JP2002165599A - Method for detecting viable microbial cell of escherichia coli and detecting kit used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detection by which a viable microbial cell of Escherichia coli in a test liquid can simply and rapidly be detected with a high accuracy and high sensitivity and a detecting kit usable therefor. SOLUTION: This method for detecting the viable microbial cell of Escherichia coli is characterized by having at least a step of developing the test liquid on a detecting tool in which a stationary phase fixing a specific binding component specifically bindable to the Escherichia coli is formed in an optional region on the surface of a water absorbent base material and a step of developing a substrate liquid containing a color developing substrate component capable of specifically binding to an alkaline phosphatase and developing a color on the water absorbent substrate after developing the test liquid. The kit is used for the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting viable Escherichia coli cells which can detect live Escherichia coli cells in a test solution simply, quickly, with high precision and high sensitivity, and a detection kit usable therefor. About.

[0002]

2. Description of the Related Art In recent years, infectious diseases caused by pathogenic Escherichia coli have become a problem. Among them, O157 mainly enters the body from foods that become the source of infection and develops after a latency period of about 4 to 9 days, and symptoms such as hemolytic anemia, renal failure, and thrombocytopenia are produced by the action of produced verotoxin toxin. , Which can lead to haemolytic uremic syndrome (HUS), which in some cases can lead to serious situations, which is a major cause of social anxiety.

[0003] Escherichia coli, including such pathogenic ones, is regarded as a pollution indicator bacterium for foods and the like. That is, the presence or absence of Escherichia coli in food is a measure for environmental health management necessary for producing and securing high-quality food with high safety. E. coli is a member of the coliform group, for example, Citrob.
acter freundii, Klebsiella
This is because, compared to other microorganisms such as aerogenes, it is more likely to be present in feces of humans and animals, and is more likely to die in nature. From the food in which live E. coli cells are present, it can be inferred that the food was caused by relatively new contamination, and that the food was treated more filthy than when other microorganisms belonging to the E. coli group existed.

[0004] Various methods are conventionally known as a method for detecting Escherichia coli from a test solution prepared from a sample such as food or feces. For example, a method is known in which a test solution is cultured in a medium for coliform bacteria, and a multi-item test is selectively performed on the medium to determine the grown bacteria as Escherichia coli. The tests include, for example, a test for detecting lactose-decomposed gas, an Invic (IMViC) test (indole production, methyl red reaction, Forges-Proscauer reaction, utilization of citric acid), and observation of the color of colonies.

[0005] A method is also known which utilizes β-glucuronidase, which is an enzyme specifically found in Escherichia coli, Shigella, and Salmonella out of 100 types of intestinal bacterial species. Since β-glucuronidase is found in 95% or more of Escherichia coli strains, detection of the presence or absence of β-glucuronidase in the above medium is an indicator of the presence or absence of Escherichia coli in the sample. β-glucuronidase is available from Feng a
nd Hartman, Appl. Enriron. M
microbiol. , 43, 1320, 1982, etc., for example, 4-methyl
umbelliferyl-β-D-glucroni
Detection can be performed using a medium for coliform bacteria to which de is added.

Further, as a method utilizing an immunochemical reaction, there is known a method of aggregating and detecting Escherichia coli using an antibody which specifically binds Escherichia coli as an antigen.
Above all, immunochromatography has attracted attention because it allows quick and simple high-sensitivity tests. The immunochromatography method involves, for example, the following steps. The test solution is developed on a water-absorbing substrate having a stationary phase on which a first antibody capable of specifically binding to Escherichia coli is immobilized,
Capturing E. coli with the first antibody. Labeled immunity (second antibody-
(Development of a labeling agent) and binding to E. coli captured on the stationary phase. A step of visually confirming whether or not an immune complex (first antibody-E. Coli-second antibody-labeling agent) is formed on the stationary phase.

In the detection of Escherichia coli using the immunochromatography method, for example, an anti-Escherichia coli antibody is conventionally used as the first antibody and the second antibody. As the labeling agent, conventionally known colloidal gold, colored latex, silicone, silica and the like are used.

[0008]

However, the method of performing a multi-item test on the above-mentioned medium requires a lot of time and labor to detect Escherichia coli, and is not suitable for the case where quick detection is required. .

