JPH0534349A - Detecting method of food-poisoning bacteria and sensor using the same - Google Patents

Abstract

PURPOSE:To detect a food-poisoning bacteria in a food constituent simply and quickly by bringing the food constituent into contact with a surface acoustic wave element on which an anti-food-poisoning bacteria antibody is stuck and by measuring a change in a frequency before and after the contact. CONSTITUTION:A surface acoustic wave oscillator 17 having transducers 13 and 15 provided on a crystal plate 11 of ST cut is covered with polystyrene and an anti-food-poisoning-bacteria antibody 19 is stuck between the two transducers 13 and 15, whereby a sensor 21 is prepared. This sensor 21 is connected with amplifiers 23 and 25 and a frequency counter provided between them. A food constituent is brought into contact with the sensor 21, and based on a change in a frequency before and after the contact, a food-poisoning bacteria in the food constituent is detected. The sensor 21 is subjected to washing in three times by a phosphoric acid buffer and protection of a nonspecific adsorption site by calf serum albumin, so as to prevent fluctuation in the frequency due to a cause other than combination of the food-poisoning bacteria.

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 food poisoning bacteria and a sensor suitable for use in this method.

[0002]

2. Description of the Related Art Food poisoning includes bacterial food poisoning and chemical food poisoning. Bacterial food poisoning is further classified into an infectious type (for example, Vibrio parahaemolyticus, Salmonella, etc.) and a toxin type (for example, poisoning due to staphylococcal venom, botulism, etc.). Toxin-type bacterial food poisoning, in which toxins are already produced in large amounts in contaminated foods, has the property that the time required for onset of disease is short.

[0003] Bacterial food poisoning, regardless of infection type or toxin type, causes a large number of victims, especially in areas where the living environment is poor and the idea of hygiene is low. In such areas, the chances of food being contaminated with bacteria are extremely high, and it is common practice to eat food that is contaminated with bacteria or is spoiled due to chronic food shortage. is there.

In order to prevent the occurrence of bacterial food poisoning, it is effective to simply and quickly detect whether food poisoning bacteria causing food poisoning are present in food.

[0005]

However, at the present time, the inventors of the present application did not know of any suitable method for easily and quickly detecting food-toxic bacteria.

The present invention has been made in view of the above points, and therefore an object of the present invention is to provide a method capable of simply and rapidly detecting food poisoning bacteria and a sensor suitable for use in the method. .

[0007]

In order to achieve this object, the inventor of this application has made various studies. As a result, we paid attention to the fact that the oscillation frequency and resonance frequency of surface acoustic wave elements such as surface acoustic wave oscillators and surface acoustic wave filters decrease in proportion to the amount of substances adhering to the surface acoustic wave elements. Then, they have found that the above object can be achieved by further introducing an antigen-antibody reaction into this technique, and completed the present invention.

Therefore, according to the first invention of this application, in detecting the presence or absence of food poisoning bacteria, an anti-food poisoning bacterial antibody is attached to the surface acoustic wave element, and the surface acoustic wave element to which the antibody has been attached is attached. It is characterized in that a food component is contacted, and food poisoning bacteria in the food component are detected from the frequency change of the surface acoustic wave device before and after the contact.

Here, the attachment of the anti-food poisoning bacterial antibody attached to the surface acoustic wave element means that the antibody is removed from the surface acoustic wave element when the surface acoustic wave element with the antibody attached thereto is brought into contact with a food component. The form does not matter as long as it does not come off. It may be either physically attached or chemically attached. In Examples described later, the surface acoustic wave device is brought into contact with a liquid containing the antibody for several hours to adsorb the antibody to the surface acoustic wave device.

Further, the contact of contacting the food component with the surface acoustic wave element to which the anti-food poisoning bacterial antibody has been adhered means that the anti-food poisoning bacterial antibody adhered to the surface acoustic wave element has a food component (specifically, a food ingredient). Any means can be used as long as it can effectively contact the bacteria in the components. In the examples described later, the surface acoustic wave device to which the anti-food poisoning bacterial antibody has been attached is brought into contact with the bacterial culture for several minutes.

The term "food component" means that food poisoning bacteria as an antigen are in a state of easily reacting with an anti-food poisoning bacterial antibody. Specifically, for example, a solution obtained by dissolving food in a culture solution or buffer of food-toxic bacteria can be mentioned.

