KR20160126652A - Method for highly sensitive immunosorbent assay based on modified phage infectivity readout - Google Patents

Abstract

The present invention relates to a high-sensitivity immunoabsorption assay method, and more particularly, to a method for assaying a high-sensitivity immunoabsorption assay, comprising the steps of: attaching an antibody capable of performing an antigen-antibody reaction with an antigen to be detected on a surface of a support; Performing an antigen-antibody reaction between the antibody and the antigen to be detected by adding an antigen to be detected to the surface; Preparing a bacteriophage transplanted with a gene encoding a protein capable of binding to the antibody; Applying the bacteriophage to the surface to which the antibody is attached; Infecting the bacterium with the bacteriophage by applying the bacteria to the surface; Culturing the bacteriophage-infected bacterium; And detecting the antigen to be detected by detecting the infected bacteria.
According to the present invention, it is possible to provide a high-sensitivity immunoabsorption analysis method capable of detecting a target substance present in a low concentration at a low cost with a high sensitivity, and performing it by a simple and easy method. The method according to the present invention can be widely employed in clinical and medical analysis fields including disease diagnosis, food and environmental fields requiring pollutant detection, and other industrial chemistry fields.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a highly sensitive immunosorbent assay based on modified phage infectivity readings,

The present invention relates to a high-sensitivity immunoabsorbent assay method for replacing the conventional enzyme-linked immunosorbent assay (ELISA), wherein the infectivity using the modified phage is read, and sample analysis is performed using the same Sensitive immunoabsorption assay.

Conventionally, techniques for detecting and identifying various proteins through enzymatic analysis have been extensively developed. Of these technologies, enzyme-linked immunosorbent assay (ELISA) is an analytical technique for trace samples using an antigen-antibody reaction, and is being used as a representative analytical technique. For example, the ELISA method can be used to analyze various viruses such as human immunodeficiency virus (HIV) and West Nile virus by detecting the serum antibody concentration of a sample, to be used for pregnancy test, It is also used in the food industry for the detection and quality control of the presence of the same allergen.

Conventional ELISA methods generally require three steps: first, the primary antibody or antigen is attached to a microtiter plate, a microcentrifuge tube, or other solid support, and the second An antigen or a primary antibody is added to perform an antigen-antibody reaction, and then a second secondary antibody is added to perform a secondary antigen-antibody reaction with the antigen of the antigen-antibody complex. In general, the secondary antibody added is conjugated to a signal generating enzyme such as horseradish peroxidase (HRP), which is a 3,3 ', 5,5'-tetramethylbenzidine (3, 3 ', 5,5'-Tetramethylbenzidine, TMB).

However, these conventional conventional ELISA methods have severe disadvantages. First, there is a problem in that a high effort, time, and cost are required to prepare the primary and secondary antibodies necessary for performing the antigen-antibody reaction. Next, since the color change observed in the conventional ELISA assay method can not be stably maintained for a long time, a colorimetric readout must be performed immediately. Finally, conventional ELISA methods generate very weak signals when small amounts of antigen are used, which means that the sensitivity of ELISA assays is very low when detecting low concentrations of substrate.

In recent years, several studies have been reported to improve the problems of the conventional ELISA method. For example, researchers at the University of Colorado, such as Michael Brasino, have reported a highly amplified ELISA method using genetically engineered bacteriophages to improve the sensitivity of conventional ELISA methods (Michael Brasino et al., Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage, Analytical Biochemistry 470 (2015) 7-13). In this method, a double-modified version of a filamentous bacteriophage Fd is used which can generate a significantly improved color signal as compared with the case of using an antibody alone.

FIG. 1 shows a schematic process flow chart for an ELISA method using the processed bacteriophage. Referring to FIG. 1, an antibody is immobilized on a surface of a plate or the like, and a genetically modified phage is bound to the antibody. At this time, the phage is genetically modified to bind to the Fc region of the detection antibody, so that the phage can bind to a specific antigen with higher specificity than single peptide sequences. Next, the secondary antibody is bound to the viral coat protein, which is conjugated to a signal generating enzyme such as HRP. Finally, when the substrate of the signal generating enzyme such as TMB is added, the color reaction is performed by the HRP conjugated to the secondary antibody, and the degree of the color reaction is quantitatively measured using a colorimetric system. According to the description of the prior art, it has been disclosed that the ELISA method using the bacteriophage thus processed improves detection sensitivity by 3-4 times as compared with the conventional ELISA method using an antibody alone.