In the method of detecting the presence or absence of β-glucuronidase, if the specimen (test object) is shellfish such as oysters, animal meat or fish meat, these foods contain β-glucuronidase, and these foods are inspected. In such a case, there is a concern that the test result will be a false positive, and it is necessary to confirm that fact in advance. Furthermore, since β-glucuronidase is an enzyme present in the cells,
-When the permeability of the substrate to glucuronidase is low, it is necessary to crush the cells and enhance the reaction, which makes it difficult to detect live Escherichia coli cells.

In the above-mentioned immunochromatography method, since an antibody which specifically binds to lipopolysaccharide present on the outer membrane of Escherichia coli is used, it can react even when Escherichia coli is dead cells or bacterial fragments. I will. If Escherichia coli is detected without distinguishing whether the Escherichia coli is a viable cell or a dead cell in this way, the results from the above-mentioned multi-item test will be different, which may cause confusion in the test results. Is not preferred. In addition, in terms of food hygiene, the suitability of the food is determined based on the presence or absence of live Escherichia coli cells, but if not only live cells of Escherichia coli but also dead cells and bacterial fragments are detected together, dead cells and There is a problem that a conforming product containing only bacterial fragments is regarded as a nonconforming product.

An object of the present invention is to provide a method for detecting Escherichia coli that can detect live Escherichia coli cells in a test solution simply, quickly, with high precision and high sensitivity, and a detection kit that can be used therefor. Is what you do.

[0012]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have completed the present invention. The present invention is as follows. (1) A step of spreading a test solution on a detection tool in which a stationary phase on which a specific binding component capable of specifically binding to Escherichia coli is immobilized is formed in an arbitrary region on the surface of a water-absorbing substrate. And a step of developing a substrate solution containing a chromogenic substrate component capable of specifically binding to alkaline phosphatase and developing a color on the water-absorbing substrate after the test solution has been developed. A method for detecting live cells. (2) A detection device formed by forming a stationary phase on which a specific binding component capable of specifically binding to Escherichia coli is immobilized in an arbitrary region on the surface of a water-absorbing substrate, and a detector specifically binding to alkaline phosphatase. A kit for detecting a viable Escherichia coli cell, comprising: a substrate solution containing a color-forming substrate component capable of developing a color.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
FIG. 1 is a simplified diagram showing a preferred example of a kit 1 for detecting live Escherichia coli cells of the present invention. The detection kit 1 of the present invention is for detecting whether or not viable cells of Escherichia coli are present in a test solution using a specific binding reaction, A stationary phase 4 in which a specific binding component capable of specifically binding to Escherichia coli is immobilized on the arbitrary region above.
And a substrate solution containing a chromogenic substrate component capable of specifically binding to alkaline phosphatase to form a color.

In the present invention, the "test liquid" is an object (specimen) for which it is desired to confirm whether or not viable E. coli cells are present.
Refers to those prepared by applying an appropriate treatment to the detector 2 so as to be deployable, such as food, feces, serum, blood,
Urine, a solution extracted from a sample such as saliva, a supernatant of a culture liquid obtained by culturing the sample under appropriate conditions, or any of them, for example, sterilized physiological saline or It includes a diluent obtained by diluting with a suitable diluting solvent such as a buffer. When live Escherichia coli cells to be detected are present in the test solution, they are usually measured by a dilution plate method using a desoxycholate agar medium, for example, according to a measurement method specified in a food hygiene inspection guideline or the like. The number of bacterial cells is 5 × 10 ⁷ cfu / mL to 5 × 10 ⁸ cfu / mL
To the extent, favorable positive reaction results can be observed.

The water-absorbing substrate 3 in the present invention is preferably realized so as to have an appropriate water-absorbing property with respect to the test liquid and a substrate liquid described later. The above-mentioned "moderate water absorption" specifically means that one end of the water-absorbing substrate 3 cut into a 6 mm wide strip is immersed in water, and the water absorption distance after 1 minute (from the immersed one end) , The linear distance to the point where the water spreads) is about 2 cm to 6 cm. When the water-absorbent substrate 3 is poor in water absorbency, it takes a long time for the test liquid or the substrate liquid to reach the stationary phase 4, and as a result, a rapid measurement tends to be impossible, which is not preferable. . When the water absorption of the water-absorbing substrate 3 is too high, a specific binding reaction (specifically, a reaction between Escherichia coli and a specific binding component described later, (Reaction between alkaline phosphatase and a chromogenic substrate component) is not preferable because the time required for the reaction is insufficient and accurate measurement tends to be difficult. As a method for spreading the test liquid or the substrate liquid onto the water-absorbing substrate 3, the liquid is spontaneously developed from the dripping portion on one end side of the water-absorbing substrate 3 by a capillary phenomenon.