In carrying out the first invention, it is preferable that the surface acoustic wave element is coated with polystyrene and then the anti-food poisoning bacterial antibody is attached to the polystyrene-coated surface acoustic wave element. This is because the protein is easily attached to polystyrene, which facilitates the attachment of the anti-food poisoning bacterial antibody to the surface acoustic wave device.

The sensor for detecting food poisoning bacteria of the second invention of this application is characterized in that the surface acoustic wave element is coated with polystyrene and anti-food poisoning bacterial antibody is attached to the polystyrene.

[0014]

According to the method for detecting food poisoning bacteria of the first invention of the present application, when a food component is attached to the surface acoustic wave device to which the anti-food poisoning bacterial antibody has been attached, the food poisoning bacteria are contained in the food component. If the bacterium is included, the bacterium specifically binds to the antibody of the surface acoustic wave element, and the oscillation frequency or resonance frequency of the surface acoustic wave element decreases. The above frequency reduction does not occur when food poisoning bacteria are not contained in the food ingredients.
It is possible to detect the presence or absence of food poisoning bacteria in food components by utilizing such a difference in frequency change.

Further, the sensor for detecting food poisoning bacteria of the second invention of the present application (hereinafter sometimes simply referred to as "sensor") facilitates the implementation of the first invention.

[0016]

EXAMPLES Examples of the food poisoning bacteria detection method of the present application and sensors used therefor will be described below with reference to the drawings. Here, FIG. 1 is a diagram for explaining the configuration of the sensor of the example and the measurement system, FIG. 2 is a diagram for explaining a method of manufacturing the sensor of the example, and FIGS. 3 and 4 are characteristics of the sensor of the example. FIG.

1. Preparation of Antibodies Anti-food poisoning bacterial antibodies were prepared in this example by the following immunization method. Regarding the immunization law, refer to the literature ("Biotechnology Experiment Manual" (198
7), Sankyo Publishing).

In the case of this example, the antigen used for preparing the antibody was Salmonella antigen (manufactured by Funakoshi Pharmaceutical Co., Ltd., model number 49-0634-02), which is a kind of infectious food poisoning bacterial antigen.
And a staphylococcal antigen (manufactured by Funakoshi Pharmaceutical Co., Ltd., model number 49-0636-41), which is a kind of toxic food poisoning bacterial antigen.

5 mg of the antigen (for example, Salmonella antigen) and 1.7 mg of fetal bovine serum albumin (manufactured by Sigma) were added.
Dissolve in 1 ml of H7.3 phosphate buffer. In addition,
This phosphate buffer contains 0.29% Na ₂ HPO ₄ ,
0.08% NaCl, 0.02% KH ₂ PO ₄ and 0.02% KCl. The indication of the content is% by weight (hereinafter the same in this example).

Next, 10 μl of a 20% glutaraldehyde aqueous solution is added to the phosphate buffer in which the antigen and bovine serum albumin are dissolved, and the antigen and glutaraldehyde are reacted for 24 hours. Glutaraldehyde used was manufactured by Kanto Chemical Co., Inc.

Then, in order to remove unreacted glutaraldehyde from the phosphate buffer, it is dialyzed against the phosphate buffer at a temperature of 4 ° C. overnight. Furthermore, pH 8.0 Tris buffer (0.3% -Trismbase, 0.0
Dialyze against 5% -HCl) at a temperature of 4 ° C overnight. In this way, the antigen for inoculation was obtained.

Mice are inoculated with the obtained inoculating antigen.
Salmonella antibody was obtained from this mouse.

Staphylococcal antibodies were obtained from mice by the same procedure as above using the antigen as the staphylococcal antigen.

2. Preparation of Sensor of Example As a surface acoustic wave device, in this example, as shown in FIG. 1, a known surface having a first transducer 13 and a second transducer 15 on an ST-cut quartz plate 11 was used. The acoustic wave oscillator 17 has an oscillation frequency of 10.3
The thing of MHz was prepared. The surface acoustic wave oscillator 17 in this case has a size of 1 inch × 0.5 inch (1 inch is about 2.54 cm. The same applies hereinafter) and has a thickness of 0.0.
It is a 4-inch one.

