acid refers to DNA and RNA extracted from each sample, artificially synthesized nucleic acid, and the like, but is not limited thereto. For nucleic acid extraction, a kit sold by each manufacturer may be used.

The method for collecting and preparing the sample is not particularly limited, and a known method can be used depending on the type and purpose of the sample.

[About the first step (amplification step)]
[Primer set (sequence)]
In the first step of the method of the present invention, nucleic acid amplification is performed using one primer set to obtain an amplification product. As an embodiment of the primer set used in the present invention, a primer set composed of a forward primer represented by the following (A) and a reverse primer represented by the following (B) is exemplified.
(A) an oligonucleotide represented by any one of the following nucleotide sequences (A1) to (A4): (A1) a nucleotide sequence represented by SEQ ID NO: 1 (A2) a nucleotide sequence represented by SEQ ID NO: 2 (A3) SEQ ID NO: Sequence (A4) containing the sequence of the most 3′-terminal 6 bases of No. 1 as a part thereof (A4) Following sequence (B) including the sequence of the most 3′-terminal 6 nucleotides of SEQ ID NO: 2 as a part thereof Oligonucleotide (B1) represented by any one of base sequences (B1) to (B4) (B1) base sequence represented by SEQ ID NO: 3 (B2) base sequence represented by SEQ ID NO: 4 (B3) most 3 ′ of SEQ ID NO: 3 Sequence containing the terminal 6-base sequence as a part thereof (B4) Sequence containing the most 3 ′ terminal 6-base sequence of SEQ ID NO: 4 as a part thereof

Here, each of the primers (A1), (A2), (B1) and (B2), that is, the base sequences represented by SEQ ID NOs: 1 to 4 are aureus, S.A. epidermidis and S. When alignment is performed with respect to the genome sequence of C. capitis, these regions are commonly highly identical.

1 and 2 show the nucleotide sequences represented by SEQ ID NOs: 1 to 4; aureus strain FCFHV36, complete genome (GenBank: CP011147.1), S.A. epidermidis strain 949_S8 genome (GenBank: CP010942.1) and S.e. capitis subsp. 3 shows the alignment with the genomic sequences of three types of staphylococci, ie, captis strain AYP1020, complete genome (GenBank: CP007601. 1).

Each of the primers (A3), (A4), (B3), and (B4) is a sequence containing, as a part thereof, a sequence of 6 nucleotides at the 3′-terminal side of the nucleotide sequence represented by SEQ ID NOS: 1 to 4. It is characterized by the following.

In general, it is important for a highly specific primer that the 3 'end is complementary to the target sequence. In other words, if the 3' end is complementary, it is effective even if the 5 'end is not complementary. It is supposed to work as a good primer. For this reason, it is preferable that the primer described in the present specification is also designed so that the 3 'end has a sequence complementary to the genomic sequence. That is, it is considered that the base sequence of the 3 'end of the primer described in this specification is particularly important, and that the base sequence enables detection and classification of staphylococcus.

Therefore, when each of the primers (A3), (A4), (B3) and (B4) is used, the sequence of the most 3′-terminal 6 bases of the base sequence represented by SEQ ID NOs: 1 to 4 is used It is preferable to include it in a part of the sequence of 10 bases on the 3 'end side. More preferably, it is included in a part of the sequence of 8 bases at the 3 ′ end of each primer, and even more preferably, it is included as part of the sequence of 7 bases at the 3 ′ end of each primer, Most preferably, it is included in the sequence of 6 bases at the 3 'end of each primer.

In addition, the number of bases of each of the primers (A3), (A4), (B3), and (B4) may be within the range of the number of bases that sufficiently functions as a primer. The number of bases of the primer used for nucleic acid amplification varies depending on the nucleic acid amplification method, but the preferred upper limit is 50 bases, more preferably 30 bases, and further preferably 25 bases. On the other hand, a preferred lower limit of the number of bases is 10 bases, and more preferably 15 bases. Short primers may not provide sufficient specificity and long primers may increase non-specific products and decrease annealing efficiency.

The above-mentioned primer set is also one aspect of the present invention.