However, even in the case of the above-mentioned prior arts, it can be judged that the effect has been obtained to some extent in terms of improvement in the detection sensitivity. However, in addition to the production of primary and secondary antibodies, expensive chromogenic substrates such as TMB And it is impossible to maintain a stable color change for a long time, so that a colorimetric reading must be immediately carried out. Thus, the conventional ELISA method has problems.

Accordingly, it is an object of the present invention to solve the above-described problems of the prior art and to provide an ELISA assay capable of performing ELISA analysis at a remarkably low cost, and in some cases, a colorimetric reading process can be omitted, A high sensitivity immunoabsorption analysis method capable of detecting a high sensitivity.

In order to solve the above problems,

Attaching an antibody capable of performing an antigen-antibody reaction with an antigen to be detected on a surface of a support;

Performing an antigen-antibody reaction between the antibody and the antigen to be detected by adding an antigen to be detected to the surface;

Preparing a bacteriophage transplanted with a gene encoding a protein capable of binding to the antibody;

Applying the bacteriophage to the surface to which the antibody is attached;

Infecting the bacterium with the bacteriophage by applying the bacteria to the surface;

Culturing the bacteriophage-infected bacterium; And

And detecting the antigen to be detected by detecting the infected bacteria.

According to one embodiment of the present invention, the support may be a microtiter plate or a microcentrifuge tube.

According to another embodiment of the present invention, the antibody is selected from the group consisting of an immunoglobulin G antibody (IgG antibody), an immunoglobulin E antibody (IgE antibody), an immunoglobulin A antibody (IgA antibody) and an immunoglobulin M antibody ≪ / RTI > Or antigen binding fragments (Fab '), antigen binding dimers (F (ab') 2), single chain variable fragment (scFv) and single chain variable dimers (di-scFv), single domain antibodies (sdAb) and affibody ). ≪ / RTI >

According to another embodiment of the present invention, the protein capable of binding to the antibody may be a protein capable of specifically binding to an antibody or a region other than the complementarity determining region of the antibody fragment.

According to another embodiment of the present invention, the protein capable of binding to the antibody is selected from the Z domain of Staphylococcus aureus protein A, protein G or proteins A and G binding to the Fc domain of the antibody or antibody fragment, Lt; / RTI >

According to another embodiment of the present invention, the step of culturing the bacteria infected with the bacteriophage can be carried out at a temperature of 30 ° C to 37 ° C for 12 hours to 16 hours.

According to another embodiment of the present invention, the bacteriophage is a filamentous bacteriophage, and may be at least one bacteriophage selected from the group consisting of f1 phage, fd phage and M13 phage.

According to another embodiment of the present invention, the bacterium is a gram negative bacterium, and may be a F-pilus and a bacteria expressing the TolA protein.

According to another embodiment of the present invention, the bacteria are selected from the group consisting of Escherichia, Salmonella, Pseudomonas, Xanthomonas, Vibrio, Thermus, (Neisseria). ≪ / RTI >

According to another embodiment of the present invention, the step of detecting the infected bacteria comprises:

Washing the culture resultant to remove uninfected bacteria;

Diluting the washed resultant with a predetermined magnification;

Inoculating and culturing the resultant dilution in a second culture medium; And

And counting the bacterial community formed by the culture.

According to another embodiment of the present invention, the method may further comprise the step of transplanting a gene conferring antibiotic resistance to the bacteriophage and removing the uninfected bacteria by adding the antibiotic to the second culture medium .

According to another embodiment of the invention, the antibiotic is selected from the group consisting of tetracycline, ampicillin, kanamycin, gentamicin, chloramphenicol, neomycin, hygromycin, and triclosan May be one or more antibiotics.