The water-absorbent substrate 3 of the present invention has a pore size (preferably 3 μm to 15 μm, more preferably 8 μm) that allows Escherichia coli to pass through and has an appropriate liquid spreading property.
It is necessary to have a large number of pores (μm to 10 μm).
Preferred materials for forming such a water-absorbent substrate 3 include nitrocellulose membranes such as polyester,
Nonwoven fabrics formed of rayon, polypropylene, nylon, glass fiber, and the like, filter paper, and the like can be given. When the water-absorbing substrate 3 is formed of the nonwoven fabric, the basis weight is 20 g / m2.
^It is preferably from ^{2 to} 200 g / m ² . By forming from these materials, it is possible to realize the water-absorbing substrate 3 which has an appropriate absorption rate, and has an advantage of being excellent in visual confirmability when a color-forming substrate component binds to alkaline phosphatase and forms color. .

As described above, the detector 2 of the present invention
Has a stationary phase 4 formed in an arbitrary region on the surface of the water-absorbing substrate 3. The stationary phase 4 has a specific binding component capable of specifically binding to Escherichia coli immobilized thereon. Such specific binding components include monoclonal antibodies, polyclonal antibodies or ligands having high specificity and affinity for E. coli.

A monoclonal antibody that recognizes pathogenic Escherichia coli, which is suitably used as a specific binding component in the present invention, can be obtained by a conventionally known cell fusion method (an antibody-producing cell derived from an immunized animal is fused with a myeloma (myeloma) cell). Method using cloned hybridoma).

The polyclonal antibody suitably used as the specific binding component in the present invention can be obtained by a conventional technique for purifying antibodies in the blood of immunized animals, which is widely used.

The ligand preferably used as the specific binding component in the present invention can be obtained by a conventionally widely used technique for purifying by antigen-immobilized affinity chromatography.

Further, monoclonal antibodies, polyclonal antibodies and ligands that specifically recognize pathogenic Escherichia coli are
It may be obtained by purchasing a commercially available product.

The method for immobilizing the specific binding component thus obtained on the water-absorbent substrate 3 (the method for producing the stationary phase 4) is not particularly limited, but is conventionally known. It is preferable to use the physical adsorption method or the covalent bonding method described above, and it is particularly preferable to use the covalent bonding method in that the specific binding component is hardly detached from the substrate.

Further, after a solution containing a specific binding component and a hydrophilic polymer is applied to the water-absorbent substrate 3, the stationary phase 4 is formed by immersing the solution in a coagulating solvent for coagulating the hydrophilic polymer. Can also. Examples of such a hydrophilic polymer include hydroxypropylmethylcellulose, polyvinyl alcohol, and hydroxyethylcellulose. Examples of the coagulating solvent include acetone, ethanol, methanol, and ether.

After immobilizing the specific binding component, the water-absorbing substrate 3 is preferably used after being treated with a solution containing a protein, a surfactant and a saccharide. Examples of the protein include bovine serum albumin, casein, gelatin, skim milk and the like. As the above surfactant,
Examples thereof include polyoxyethylene (10) octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, and polyoxyethylene alkyl phenyl ether. Examples of the sugar include glucose, trehalose, and saccharose. The above process is
This is carried out by dissolving the above components in water and immersing the water-absorbing substrate after fixing in the water. The protein prevents unnecessary proteins from adsorbing on the surface of the water-absorbing substrate, the surfactant facilitates the development, and the sugar improves the stability of the specific binding component applied to the surface of the water-absorbing substrate. This has the effect.

The content of the protein in the treatment solution is preferably 0.1% by weight to 10% by weight. If the amount is too small, the effect is hardly obtained, and if it is too large, the reactivity in the stationary phase tends to decrease. . The content of the surfactant is preferably 0.0
The content is 1% by weight to 1% by weight. When the amount is too small, the effect is hardly obtained. The content of the saccharide is 0.1% by weight to 10% by weight. If the amount is too small, the effect is hardly obtained, and if it is too large, it is difficult to develop.