[For amplification, except for primer set]
The nucleic acid amplification is not particularly limited in the present invention except for the matters described in the claims, and a known method can be used. For example, PCR, real-time PCR, digital PCR, quantitative PCR (qPCR), LA-PCR (Long and Accurate PCR), competitive PCR including ABC-PCR, in situ PCR, RNA-primed PCR, multiplex PCR, shuttle PCR, PCR / GC-calmp method, Immuno PCR, reverse transcription polymerase chain reaction, NASBA method, TMA method, SDA method, LAMP method and the like. In the present invention, the conditions for the amplification reaction in each method are not particularly limited, and the amplification reaction can be performed under the conditions of each method.

The composition of the present invention requires several components depending on the nucleic acid amplification method and detection / classification method to be performed. As an example, a nucleic acid amplification method using PCR generally requires a measurement sample, a DNA polymerase, a primer, deoxyribonucleoside triphosphates (dNTPs), a reaction buffer, and a magnesium salt. Depending on the purpose, UNG (Uracil-N-Glycosylase), BSA, DMSO, betaine, glycerol, saccharides, amino acids, peptides, amines, potassium salts, and the like may be included as additives, but the present invention is not limited thereto. Not done.

When PCR is used for the nucleic acid amplification method, the DNA polymerase to be used is not particularly limited. Examples include, but are not limited to, Taq polymerase, Tth polymerase, KOD polymerase, Pfu polymerase, and the like. As the DNA polymerase, not only a naturally occurring enzyme but also a recombinant enzyme having an artificially modified amino acid sequence may be used.

By performing nucleic acid amplification using the forward primer and the reverse primer designed according to the above concept as one primer set, as illustrated in Example 1 described below, S. aureus can be used. aureus, S.A. epidermidis, S.P. Capitis can confirm amplification products of about 280 bp, about 400 bp, and about 180 bp, respectively.

[Second Step (Step of Differentiating and Detecting Amplified Products)]
The second step of the method of the present invention is a step of distinguishing and detecting the amplification product obtained in the first step.

In the present invention, the method for distinguishing and detecting the amplification products is not particularly limited except for the matters described in the claims, and a known nucleic acid detection method can be used. For example, a method of distinguishing amplification products by a difference in length or a method of distinguishing by a difference in base sequence can be used. Specific examples include melting curve analysis, electrophoresis, DNA probe method, intercalator method, nucleic acid hybridization, Northern blotting, mass spectrometry, DNA sequencing, next-generation sequencing, autoradiography, etc. However, it is not limited to these.

When the DNA probe method is used as a method for distinguishing and detecting amplification products from each other, a DNA probe may be further added to the composition. DNA probes include, but are not limited to, Taqman® probes, FRET probes, molecular beacons, Scorpions, QProbes, and the like.

When the real-time PCR, the SDA method, and the LAMP method are adopted, “amplification” and “detection by distinction” can be performed simultaneously.

As an example, when performing real-time PCR using a Taqman® probe, the concentration of Taq polymerase is 0.001 to 1.0 U / μL, more preferably 0.01 to 0.1 U / μL, and the primer concentration is 0.01 to 10 μM, more preferably 0.1 to 2 μM, dNTPs concentration is 0.01 to 2 mM, more preferably 0.1 to 0.5 mM, and the pH of the reaction buffer is 2 to 13, more preferably Is 4 to 10, the magnesium salt concentration is 0.1 to 10 mM, more preferably 1.0 to 5 mM, and the Taqman® probe concentration is 0.01 to 1 μM, more preferably 0.02 to 0 μM. 0.6 μM. Real-time PCR can be performed under known conditions. Further, a plurality of Taqman® probes may be added according to the measurement.