Next, the above-described surface acoustic wave oscillator 17 is immersed in acetone in which polystyrene is dissolved. next,
The surface acoustic wave oscillator 17 is pulled out from the acetone solution of polystyrene and then left to stand to volatilize the solvent acetone. As a result, the surface acoustic wave oscillator 17
The surface is coated with polystyrene (not shown). The polystyrene coating amount is such that the surface acoustic wave oscillator 17 can be covered and the characteristics of the surface acoustic wave oscillator 17 are not significantly deteriorated. The coating amount of polystyrene can be adjusted by the polystyrene concentration in an acetone solution of polystyrene, the speed of pulling up the surface acoustic wave oscillator 17 from this solution, and the like.

Next, the surface acoustic wave oscillator 17 coated with polystyrene is set in the recess 31a of the pedestal 31 of the apparatus shown in FIG.

Here, FIG. 2 shows that in order to attach the anti-food poisoning bacterial antibody to the surface acoustic wave oscillator 17 and to bring the food component into contact with the surface acoustic wave oscillator 17 having the anti-food poisoning bacterial antibody attached thereto. It is sectional drawing of the apparatus prepared in the Example. In FIG. 2, 31 is the above-mentioned pedestal, 31a is the above-mentioned recessed portion, 33 is an O (o-ring), 35 is an upper block that is used by being stacked on the pedestal 31, and 35a is an opening for storing a sample solution provided in the upper block It is a department. A hole 31b for inserting the bolt 37 is provided at a predetermined position of the pedestal. A female screw portion 39 for the bolt 37 is formed in a portion of the upper block 35 facing the hole portion 31b.

The surface acoustic wave oscillator 17 is installed in the recess 3 of the pedestal 31.
After setting to 1a, the upper block 3 via the O-ring 33
5 is placed on the pedestal 31, and the upper block 35 and the pedestal 31 are fixed by bolts. With this configuration, the portion of the surface acoustic wave oscillator 17 between the first transducer 13 and the second transducer 15 is located inside the O-ring 33 and faces the opening 35 a of the upper block 35. (State of FIG. 2).

Next, the salmonella antibody and staphylococcal antibody prepared as described above are placed in the sample solution reservoir opening 35a of the upper block 35 of the apparatus shown in FIG. 2 in the state where the surface acoustic wave oscillator 17 is set. Is added, for example, in a phosphate buffer in which equimolar amounts are dissolved. Since the phosphate buffer in which the antibody has been dissolved collects in the space defined by the O-ring 33 and the opening 35a, it comes into contact with the portion of the surface acoustic wave oscillator 17 between the transducers 13 and 15. Then, the entire apparatus in this state is placed in a refrigerator set at a temperature of 4 ° C. overnight and incubated. In this process, the anti-food poisoning bacterial antibody can be attached to the portion of the surface acoustic wave oscillator 17 between the transducers 13 and 15.

Next, the bolt 37 of the device in which the surface acoustic wave oscillator 17 is set is loosened, the pedestal 31 and the upper block 35 are separated, and the phosphate buffer in which the antibody is dissolved is poured out from the device and removed. Then, the sample liquid reservoir opening 35a and the surface acoustic wave oscillator 17 of the apparatus are washed three times with a phosphate buffer. This is to remove the anti-food poisoning bacterial antibody that is unstablely attached to the surface acoustic wave oscillator 17.

Next, the bolts 37 of the device in which the surface acoustic wave oscillator 17 is set are tightened again, and the pedestal 31 and the upper block 35 are re-laid on each other via the O-ring. Then, 1% fetal bovine serum albumin-phosphate buffer solution was put into the opening 35a for storing the sample solution at room temperature under this condition.
Leave for hours. This is because the area of the surface acoustic wave oscillator 17 where the anti-food poisoning bacterial antibody is not adsorbed (non-specific adsorption site) is protected by calf serum albumin.

In this way, the sensor 21 of the embodiment obtained by attaching the anti-food poisoning bacterial antibody 19 to the surface acoustic wave oscillator 17 coated with polystyrene is obtained (see FIG. 1). Of course, it should be understood that FIG. 1 is a schematic diagram.

Since the above-mentioned washing with the phosphate buffer was performed three times and the non-specific adsorption site was protected with bovine serum albumin, the sensor of the example causes frequency fluctuations due to causes other than the binding of food poisoning bacteria. Can be prevented as much as possible.

3. Characteristics of Sensor of Example Next, changes in the oscillation frequency of the sensor of the example with respect to Salmonella and Staphylococcus were measured as follows.