According to the present invention, it is possible to provide a high-sensitivity immunoabsorption analysis method capable of detecting a target substance present in a low concentration at a low cost with a high sensitivity, and performing it by a simple and easy method. The method according to the present invention can be widely employed in clinical and medical analysis fields including disease diagnosis, food and environmental fields requiring pollutant detection, and other industrial chemistry fields.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic flow diagram of an ELISA method using a bacteriophage according to the prior art.
Figure 2 is a schematic flow diagram of a method for high sensitivity immunoabsorption analysis based on modified phage infectivity readings according to the present invention.
Fig. 3 is a schematic diagram of a bacteriophage into which a gene that produces protein A is introduced.
Fig. 4 is a diagram showing an amino acid sequence of P3 envelope protein of virus processed according to the present invention. Fig.
Figures 5A and 5B are graphs showing the results of a test comprising a sample containing 0.1 mu g of mouse monoclonal IgG biotin bound to an antibody and a sample containing no mouse monoclonal IgG biotin as a control group, .
6A and 6B are graphs showing the obtained primary data (OD450 and the number of viruses) in the phage ELISA 6a and the present invention 6b, respectively.
Figure 7 graphically shows the relative signal intensities for the target protein ([mu] g) in the ELISA, phage ELISA and methods of the present invention.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may appropriately define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention. Therefore, the embodiments described in the present specification and the constitutions described in the drawings are merely the most preferred embodiments, and not all of the technical ideas of the present invention are described. Therefore, various equivalents which can be substituted at the time of the present application It should be understood that variations can be made.

Hereinafter, the present invention will be described in detail with reference to the drawings and examples.

As described above, the present inventors have proposed the present invention to solve the problems of the conventional ELISA method, and accordingly, in the present invention,

Attaching an antibody capable of performing an antigen-antibody reaction with an antigen to be detected on a surface of a support;

Performing an antigen-antibody reaction between the antibody and the antigen to be detected by adding an antigen to be detected to the surface;

Preparing a bacteriophage transplanted with a gene encoding a protein capable of binding to the antibody;

Applying the bacteriophage to the surface to which the antibody is attached;

Infecting the bacterium with the bacteriophage by applying the bacteria to the surface;

Culturing the bacteriophage-infected bacterium; And

And detecting the antigen to be detected by detecting the infected bacteria.

FIG. 2 shows a schematic process diagram of the immunoabsorption analysis method according to the present invention. Referring to FIG. 2, an antibody capable of performing an antigen-antibody reaction with the antigen to be detected is first attached on the surface of the supporter. As such a support on which the antibody is adhered, various supports commonly used in conventional ELISA methods can be used, and a support such as, but not limited to, a microtiter plate or a microcentrifuge tube can be used. In the next step, the antibody attached to the support is bound to the detection target antigen in the sample by an antigen-antibody reaction when the sample containing the antigen to be detected is added.

In the present invention, in addition to the above process, a gene encoding a protein capable of binding to an antibody attached to the surface of the support is introduced into the DNA of the bacteriophage using DNA recombination technology. This new bacteriophage incorporates a new protein, which, unlike the natural bacteriophage, produces a new protein by the expression of the newly introduced gene and allows the newly produced protein to bind to the antibody attached to the surface of the support.

For example, FIG. 3 shows a schematic view of a bacteriophage into which a gene that produces protein A is introduced as an example of such a protein. Referring to FIG. 3, the genetically modified bacteriophage further includes Protein A is further retained. Protein A is well known to bind strongly to the Z domain of immunoglobulin G (IgG) derived from a variety of species such as human, mouse, rat, goat, rabbit and the like and thus attach IgG as an antibody to the surface of the support, When a bacteriophage genetically modified to express A is applied to the surface of the support, a strong bond is formed between the antibody IgG and protein A.

On the other hand, in the above description, although IgG as an antibody and protein A as a binding protein have been exemplified, it is possible to adopt various antibody-protein pairs in addition to the above pair, if specific binding between an antibody and a specific protein is possible. Thus, any monoclonal antibody or antibody fragment may be used, including, but not limited to, an immunoglobulin G antibody (IgG antibody), an immunoglobulin E antibody (IgE antibody), an immunoglobulin A antibody At least one antibody selected from the group consisting of immunoglobulin M antibodies (IgM antibodies); Or antigen binding fragments (Fab '), antigen binding dimers (F (ab') 2), single chain variable fragment (scFv) and single chain variable dimers (di-scFv), single domain antibodies (sdAb) and affibody ). ≪ / RTI >

In addition, the protein capable of binding to the antibody may be any protein capable of specifically binding to an antibody or a region other than the complementary determining region of the antibody fragment, and includes, but is not limited to, Fc Staphylococcal protein A binding to the domain, the Z domain of protein G or proteins A and G, or a peptide or protein that performs a similar function.