Further, as an example, when performing a melting curve using QProbe, the concentration of KOD polymerase is 0.001 to 1.0 U / μL, more preferably 0.01 to 0.1 U / μL, and the primer concentration is 0.01. 10 μM, more preferably 0.1 μM to 8 μM, the dNTPs concentration is 0.01 to 2 mM, more preferably 0.1 to 0.5 mM, and the pH of the reaction buffer is 2 to 13, more preferably 4 to 10, the magnesium salt concentration is 0.1 to 10 mM, more preferably 1.0 to 5 mM, and the QProbe concentration is 0.01 to 2 μM, more preferably 0.1 to 0.8 μM. PCR and melting curve analysis can be performed under known conditions. Further, a plurality of types of QProbe may be added according to the measurement.

Prior art (Patent Literature 2) discloses S.S. aureus and S. aureus. In contrast to the multiplex assay which requires at least two primer sets to detect two types of staphylococci of E. epidermidis, the present invention distinguishes between the two types of staphylococci with one primer set. it can. Further, according to the present invention, at least S.p. aureus, S.A. epidermidis and S. The present invention has made it possible to more easily detect and classify staphylococci, since three types of staphylococci can be distinguished and detected.

[Composition and Kit for Differentiating and Detecting Multiple Types of Staphylococci]
Another embodiment of the present invention includes a composition or kit for detecting a plurality of types of staphylococci in a sample by distinguishing each other, comprising the above-mentioned primer set.

The composition for carrying out the method of the present invention or contained in the composition or kit of the present invention is not particularly limited as long as reagents necessary for amplification and detection of nucleic acids are appropriately used, other than the primer set. As the nucleic acid amplification / detection method, the various methods described above can be used, and these methods have already been established in the art. Therefore, according to a known method, a plurality of types of staphylococci in various samples can be distinguished from each other and detected using the primer set.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to Examples.

Example 1 Detection and Classification of Staphylococcus (1) Method Staphylococci and the like were detected and classified using one primer set, the primer shown in SEQ ID NO: 2 and the primer shown in SEQ ID NO: 4. Specifically, after performing PCR using Blend Taq (R) -Plus- (Toyobo Co., Ltd.) using the primers to amplify the nucleic acid, the presence or absence of the amplification product and its size were compared by electrophoresis. As a measurement sample, colonies separated and cultured (S. aureus, S. epidermidis, S. captis, Enterococcus faecalis) were used.
(A) Preparation of reaction solution The reaction solution was prepared as follows according to the instruction manual of Blend Taq (R) -Plus-. Further, the measurement sample was added in an amount corresponding to 1/40 volume of the reaction solution.
PCR Buffer for Blend Taq 1 ×
Blend Taq (R) -Plus- 0.025 U / μL
Primer 0.2μM each
dNTPs 0.2 mM
(B) PCR cycle conditions PCR was performed under the following conditions.
94 ° C. 2 minutes 94 ° C. 1 minute-55 ° C. 30 seconds-72 ° C. 1 minute 30 seconds 37 cycles (c) Electrophoresis The obtained amplification product was mixed with 6 × Loading Dye (Toyobo) and agarose electrophoresis was performed. Was. The agarose gel used was 3%, the electrophoresis conditions were 100 V for 30 minutes, and the marker used was 100 bp DNA Ladder (Toyobo). The agarose gel was stained with ethidium bromide, and the presence and size of amplification products were confirmed by UV irradiation.
(2) Results The measurement results are shown in FIG. S. aureus is about 280 bp, epidermidis is about 400 bp; captis confirmed a band at about 180 bp. E. coli that is not staphylococcal. faecalis did not show any band. From these results, S.p. aureus, S.A. epidermidis, S.P. Captis could be detected and classified. It was also confirmed that bacteria other than staphylococci were not detected.