The shape of the water-absorbing substrate in the present invention is not particularly limited as long as it can expand the test liquid and the substrate liquid, and is preferably, for example, a rectangular sheet (piece) or rod. . When realized by the sheet-shaped water-absorbing substrate 3 as in the embodiment shown in FIG. 1, the length, width, and thickness are not particularly limited, but the length is usually 15
mm to 100 mm, width 1 mm to 20 mm, and thickness 0.
Each is selected within a range of 03 mm to 0.3 mm. The water-absorbing substrate 3 may be made of one kind of material, or may be obtained by joining members made of different kinds of materials by an arbitrary bonding means.

In the present invention, the distance between the stationary phase 4 and the site where the absorption of the test liquid or the substrate liquid of the water-absorbent substrate 3 starts (ie, the dropping portion) is not particularly limited.
The above-mentioned distance is too far, the test liquid does not reach the stationary phase, the detection signal sensitivity is too strong, or the measurement takes a long time.On the contrary, the above-mentioned distance is too close to the stationary phase. The chromogenic substrate component is appropriately selected so as not to cause a problem that the captured chromogenic substrate component is not uniform but sparse, and the detection signal sensitivity is too low.

Further, for example, polyester,
You may provide a pad excellent in water absorption, such as a nonwoven fabric or filter paper formed of a material such as polypropylene, rayon, nylon, or glass fiber. This makes it easier for the test liquid and the substrate liquid to be added onto the water-absorbing substrate 3. FIG. 1 shows an embodiment in which two dropping portions 5a and 5b are provided on one end 3a side of the water-absorbent substrate 3 and on the stationary phase 4 side thereof, respectively.

As shown in FIG. 1, the other end 3b on the opposite side of the stationary phase 4 is provided with the above-mentioned dropping portion 5a.
For example, an absorbent sheet material made of a material such as glass fiber or cellulose may be provided as the water absorbing pad 6. As a result, the liquid dropped on the water-absorbing substrate 3 is absorbed in the traveling direction, so that the liquid can be developed smoothly.

The substrate solution in the present invention contains a chromogenic substrate component capable of specifically binding to alkaline phosphatase to form a color, as described above. Alkaline phosphatase is widely distributed in the living world, but is known to be present in bovine intestine and Escherichia coli with high activity. This enzyme is present on the cell membrane in microorganisms. Therefore, by specifically binding to the color-forming substrate component to form a color, the presence or absence thereof can be easily confirmed. By using a chromogenic substrate component that specifically binds to this enzyme, viable cells can be specifically detected in Escherichia coli, and it is not a substrate for enzymes present in the cells such as β-glucuronidase. In addition, even when a substrate having low cell membrane permeability is used, there is no need to perform an operation for disrupting the cells and enhancing the reaction as in the case of the above-mentioned intracellular enzyme, and thus it is possible to reliably detect live E. coli cells.

As such a chromogenic substrate component for alkaline phosphatase, conventionally known p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indoyl phosphate / nitroblue tetrazolium, fast red / naphthol AS-TR phosphate And the like.

The substrate solution contains 0.001% of a coloring substrate component.
It is preferable to contain it in the range of 〜1% from the viewpoint of its detection sensitivity and background color development. The substrate solution is realized by, for example, a 0.1 M Tris buffer (pH 9.8) containing 0.02% of 5-bromo-4-chloro-3-indoyl phosphate and 0.04% of nitroblue tetrazolium. Is preferred.

The detection method of the present invention is applied to a detection tool in which a stationary phase on which a specific binding component capable of specifically binding to Escherichia coli is immobilized is formed in an arbitrary region on the surface of a water-absorbing substrate. A step of developing a test solution, and at least a step of developing a substrate solution containing a chromogenic substrate component capable of specifically binding to alkaline phosphatase and developing a color on the water-absorbing substrate after developing the test solution. Is the way. The detection method of the present invention is preferably performed using the above-described detection kit of the present invention. Hereinafter, the detection method of the present invention will be described with reference to an example in which the detection kit 1 of the embodiment shown in FIG. 1 is used.