As described above, in the present invention, a modified virus that can be specifically targeted to a specific antigen, antibody or protein is used in place of the secondary antibody conjugated with an enzyme such as HRP used in the conventional ELISA method, and The modified viruses may replace the functions performed by the conventional secondary ELISA antibody. That is, the modified virus can specifically bind to the primary antibody, which enables a detection method based on virus infection, replacing the conventional detection method of detecting the presence of a specific protein and an antigen. Modified viruses can be produced in large quantities by inexpensive and simple synthetic methods, so that they are more economical than conventional methods using expensive antibodies, and are also useful for detecting proteins and peptides present in the envelope proteins of viruses in order to detect antigens or specific target substances Can be variously modified or newly designed, so that it can be applied to various fields.

1 and FIG. 2, it can be understood that the processes described above are substantially similar. Therefore, there are considerable similarities to the method of ELISA using bacteriophage by the researchers of the present invention and the inventors of the University of Colorado, such as Michael Brasino, until the process of "immobilization of the antibody on the support → antigen-antibody reaction → addition of the genetically modified bacteriophage". However, the conventional bacteriophage ELISA method involves a secondary antibody addition to the signal generating enzyme, a substrate addition, a coloring reaction by a signal generating enzyme, and the like in a subsequent step. In contrast, in the present invention, Instead of adding an antibody or a substrate, bacteria that are infected by the bacteriophage are added.

In the present invention, the bacterium should be a type of bacteria capable of transferring DNA into a cell with bacteriophage attached thereto, and thus any gram-negative bacteria such as F-pilus and bacteria expressing the TolA protein Can be used. Thus, the bacteriophage that infects the bacteria includes, but is not limited to, filamentous bacteriophage, but one or more bacteriophages selected from the group consisting of f1 phage, fd phage and M13 phage can be used, Specific examples of bacteria that may be present include Escherichia, Salmonella, Pseudomonas, Xanthomonas, Vibrio, Thermus and Neisseria. At least one bacterium selected from the group consisting of:

The bacteria once infected by the bacteriophage should be cultured under a predetermined culture condition, and preferably at a temperature of 30 to 37 DEG C for 12 to 16 hours. The culture conditions are adjusted to conditions that facilitate the detection of the bacteria at a later stage, while being suitable for propagation of the bacteria.

In the final stage of the present invention, only the infected bacteria are selectively detected as described above. As a result, the present inventors have found that a single bacteriophage infects a single bacterium when the bacteria to be infected are not excessively added The number of bacteria detected corresponds to the number of bacteriophages correctly bound to the antibody and further the number of antibodies corresponds to the number of antigens to be detected so that the number of cultured bacteria By quantification, it becomes possible to quantify an antigen to be detected.

The step of detecting the infected bacteria can be performed by counting colonies formed by culturing the infected bacteria.

Thus, the step of detecting the infected bacteria comprises:

Washing the culture resultant to remove uninfected bacteria;

Diluting the washed resultant with a predetermined magnification;

Inoculating and culturing the resultant dilution in a second culture medium; And

And counting the bacterial community formed by the culture.

In this method, first, a bacterium that has not been infected by a bacteriophage, that is, a bacterium (bacteriophage) not bound to an antibody attached to a surface thereof is removed through a process such as washing and the resultant is diluted to a predetermined magnification, Inoculation and culture. As shown in FIG. 2, when the population was diluted to 1/10 in the population counting step, if the population was too dense to be counted, it was diluted to a concentration of 1/100 to facilitate the population count I can do it. The degree of dilution can be adjusted to such an extent as to facilitate the counting of the population, and when measuring the concentration of the antigen to be detected finally, the count value measured by the naked eye can be multiplied by a dilution factor.

On the other hand, as another modified example, when a gene that confers antibiotic resistance to the bacteriophage is introduced, the bacterium infected with the bacteriophage possesses the corresponding antibiotic resistance, so that the bacteria are cultured in a second culture medium containing the antibiotic , Only bacteria that are infected with bacteriophage can be easily counted. In this case, bacteria not infected with the bacteriophage, that is, bacteria not associated with the antibody attached to the support, are selectively killed by the antibiotic contained in the second culture medium. Antibiotics that can be used to carry out this method may be selected variously depending on the species of bacteria selected, including but not limited to tetracycline, ampicillin, kanamycin, gentamicin, chloramphenicol, , Neomycin, hygromycin, and triclosan can be used.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to assist the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Example

PCR  And recombination DNA ≪ / RTI >

The pCanta-b6 plasmid was used as the vector template and the used insit was the Z domain dimer of the protein A coding region in the PFA6A-PA-GFP-HIS3MX6 plasmid. The pCanta-b6 vector was prepared by introducing PstI and NotI restriction sites into the P3 coding region by PCR. The PCR conditions were as follows:

25 cycles

Denaturation at 92 [deg.] C (50 sec)

Annealing at 56 ° C (20 seconds)

Extended at 72 ° C (10 min).

