JP2007300817A - Method for detecting bacteria, detection reagent and detection kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting bacteria in which highly pathogenic methicillin resistant Staphylococcus aureus or sensitive Staphylococcus aureus is simply and rapidly detected, a reagent for the detection and a kit for the detection. <P>SOLUTION: Panton-Valentine leukocidin or an adhesive factor separated from a biological sample is detected. Preferably, the detection of Panton-Valentine leukocidin or the adhesive factor separated in the biological sample is immunochemically carried out. The immunochemical method uses either one of a reversed passive latex agglutination method, an enzyme immunochemical method or an immunochromatography method. Preferably, the reversed passive latex agglutination method is used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a bacteria detection method, a detection reagent, and a detection kit. More specifically, the present invention relates to a serological rapid detection method, a detection reagent, and a detection kit for highly pathogenic methicillin-resistant Staphylococcus aureus or susceptible Staphylococcus aureus.

Staphylococcus aureus, a Gram-positive bacterium resident on the body surface of the human nasal cavity and skin, is usually harmless, but various epidermal infections and food poisoning such as human abscesses, pneumonia, meningitis, It is one of the causative agents of infectious diseases such as sepsis. The Staphylococcus aureus in terms of antimicrobial resistance, methicillin-resistant Staphylococcus aureus methicillin-resistant (Methicillin Resistant Staphylococcus aureus, hereinafter MRSA) and methicillin-sensitive Staphylococcus aureus conventional methicillin-sensitive (Methicillin Susceptible Staphylococcus aureus, hereinafter, MSSA).

  In particular, MRSA is resistant not only to methicillin but also to many drugs, and its appearance is based on the heavy use of antibiotics. MRSA is a major pathogenic bacterium of hospital infections that was raging from the 1980s to the 1990s. Even now, nosocomial infections due to MRSA and the like are serious, and the number of deaths is comparable to the number of deaths from AIDS, lung cancer, and traffic accidents in the United States. From 1997 to 1999, childhood deaths due to necrotizing pneumonia and sepsis occurred frequently in the United States, and community-acquired highly pathogenic MRSA was newly identified. In recent years, community-acquired highly pathogenic MRSA has been spreading worldwide, and hospital infections have been rapidly increasing, so countermeasures are being considered from various directions.

A feature of community-acquired highly pathogenic MRSA is that it produces Panton-Valentine Leukocidin (hereinafter referred to as PVL) and has strong adhesion to human tissues such as collagen.
PVL is a leukocyte-destructing toxin that acts specifically on leukocyte cells and requires two components, luk _PV S (S component) and luk _PV F (F component), for its activity expression. It is a toxin. The genes encoding these ( luk _PV SF ) form an operon. The S-type component is involved in target cell recognition, and monosialoganglioside GM1 on the leukocyte membrane is considered to be a receptor. Then, it binds to the target cell membrane together with the F-type component to form a complex, and forms a water-insoluble membrane pore through a hetero 7-quantum intermediate while causing a structural change. Further, phosphorylation of the S component by protein kinase A is important for the membrane pore, and cell disruption is established only when there is phosphorylation. PVL positive Staphylococcus aureus is thought to be associated with skin and soft tissue diseases in children and adolescents. At present, there are very few reports of PVL positive strains in Japan. Even when only skin / soft tissue diseases are examined, the positive rate is about 0.9%. However, in the United States, Europe, Australia, etc., PVL positive S. aureus is isolated with high frequency. The positive rate of PVL positive strains is high, followed by cellulitis, abscess, and hail cancer. Here, “settlement” refers to inflammation of pores (follicles) caused by bacterial infection (deep purulent infection). It refers to the name of the disease corresponding to acne and dexterity. In the case of a single pore, it becomes “set”, and in the case of two or more adjacent pores, it is called “yo;

  PVL is also associated with deep infections and has been isolated from osteomyelitis and community-acquired pneumonia. Many bacteria settle on host cells by adhering specifically or non-specifically to the host cell surface. In the case of specific adhesion, a bacterial cell surface layer component (adhesion factor) is involved. In the case of Staphylococcus aureus, there are cna (collagen binding protein) targeting collagen as an adhesion factor and bbp (bone sialoprotein binding protein) targeting bone sialoprotein. Strains possessing these adhesion factors are considered highly virulent strains because they can adhere and infect host cells efficiently.