First, a test solution prepared and prepared as appropriate from a specimen which is an object to be checked for the presence or absence of viable Escherichia coli cells is dropped into the dropping portion 5b of the detection kit 1. The drop amount of the test liquid is preferably 0.01 mL to 0.2 mL. If the dropping amount is less than 0.01 mL, the reaction with the stationary phase is insufficient, and there is a tendency that sensitivity tends to decrease and uneven coloring occurs. In addition, the amount of the drop is 0.
If it exceeds 2 mL, it takes time to develop the test liquid, and the detection time tends to be long, which is not preferable.

The dropped test liquid is developed in the water-absorbing substrate 3 so that the above-mentioned specific binding component is directed to the stationary phase 4 to which the above-mentioned specific binding component is fixed. When the test liquid reaches the stationary phase 4,
When Escherichia coli is present in the test solution, the Escherichia coli is captured on the stationary phase 4 by the specific binding component, regardless of whether the cells are live cells or dead cells.

The substrate liquid is dropped after the test liquid is dropped. When the substrate solution reaches the stationary phase before the test solution,
This is because the chromogenic substrate component for alkaline phosphatase in the substrate liquid is not captured by the stationary phase and passes through the stationary phase as it is. However, in the embodiment as shown in FIG. 1, the substrate liquid is dropped from the dropping portion 5a at a position separated from the stationary phase 4, and the test liquid is dropped onto the dropping portion 5b before the substrate liquid reaches the dropping portion 5b. You can also.

When the substrate solution thus dropped reaches the stationary phase 4, if live Escherichia coli cells are present in the test solution, the substrate solution captured by the stationary phase by the specific binding component. The chromogenic substrate component specifically binds to the alkaline phosphatase possessed by the bacterial cells and develops color. This makes it possible to determine that a specific binder has been formed in the stationary phase 4, and to detect the presence of live E. coli cells in the test solution. If Escherichia coli is not present in the test liquid (including the presence of microorganisms other than Escherichia coli), and if Escherichia coli is present as dead cells or bacterial fragments, the chromogenic substrate component is the stationary phase. , The color does not develop on the stationary phase 4.

As described above, the present invention is a method in which Escherichia coli is first selectively captured on a stationary phase, and the presence or absence of an enzyme derived from live cells is selectively detected in the Escherichia coli. With such a detection method, it is possible to reliably detect whether or not viable cells of Escherichia coli are present. Confusion can be reliably prevented, and a conforming product containing only dead cells or bacterial fragments will not be regarded as a nonconforming product.

The detection kit and the detection method of the present invention
It can be particularly suitably used for food inspection and environmental hygiene inspection. First, a culture liquid obtained by culturing a sample (eg, a food obtained in a food test, or a sample obtained by wiping the surface of a skin, a worktable, a cutting board, or the like in an environmental health test) in a liquid medium is applied. It may be used as a test solution, or after culturing the sample in a liquid medium, then culturing it in an agar medium, and diluting the obtained colonies with sterile water or a buffer such as phosphoric acid as a test solution. You may. In addition, a specimen may be cultured on an agar medium without using a liquid medium, and the resulting colony diluted with sterile water or the above buffer may be used as a test liquid.

The medium such as a liquid medium and an agar medium used for preparing the above-mentioned test solution may be used for testing Escherichia coli and Escherichia coli in accordance with guidelines for inspection of food hygiene, for example, EC medium, lactose broth medium, BGLG medium. It is desirable to appropriately select and use conventionally known various media such as desoxycholate agar medium, TSB medium, and EMB agar medium.

Further, the substrate solution in the detection kit of the present invention may be realized by being preliminarily impregnated into the dropping portion on the side remote from the stationary phase and then air-dried. In this case, at the time of inspection, the substrate liquid is developed in the water-absorbing substrate 3 by dropping water on the dropping portion.