DpnI was introduced into the PCR product and the original template was removed by incubation at 37 DEG C for 2 hours. PZ6A-PA-GFP-HIS3MX6 was used as a template to prepare a protein A ZZ domain inducer by PCR, and PstI and NotI restriction enzyme sites were introduced at one end of the protein A coding region. The PCR conditions were as follows:

20 cycles

Denaturation at 92 ° C (20 sec)

Annealing at 56 < 0 > C (15 sec)

Extension at 72 ° C (2 min).

The PCR product was treated with DpnI at 37 < 0 > C for 2 hours to remove the template. The vector and inshut were then cut into PstI and NotI at 37 DEG C for 2 hours, respectively. The vector and insights were purified by gel extraction. The protein A ZZ domain coding region in situ was ligated into the P3 coding region of pCanta-b6 and named ProA pCanta-b6. The ProA pCantab-6 plasmid was transformed into TG1 competent cells and plated on ampicillin LB agar plates. ProA pCantab-6 was grown in LB broth and the plasmid was packaged into phages using helper phage M13K07.

pCanta - b6  Modified plasmids p3  Into the region of protein A ZZ  Verify that an encryption zone has been inserted for the domain

 From the ampicillin plate, the community was diluted in water. In order to analyze the modified P3-encoding region, PCR was performed on the sample using a sequencing primer. As a result of sequencing, the results shown in FIG. 4 were obtained.

Analysis by visual cluster coefficient

0.1 μg of mouse monoclonal IgG biotin conjugate antibody (PROGEN biotechnik, 100 μg / mL) was diluted in 200 μl of 1 × phosphate buffered saline and then added into 96-well plates. The antibody was immobilized on the surface of the plate for 2 hours at room temperature. The solution containing the antibody was removed and 300 [mu] l of blocking buffer (0.02 g milk powder dissolved in phosphate buffered saline in 1 mL with Tween 20) was added into each well of the plate. The plate was stored at room temperature for 2 hours. Blocking buffer solutions were used to block the exposed polystyrene regions on the polystyrene plate to prevent nonspecific binding of the modified virus to the plate itself due to the absence of antibodies in these regions. Each well was washed three times with blocking buffer (0.01 g milk powder dissolved in phosphate buffered saline in 1 mL with Tween 20). Then 100 쨉 l of modified phage was added to each well and the plate was stored at room temperature for 10 minutes to 30 minutes. After each well was washed five times with blocking buffer, 100 [mu] l E. coli (TG1) culture was added and cultured at 37 [deg.] C for 1 hour for phage infection. The culture was made up to 1:10 ⁵ by continuous dilution (1:10) in LB medium and plated on selective (ampicillin) agar plates. The plates were incubated overnight at 37 ° C. The following day, platelet populations were counted, and the number of these populations represents the number of combined viruses

Figures 5a and 5b are graphs showing the results of experiments on a sample containing 0.1 占 퐂 of mouse monoclonal IgG biotin bound to an antibody according to the above method and a sample containing no mouse monoclonal IgG biotin as a control. The left side plate of the drawings depicts the result of a pure sample, the sample was diluted 1/10, 1/10 ^second diluted sample from each of the left and right plates ³ 1/10 diluted sample, the sample was diluted 1/10 ^4, 1/10 ⁵ dilution sample. Referring to Figure 5a and 5b, and the antibody bound sample containing a mouse monoclonal IgG Biotin 0.1 ㎍ is opposed to the colonies formed in 1/10 ⁵ diluted samples, control samples are formed at all the communities in the 1/10 dilution sample ³ .

Comparative analysis with conventional methods

In order to compare the results of the conventional ELISA and phage ELISA with the method according to the present invention, a detection experiment was carried out using glucose oxidase as a target protein. The method according to the present invention was carried out by ELISA and phage ELISA (Michael Brasino et al., &Quot; Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage, Analytical Biochemistry 470 (2015) 7-13).