As a rapid diagnostic method for such highly pathogenic factors, a DNA diagnostic method such as real-time PCR has been developed (Non-patent Document 1).
Saori Nakagawa, Ikue Taneike, et al., Gene sequences and specific detection for Panton-Valentine leukocidin., “Biochemical and Biophysical Research Communications” 2005; 328: p.995-1002.

  However, the method for amplifying and detecting DNA requires equipment such as a PCR device and a skilled engineer, and it takes time until results are obtained, so that there is a problem of lack of speed. Accordingly, there is a need for the development of a simple MRSA and MSSA highly pathogenic factor detection method that can be tested without the need for special equipment or equipment and without the need for skilled techniques.

  Therefore, the present invention creates PVL and adhesion factor antibodies that are highly pathogenic MRSA and MSSA virulence factors, and rapidly converts highly pathogenic MRSA and MSSA by an immunochemical method, particularly the reverse passive latex agglutination method. Development of a reagent for rapid detection of serology for detection, rapid detection method for serology, detection reagent and detection kit capable of detecting bacteria, particularly highly pathogenic MRSA and MSSA, easily and quickly The purpose is to provide.

  In order to solve the above-mentioned problems, the present inventors have intensively studied and developed a reverse passive latex agglutination method using an anti-PVL antibody or an anti-adhesion factor antibody. By enabling the measurement of factors, the present invention has been completed.

  That is, the method for detecting a bacterium according to claim 1 of the present invention is characterized in that the bacterium is detected by PVL or an adhesive factor separated from a biological sample.

  The method for detecting bacteria according to claim 2 detects bacteria by quantifying the production amount of PVL or adhesion factor separated from a biological sample and determining the virulence level of the bacteria.

  The method for detecting a bacterium according to claim 3 is the method according to claim 1 or 2, wherein the biological sample is human blood, pus or wiping liquid at an infected site, or tissue.

  The method for detecting a bacterium according to claim 4 is the bacterium according to any one of claims 1 to 3, wherein the bacterium produces Panton-Valentine leukocidin or has an adhesive factor.

  The method for detecting a bacterium according to claim 5 is the method according to any one of claims 1 to 4, wherein the bacterium is highly pathogenic methicillin-resistant Staphylococcus aureus or susceptible Staphylococcus aureus.

  The method for detecting bacteria according to claim 6 is the method according to any one of claims 1 to 5, wherein the detection of PVL or adhesion factor is performed by an immunochemical method.

  The method for detecting bacteria according to claim 7 is the method according to claim 6, wherein the immunochemical method is any one of a reverse passive latex agglutination method, an enzyme immunochemical method and an immunochromatography method.

  The method for detecting bacteria according to claim 8 is the method according to claim 7, wherein the immunochemical method is a reverse passive latex agglutination method.

  The reagent for detecting a bacterium according to claim 9 includes an anti-PVL antibody or an anti-adhesion factor antibody.

  The reagent for detecting a bacterium according to claim 10 includes a solid-phased anti-PVL antibody or a solid-phased anti-adhesion factor antibody and a labeled anti-PVL antibody or a labeled anti-adhesion factor antibody.

  The reagent for detecting a bacterium according to claim 11 is the reagent according to claim 9 or 10, wherein the antibody is a monoclonal antibody.

  The kit for detecting bacteria according to claim 12 is constituted by the reagent for detecting bacteria according to any one of claims 9 to 11.

  According to the bacteria detection method, detection reagent, and detection kit of the present invention, bacteria, particularly highly pathogenic MRSA and MSSA, can be easily and quickly detected by using antibodies against PVL and an adhesion factor. Therefore, all infectious disease inspection organizations and hospital laboratories all over the world or in Japan can use this detection kit to test a patient for highly pathogenic MRSA or MSSA infection and monitor infection and epidemic. . At the same time, the level of bacterial virulence can be determined by quantifying the production amount of pathogenic factors such as PVL and adhesive factors. Furthermore, due to the characteristics of the kit, the diagnostic results can be standardized and compared with the results of other facilities.

  The method for detecting a bacterium according to the present invention is characterized in that the bacterium is detected by PVL or an adhesive factor separated from a biological sample.