[0042]

EXAMPLES Examples of the present invention will be described below in more detail, but the present invention is not limited to these examples. Example 1) Production of detection kit A nitrocellulose membrane (manufactured by Whatman) having a length of 40 mm, a width of 6 mm, a thickness of 0.2 mm, and a pore diameter of 12 μm was used as the water-absorbing substrate 3. In the vicinity of the center of No. 3, a rabbit anti-E. Coli antibody (1 mg / mL, 0.1 M phosphate buffer, pH 7.2, manufactured by Biogenesis)
Was applied in a line, immersed in an aqueous solution consisting of 1% bovine serum albumin, 1% saccharose and 0.1% polyoxyethylene sorbitan monolaurate for 20 minutes, and then air-dried overnight to form a stationary phase 4. . One end 3a of the water-absorbent substrate 3, and between the one end 3a and the stationary phase 4,
A 6 mm × 6 mm polyester nonwoven fabric (Hibon 5880C, manufactured by Shinwa Corporation) was placed, and the
a and 5b were provided. Also, 30 is attached to the other end 3b of the water-absorbent substrate 3.
A water-absorbing material 6 (mm / 15 mm) made of glass fiber (GF / B, manufactured by Whatman Japan) was provided as the water-absorbing pad 6.
The same number of such detectors 2 as the types of test liquids described in Examples and Comparative Examples were produced. A liquid (BCIP / NBT Phosphatase Sub) containing an alkaline phosphatase chromogenic substrate component is applied to such a detector 2.
state 1 Component, Kirkeg
aard & Perry Laboratories
Was prepared as a substrate solution to obtain a detection kit 1 of the present invention as simplified in FIG.

2) Inspection A solution obtained by culturing Escherichia coli K12 (obtained from Osaka University) in a conventionally known tryptic soybean (TSB) medium and diluting it with a physiological saline solution was used as a test liquid to prepare a dropping portion 5b.
50 μL was dropped. According to the measurement method specified in the Food Sanitation Inspection Guideline, the number of cells in each of the test liquids to the test liquid measured by the dilution plate method using desoxycholate agar medium is as shown in Table 1. Was. Further, 100 μL of the above-mentioned substrate solution was dropped on the dropping portion 5a. Fifteen
After a minute, the color development of the stationary phase 4 was examined. Positive (+) was determined when there was linear coloring, and negative (-) when no linear coloring was present.
Table 1 shows the results.

[0044]

[Table 1]

Comparative Example The procedure was the same as in the Example, except that each of the test liquids containing bacteria other than Escherichia coli was dropped. The bacteria contained in each of the test liquids are as shown in Table 2.
The number of bacterial cells measured in the same manner as in the Examples was as follows.
0 ⁸ cfu / mL, the test liquid is 4 × 10 ⁸ cfu / mL,
Test liquid is 8 × 10 ⁸ cfu / mL, test liquid is 6 × 1
0 ⁸ cfu / mL, and the test liquid was 8 × 10 ⁸ cfu / mL. Table 2 shows the results.

[0046]

[Table 2]

[0047]

As is clear from the above description, according to the present invention, a method for detecting Escherichia coli which can detect live Escherichia coli in a test solution simply, quickly, with high precision and high sensitivity, and A detection kit that can be used can be provided.

[Brief description of the drawings]

FIG. 1 is a simplified diagram showing a preferred example of a kit 1 for detecting live Escherichia coli cells of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Detection kit 2 Detector 3 Water-absorbing base material 4 Stationary phase

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) // C12M 1/04 C12M 1/04 (72) Inventor Shuji Senda 1-1-1 Shimohozumi, Ibaraki-shi, Osaka No. 2 Nitto Denko Corporation F-term (reference) 4B029 AA07 BB02 CC03 FA03 4B063 QA01 QA18 QQ06 QR13 QR48 QR58 QR82 QS02 QS33 QX02

Claims (2)

[Claims] 1. A test solution is developed on a detection tool in which a stationary phase on which a specific binding component capable of specifically binding to Escherichia coli is immobilized is formed in an arbitrary region on the surface of a water-absorbing substrate. And a step of developing a substrate solution containing a chromogenic substrate component capable of specifically binding to alkaline phosphatase and developing a color on the water-absorbing substrate after developing the test solution. For detecting live Escherichia coli. 2. A detection device comprising a stationary phase on which a specific binding component capable of specifically binding to Escherichia coli immobilized is formed in an arbitrary region on the surface of a water-absorbing substrate, and a detector specifically comprising alkaline phosphatase. A kit for detecting a viable Escherichia coli cell, comprising: a substrate solution containing a chromogenic substrate component capable of binding to form a color and developing on a water-absorbing substrate.