Table 1 summarizes the differences in composition of the three experiments.

variable ELISA Phage ELISA Invention Deformed phage X O O Antibodies conjugated with HRP O O X TMB substrate O O X E. coli TG1 X X O Colorimetric reading O O X Phage infectivity reading X X O

6A and 6B show graphs of the obtained primary data (OD450 and number of viruses) in the phage ELISA (6a) and the present invention (6b), respectively, and FIG. 7 shows the target proteins ≪ / RTI > < RTI ID = 0.0 > g) < / RTI > Relative signal intensities were calculated by the following formula 1 for ELISA and phage ELISA, and by equation 2 for the present invention:

[Formula 1]

Relative signal intensity = ELISA signal intensity of sample / sound ELISA signal intensity of control (background signal)

[Formula 2]

Relative signal intensity = phage infectivity (ie, number of colonies) of the sample / phage infectivity of the control group (background signal)

Referring to FIG. 7, it can be seen that the method according to the present invention shows significantly superior relative signal intensity as compared to conventional ELISA or phage ELISA, and thus the present invention can analyze a sample with excellent sensitivity .

Claims (12)

Attaching an antibody capable of performing an antigen-antibody reaction with an antigen to be detected on a surface of a support;
Performing an antigen-antibody reaction between the antibody and the antigen to be detected by adding an antigen to be detected to the surface;
Preparing a bacteriophage transplanted with a gene encoding a protein capable of binding to the antibody;
Applying the bacteriophage to the surface to which the antibody is attached;
Infecting the bacterium with the bacteriophage by applying the bacteria to the surface;
Culturing the bacteriophage-infected bacterium; And
And detecting the antigen to be detected by detecting the infected bacteria. The method of claim 1, wherein the support is a microtiter plate or a microcentrifuge tube. The antibody of claim 1, wherein the antibody is selected from the group consisting of an immunoglobulin G antibody (IgG antibody), an immunoglobulin E antibody (IgE antibody), an immunoglobulin A antibody (IgA antibody) and an immunoglobulin M antibody Antibody; Or antigen binding fragments (Fab '), antigen binding dimers (F (ab') 2), single chain variable fragment (scFv) and single chain variable dimers (di-scFv), single domain antibodies (sdAb) and affibody Wherein the antibody fragment is an antibody fragment selected from the group consisting of: 2. The immunoabsorbing assay method according to claim 1, wherein the protein capable of binding to the antibody is a protein capable of specifically binding to a region other than the complementarity determining region of the antibody or the antibody fragment. The protein according to claim 4, wherein the protein capable of binding to the antibody is the Z domain of Staphylococcus aureus protein A, protein G or proteins A and G binding to the Fc domain of the antibody or antibody fragment, or a functional analog thereof Lt; / RTI > The method of claim 1, wherein culturing the bacteria infected with the bacteriophage is carried out at a temperature of 30 ° C to 37 ° C for 12 hours to 16 hours. The method of claim 1, wherein the bacteriophage is filamentous bacteriophage, and the bacteriophage is at least one bacteriophage selected from the group consisting of f1 phage, fd phage, and M13 phage. 2. The method of claim 1, wherein the bacteria is gram-negative bacteria and is a bacteria expressing F-kills (F-pilus) and TolA protein. The method of claim 1, wherein the bacteria is selected from the group consisting of Escherichia, Salmonella, Pseudomonas, Xanthomonas, Vibrio, Thermus and Neisseria. ≪ RTI ID = 0.0 > 1, < / RTI > The method of claim 1, wherein detecting the infected bacteria comprises:
Washing the culture resultant to remove uninfected bacteria;
Diluting the washed resultant with a predetermined magnification;
Inoculating and culturing the resultant dilution in a second culture medium; And
And counting the bacterial community formed by said culturing. 2. The method of claim 1, further comprising the step of transplanting the gene that confers antibiotic resistance to the bacteriophage and then removing the uninfected bacteria by adding the antibiotic to the second culture medium Analysis method. 12. The method of claim 11, wherein the antibiotic is one or more antibiotics selected from the group consisting of tetracycline, ampicillin, kanamycin, gentamicin, chloramphenicol, neomycin, hygromycin, and triclosan ≪ / RTI >

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