Moreover, bacteria can be detected by quantifying the production amount of PVL or adhesion factor separated from a biological sample and determining the level of bacterial virulence.
In the present invention, human blood, pus or wipes of infected sites, and tissues can be used as the biological sample. This operation has various methods such as blood collection with an injection needle and wiping the affected area with a sterile cotton swab. As appropriate, it can be carried out according to conventional methods.

The target bacterium in the method for detecting a bacterium of the present invention is a bacterium that produces Panton-Valtine leucocidin or a bacterium having an adhesion factor.
Examples of the bacterium include highly pathogenic methicillin-resistant Staphylococcus aureus or susceptible Staphylococcus aureus.

Although the detection method of PVL or an adhesion factor is not limited to a specific method, an immunochemical method is used suitably.
The immunochemical method is not limited to a specific method. For example, reverse passive latex agglutination method, enzyme immunochemical method, immunochromatography method, radioimmunoassay method, fluorescent immunoassay method, luminescence immunoassay method Spin immunoassay, turbidimetry, enzyme sensor electrode method, immunoelectrophoresis, Western blotting, and the like can be used. Among these, the reverse passive latex agglutination method, enzyme immunochemical method, or immunochromatography method is preferably used because of its high sensitivity.

The reverse passive latex agglutination (RPLA) is a method utilizing an agglutination reaction between antibody-sensitized latex particles and an antigen. The principle is that when a measurement sample containing an antigen is mixed with a latex particle sensitized with a polyclonal antibody or several types of monoclonal antibodies, the antibody on the particle reacts with the antigen, and the antibody on another particle detects another epitope on that antigen. Recognize and join. Then, the latex particles are bound to each other with the antigen in between. Since the antibodies are evenly bound to the surface of the particles, the latex particles are successively bound via the antigen to form large aggregates. When this reaction is allowed to stand overnight in a V-shaped bottom microplate, in the case of negative, latex particles roll as they are and collect on the V-shaped bottom. In the positive case (formation of agglomerates), the latex particles cannot roll on the slope, but settle and adhere to the entire slope on the V-shaped bottom. When the plate is viewed from above, negative is observed as a small spot where latex particles gather in the center of the well, and positive is observed as latex particles diffuse throughout the well bottom. Therefore, by using this method, by measuring the degree of aggregation of latex particles generated by the immune reaction between anti-PVL antibody-sensitized latex particles and PVL, or anti-adhesive factor antibody-sensitized latex particles and adhesive factor, PVL Alternatively, the adhesion factor concentration can be measured. Conventionally, there has been a passive hemagglutination method using red blood cells as antigens instead of using latex particles. However, since the reverse passive latex agglutination method is now common, the reverse passive latex agglutination method is sometimes referred to as the latex agglomeration method.
The enzyme immunochemical method may be a competitive method or a non-competitive method. Among them, the sandwich enzyme immunoassay method, which is a kind of non-competitive method, is more preferably used because it can measure the PVL or adhesive factor concentration with higher sensitivity and accuracy with a simple operation.

  Sandwich-type enzyme immunoassay sandwiches PVL between two types of antibodies that recognize different epitopes present in PVL or adhesion factor, ie, immobilized anti-PVL antibody and labeled anti-PVL antibody, or An adhesion factor is sandwiched between the immobilized anti-adhesion factor antibody and the labeled anti-adhesion factor antibody. And the PVL or adhesive factor density | concentration is measured by measuring the enzyme amount of the labeled antibody couple | bonded with PVL or the adhesive factor.

  The immunochromatography method uses a sheet-like carrier in which all immunochemical reaction systems are held. Since this method does not require a special measuring device, it is an advantageous method when quick determination is required for determination outside the hospital, emergency, and the like. Further, this method is suitable for qualitatively detecting the presence or absence of PVL or an adhesion factor that is an arbitrarily set concentration, that is, a cutoff value or more.

  In immunochromatography, when a specimen purified from a biological sample is dropped on a carrier, PVL or adhesive factor in the biological sample and an anti-PVL antibody or anti-adhesive factor antibody labeled with colloidal gold or the like undergo an immunoreaction, resulting in an immune complex. The body is formed. This complex develops on the carrier, is captured by an anti-PVL antibody or an anti-adhesive factor antibody that recognizes another epitope immobilized on a specific location on the carrier, and the label is accumulated. Then, the concentration of PVL or adhesive factor is measured by visually observing the degree of accumulation.