Example 2: Measurement of biological sample (1) Method As one primer set, a biological sample was measured using the primer shown in SEQ ID NO: 2 and the primer shown in SEQ ID NO: 4. Specifically, after performing PCR using Blend Taq (R) -Plus- (Toyobo Co., Ltd.) to amplify the nucleic acid using the primers, electrophoresis was performed and sequence analysis was performed. As the biological sample, 16 types of colonies separated and cultured were used.
(A) Preparation of reaction solution The reaction solution was prepared as follows according to the instruction manual of Blend Taq (R) -Plus-. Further, the measurement sample was added in an amount corresponding to 1/40 volume of the reaction solution.
PCR Buffer for Blend Taq 1 ×
Blend Taq (R) -Plus- 0.025 U / μL
Primer 0.2μM each
dNTPs 0.2 mM
(B) PCR cycle conditions PCR was performed under the following conditions.
94 ° C. 2 minutes 94 ° C. 1 minute-55 ° C. 30 seconds-72 ° C. 1 minute 30 seconds 37 cycles (c) Electrophoresis The obtained amplification product was mixed with 6 × Loading Dye (Toyobo) and agarose electrophoresis was performed. Was. The agarose gel used was 3%, the electrophoresis conditions were 100 V for 30 minutes, and the marker used was 100 bp DNA Ladder (Toyobo). The agarose gel was stained with ethidium bromide, and the presence and size of amplification products were confirmed by UV irradiation.
(2) Results The results of the electrophoresis are shown in FIG. Bands were confirmed at about 180 bp, about 280 bp, and about 400 bp. Samples with bands were subjected to sequence analysis. The results of sequence analysis of the bands of about 280 bp, about 400 bp, and about 180 bp are shown in FIGS. 5, 6, and 7, respectively. When homology search was performed for each sequence, S. aureus, S.A. epidermidis, S.P. capitis and high homology. From these results, it was confirmed that staphylococci contained in the samples could be identified and detected using the present invention.

According to the present invention, staphylococci are detected and at least S. aureus is detected. aureus, S.A. epidermidis, S.P. Thus, it was possible to provide a method for easily classifying C. captis and a composition therefor. By using the method and the composition, it is particularly useful for selection of a therapeutic agent for staphylococcal infection, prevention of spread of infection, prevention of food poisoning, etc., and not only for research use but also for clinical tests, environmental tests, food tests, etc. Can also be used.

Claims (9)

The following (1) and comprising the step of (2), S. in a sample aureus and S. aureus. A method for distinguishing and detecting two types of staphylococci of E. epidermidis .
(1) A step of obtaining an amplification product by performing nucleic acid amplification using one primer set consisting of a forward primer represented by the following (A 2 ) and a reverse primer represented by the following (B 2 ) :
(A 2 ) Oligonucleotide represented by base sequence represented by SEQ ID NO: 2 (B 2 ) Detection of amplification product obtained in step of oligonucleotide (2) (1) represented by base sequence represented by SEQ ID NO: 4 Process In a sample, the method comprises the following steps (1) and (2): aureus, S.A. epidermidis and S. A method for detecting three types of staphylococci of C. captis separately from each other.
(1) A step of obtaining an amplification product by performing nucleic acid amplification using one primer set including a forward primer represented by the following (A2) and a reverse primer represented by the following (B2):
(A2) an oligonucleotide represented by the base sequence represented by SEQ ID NO: 2
(B2) an oligonucleotide represented by the base sequence represented by SEQ ID NO: 4
(2) a step of detecting the amplification product obtained in the step (1) The method according to claim 1 or 2, wherein in step (2), amplification products are distinguished from each other by length differences and detected. The method according to claim 1 or 2, wherein the amplification products are detected in step (2) by distinguishing each other based on a difference in base sequence. A primer set comprising a forward primer represented by the following (A 2 ) and a reverse primer represented by the following (B 2 ).
(A 2) an oligonucleotide represented by the nucleotide sequence shown in SEQ ID NO: 2 (B 2) oligonucleotides represented by the nucleotide sequence shown in SEQ ID NO: 4 A sample containing the primer set according to claim 5 in a sample . aureus and S. aureus. A composition for detecting two staphylococci of E. epidermidis separately from each other. A sample containing the primer set according to claim 5 in a sample. aureus, S.A. epidermidis and S. A composition for detecting three types of staphylococci of C. captis separately from each other. A sample containing the primer set according to claim 5 in a sample . aureus and S. aureus. A kit for detecting two types of staphylococci of E. epidermidis separately from each other. A sample containing the primer set according to claim 5 in a sample. aureus, S.A. epidermidis and S. A kit for detecting three different types of staphylococci of C. captis.