  In the method for detecting a bacterium of the present invention, the reverse passive latex agglutination method is particularly preferably used because it can be detected more easily and rapidly among immunochemical methods.

The reagent for detecting a bacterium according to the present invention comprises an anti-PVL antibody or an anti-adhesive that reacts specifically by recognizing PVL or an adhesion factor so that the bacterium detection method of the present invention can be carried out by an immunochemical method. Includes factor antibodies.
The antibody used in the present invention can be produced by a usual method. Polyclonal antibodies can be obtained by immunizing animals such as rabbits, mice, goats and horses with PVL or adhesive factors. Monoclonal antibodies can also be obtained by fusing spleen cells of animals immunized with such PVL or adhesion factor with myeloma, selecting hybrids by cloning, and culturing them.

  Furthermore, when the detection reagent of the present invention is based on a sandwich enzyme immunoassay method or immunochromatography method which is a kind of non-competitive method of enzyme immunochemical method, a solid-phased anti-PVL antibody or a solid-phased anti-adhesion factor antibody And labeled anti-PVL antibody or labeled anti-adhesion factor antibody. Reagents using enzyme immunochemical methods and immunochromatography methods are common in this field, and can be produced by applying the anti-PVL antibody or anti-adhesion factor antibody obtained as described above to these reagents. it can.

  In addition, the anti-PVL antibody or the anti-adhesion factor antibody may be a polyclonal antibody, but a monoclonal antibody is more preferably used because it has strict specificity and can stably supply a homogeneous antibody.

  The detection kit of the present invention is composed of the bacteria detection reagent as described above.

  Hereinafter, the present invention will be described in detail with specific examples, but the technical scope of the present invention is not limited by these specific examples. In the following examples, methods for detecting MRSA, MSSA, and Staphylococcus aureus were shown. However, any bacteria can be detected in the same manner as described below if it is a bacterium producing PVL or a bacterium having an adhesion factor. .

(Preparation method of sample for patient materials, isolated bacteria, etc.)
MRSA and MSSA, blood of patients suspected of S. aureus infection, pus and wipes of infected sites, tissues, etc. are collected. This operation has various methods such as blood collection with an injection needle and wiping the affected area with a sterile cotton swab. As appropriate, it can be carried out according to conventional methods.
The collected patient material is (I) liquid culture (Brain Heart Infusion (BHI) liquid medium; manufactured by Eiken Chemical Co., Ltd.) or (II) MRSA selective medium (oxacillin-added mannitol salt (MSO) agar medium; Nissui Pharmaceutical Co., Ltd.) Or overnight on a selective medium for Staphylococcus aureus (Mannit Salt Agar; Nissui Pharmaceutical Co., Ltd.) to isolate MRSA or Staphylococcus aureus. In the case of (I), the overnight culture solution is centrifuged (3,000 rpm, 20 minutes) and the centrifuged supernatant is used as a measurement sample. In the case of (II), after further culturing the suspected colony on the selective medium by liquid culture, the culture solution is adjusted in the same manner as in (I) to obtain a measurement sample.

A. Method of estimating antigen epitope from pathogenic gene sequence and producing antigen by binding to hapten (1) Determination of pathogenic gene sequence Sequencing is performed by Sanger method using LISA384 system (manufactured by Shimadzu Corporation). Determine the gene sequence.

(2) Method for estimating antigenic epitope from pathogenic gene sequence Antigen site analysis is performed by consulting software (manufactured by Sigma-Aldrich Japan).

  In the analysis procedure, first, an amino acid sequence of a virulence factor is derived from a virulence gene sequence, and a hydrophilic site is selected. Next, the turn structure is confirmed, and the portion existing on the protein molecule surface is estimated. Then, sites that may be modified, such as phosphorylation specific sequences and sugar chain addition specific sequences, are excluded as much as possible. Thereafter, a sequence that allows easy peptide synthesis is selected.

(3) Method for producing antigen by binding to hapten First, a peptide is synthesized so that a cysteine residue is added to the terminal of the putative antigen epitope sequence. Next, the cysteine residue is thiolated and a thiol group is added. Thereafter, Keyhole-limpet hemocyanin; KLH (hemocyanin) is used as a carrier protein, and Maleimidebenzoic acid-N-hydroxy succinimide; MBS is added and bound as a cross-linking agent.

(4) Immunization method of animals with the prepared antigen Preparation of antigen Place Freund's adjuvant in one of the glass syringes (Incomplete Freund's Adjuvant for the first time, Complete Freund's Adjuvant for the next time), put the antigen solution in the other syringe, connect both joints, and alternately Push the syringe to emulsify.

2. Antigen administration After placing the rabbit in the box, the back skin on the forearm side of the rabbit (Japanese white species, female, body weight 2.1-2.5 kg) is lifted and subcutaneously injected with a 21G syringe. The peptide is administered at 10 sites in the first 200 μg and 100 μg from the next time. Antigen administration is performed 6 times per week.

(5) Specific antibody acquisition method after antibody production Whole blood collection Using an indwelling needle from the rabbit ear, leave the sample overnight in the refrigerator, and separate the serum by centrifugation.

2. Purification with specific antigen-immobilized column AF-Tresyl Toyopearl 650 (manufactured by Tosoh Corporation) is used, and 0.5 M sodium chloride solution is added to 0.1 M carbonate buffer as an adsorption buffer, and pH 8.0 and Adjust to pH 8.5 and use. As the elution buffer, 0.1 M Gly-hydrochloric acid having a pH of 2.5 is used, and as the blocking buffer, 0.1 M Tris-hydrochloric acid and a 0.5 M sodium chloride solution are mixed and adjusted to pH 8.0 and used.

a. Operation of immobilizing specific antigen to gel Weigh 5 mg of peptide, dissolve in adsorption buffer, and quickly add the gel. Next, mix by inverting at room temperature overnight. The gel is then washed to remove unadsorbed peptide with 0.5M sodium chloride solution. Then, mix by inverting at room temperature for 1 hour in Blocking buffer to block.

b. Specific antibody purification method Serum is precipitated with 40% ammonium sulfate and dialyzed with PBS. Add dialyzed serum and adsorb for 1 hour at room temperature using a rotator. The gel is allowed to settle and washed with PBS to remove unadsorbed antibodies. 0.1M Gly-hydrochloric acid (pH 2.5) 1/2 column volume was layered carefully, and it was confirmed with a pH test paper that the solution dropped from the column was not acidic, and then 0.1 M Gly-hydrochloric acid (pH 2) 5) is layered 0.5 mL at a time to elute the antibody, and 0.5 ml of each fraction is fractionated on ice in a tube containing 50 uL of 1.0 M Tris-HCl (pH 8.0).

  Thereafter, each fraction is measured with a spectrophotometer (280 nm), and a fraction of 0.2 or more is collected and dialyzed while exchanging the buffer several times with PBS to make a specific antibody.

(6) Antigen detection method using this specific antibody (RPLA method)
The antibody is prepared to 5, 10, 20, 50 mg / ml and used. The latex particles are made of polyester and have a particle size of 0.77 μm and 0.5%.

a. Sensitization method The antibody and latex particles are mixed with antibody: 0.5% latex particles = 1: 1 and incubated at 37 ° C. for 1 hour. Next, incubate at 50 ° C. for 30 minutes. Then, 1/10 volume of 5% BSA-PBS buffer is added and left at 4 ° C. overnight. Thereafter, the supernatant is replaced with 0.5% BSA-PBS to obtain a sensitized latex.

b. Operation method As a positive control in a 96-well V-type plate, a known concentration of antigen and a culture supernatant of a PVL positive strain are each diluted 2-fold. Add and mix sensitized latex into all wells after dilution. After allowing to stand overnight, observe the wells that have aggregated, and observe how many stages of wells have aggregated. At that time, the amount of antigen in the sample stock solution can be quantified by comparing with the control. That is, the amount of antigen production of the pathogenic factor can be quantified, and the virulence level can be determined.

B. A method of synthesizing and purifying a protein with a His-tag added from a pathogenic gene sequence and using the obtained protein as an antigen (1) A method of synthesizing and purifying a protein with a His-tag added from a pathogenic gene sequence 1 . Amplification of PVL-S and PVL-F genes by PCR method A sense primer and an antisense primer to which restriction enzyme sites not present in the target gene for PVL-S and PVL-F gene amplification are added are designed and synthesized. Then, PCR is performed using DNA extracted from PVL-positive Staphylococcus aureus, synthesized primers, and Pfu-turbo polymerase. Thereafter, the size and yield of the PCR product are confirmed by agarose electrophoresis, and the product is purified with a QIA quick PCR purification kit (Qiagen).

2. Cloning of PCR product The vector is digested with a restriction enzyme that recognizes the sequence added to the primer, and purified by QiA quick gel extraction kit (Qiagen). The PCR product is digested with a restriction enzyme in the same manner as in the vector and purified with a QIA quick PCR purification kit (Qiagen). Then, ligation is performed using Takara ligation kit (manufactured by Takara Bio Inc.). The ligation sol is transformed into competent cells (XL1blue). Thereafter, competent cells are cultured on an agar plate, colonies are picked up, and plasmid DNA is purified by QIAprep SpinMiniprep kit (Qiagen).

3. Confirmation of insert DNA It is confirmed by electrophoresis whether or not the insert plasmid is excised at the target gene size by digestion with purified plasmid DNA. For clones that have been confirmed to be excised, sequence analysis (only both ends) is performed on a maximum of 3 clones.

4). Plasmid DNA whose gene for expression confirmation is confirmed by sequencing is transformed into the expression host cell BL21 (DE3) RIL, IPTG (−, +) is induced on a small scale (4 ml culture), and NiTG is used in native & denaturation conditions. -After purification of NTA batch, it is confirmed by SDS-PAGE whether it is an expression soluble fraction.

5). Large scale culture & purification Incubate with 2L, induce expression, lyse, collect the supernatant, adsorb to Ni-NTA chromatography, and elute the protein of interest with an imidazole gradient. Then, a fraction containing a large amount of the target protein and a small amount of non-specifically adsorbed protein is collected by SDS-PAGE, and after dialysis, this is defined as a protein with His-tag added.

(2) Method for producing an antibody using this protein as an antigen The same procedure as in A- (4) and (5) is performed.

(3) Antigen detection method using this specific antibody (RPLA method)
Perform in the same manner as A- (6).

C.
(1) Method for producing monoclonal antibody against the above antigen Since the A antigen is a peptide, the antibody itself is a monoclonal antibody. Regarding B, since the whole protein of each of PVL-S and PVL-F is used as an antibody, several kinds of epitopes are contained, and an antibody produced therefrom is a polyclonal antibody. In the case of producing a monoclonal antibody from the B antigen, only an antibody against a certain epitope can be purified by absorbing an antibody against a certain epitope other than the B antigen polyclonal antibody.

(2) Antigen detection method using this monoclonal antibody (RPLA method)
The same as A- (6).

Claims (12)

A method for detecting a bacterium, wherein the bacterium is detected by Panton-Valtine leucocidin or an adhesive factor separated from a biological sample. A method for detecting bacteria comprising quantifying the production of Panton-Valtine leucocidin or an adhesion factor separated from a biological sample, and determining the virulence level of the bacteria to detect the bacteria. The method for detecting a bacterium according to claim 1 or 2, wherein the biological sample is human blood, pus or wipes of an infected site, or tissue. The bacterium detection method according to any one of claims 1 to 3, wherein the bacterium is a bacterium that produces Panton-Valtine leucocidin or a bacterium having an adhesion factor. The method for detecting a bacterium according to any one of claims 1 to 4, wherein the bacterium is highly pathogenic methicillin-resistant Staphylococcus aureus or susceptible S. aureus. The method for detecting a bacterium according to any one of claims 1 to 5, wherein the detection of Panton-Valentine leukocidin or an adhesion factor is performed by an immunochemical method. The method for detecting a bacterium according to claim 6, wherein the immunochemical method is one of a reverse passive latex agglutination method, an enzyme immunochemical method and an immunochromatography method. The method for detecting bacteria according to claim 7, wherein the immunochemical method is a reverse passive latex agglutination method. A reagent for detection of bacteria comprising an anti-Panthon-Valentine leukocidin antibody or an anti-adhesion factor antibody. A reagent for detection of bacteria comprising a solid-phased anti-Panton-Valentine leucocidin antibody or a solid-phased anti-adhesion factor antibody and a labeled anti-Panton-Valtine leucosidine antibody or labeled anti-adhesive factor antibody. The reagent for detecting bacteria according to claim 9 or 10, wherein the antibody is a monoclonal antibody. A kit for detecting bacteria comprising the reagent for detecting bacteria according to any one of claims 9 to 11.