First, the sensor 21 of the embodiment is connected to the amplifiers 23 and 25 as shown in FIG. A frequency counter (not shown) is connected between the amplifiers 23 and 25 to measure the oscillation frequency of the sensor of the embodiment. Note that this measurement system is known.

Next, the sensor 21 of the embodiment is first immersed in a buffer solution having a predetermined concentration of Salmonella, and the oscillation frequency is measured 10 minutes after the immersion, and the sensor is immersed in the buffer from this oscillation frequency. The amount of frequency change was obtained by subtracting the previous oscillation frequency. The oscillation frequency was measured 10 minutes after the sensor was immersed in the buffer.
This is because the frequency has become sufficiently stable at such an elapsed time.

The change in the oscillation frequency of the sensor when the concentration of Salmonella was varied was determined by the same procedure as the above procedure.

FIG. 3 is a characteristic diagram in which the concentration of Salmonella in the buffer in which the sensor is immersed (unit: mg / ml) is plotted on the horizontal axis, and the oscillation frequency change (KHz) of the sensor is plotted on the vertical axis, and the relationship between the two is plotted. is there.

Further, the characteristics of the frequency change with respect to the staphylococcal concentration of the sensor are similarly obtained in the case of Salmonella. This characteristic is shown in FIG.

As is apparent from FIGS. 3 and 4, it is understood that the oscillation frequency of the sensor decreases in a specific relationship as the bacterial concentration increases. From this, it can be understood that the method of the present invention can detect food poisoning bacteria.

In the above description, the examples have been described for Salmonella and Staphylococcus, but the inventions of this application can also be used for the detection of other food-toxic bacteria.

Further, in the above-mentioned embodiment, the surface acoustic wave element is composed of the surface acoustic wave oscillator using the ST cut quartz plate, but it may be composed of other materials. Also,
When the surface acoustic wave filter is made to adsorb the anti-food poisoning bacterial antibody, the same effect as that of the embodiment can be expected.

Further, in the above embodiment, the anti-food poisoning bacterial antibody is attached to the surface acoustic wave oscillator 17 between the first and second transducers 13 and 15, but the anti-food poisoning bacterial antibody is attached. The area is not limited to this and can be changed according to the design.

[0044]

As is apparent from the above description, according to the method for detecting food poisoning bacteria of the first invention of this application,
Food poisoning bacteria can be detected easily and quickly.

Further, according to the pollen sensor of the second invention of this application, the presence or absence of food-toxic bacteria in the food component can be detected only by immersing the pollen sensor in the buffer in which the food component is dissolved and measuring the frequency change. The method for detecting food poisoning bacteria of the first invention can be easily carried out.

[Brief description of drawings]

FIG. 1 is a diagram for explaining a sensor and a measurement system of an example.

FIG. 2 is a diagram for explaining a method of manufacturing the sensor according to the example.

FIG. 3 is a view showing characteristics of oscillation frequency change with respect to Salmonella, in the sensor of the example.

FIG. 4 is a graph showing characteristics of oscillation frequency change with respect to staphylococci in the sensor of the example.

[Explanation of symbols]

11: ST-cut crystal plate 13: First transducer 15: Second transducer 17: Surface acoustic wave oscillator 19: Anti-food poisoning bacterial antibody 21: Sensor of Example 23, 25: Amplifier 31: Pedestal 31a: concave portion 33: O-ring 35: Upper block 35a: sample liquid reservoir opening 37: Bolt 39: Female thread

Continued front page    (72) Inventor Masaaki Kaifu             1-7-12 Toranomon, Minato-ku, Tokyo Oki Electric             Industry Co., Ltd. (72) Inventor Masakazu Kato             1-7-12 Toranomon, Minato-ku, Tokyo Oki Electric             Industry Co., Ltd.

Claims (3)

[Claims] 1. When detecting the presence or absence of food poisoning bacteria, an anti-food poisoning bacterial antibody is attached to a surface acoustic wave element, and a food component is brought into contact with the surface acoustic wave element to which the antibody has been attached, and before and after the contact. A method for detecting food poisoning bacteria, which comprises detecting food poisoning bacteria in the food component from the frequency change of the surface acoustic wave device in step 1. 2. The method for detecting food poisoning bacteria according to claim 1, wherein the surface acoustic wave element is coated with polystyrene, and the anti-food poisoning bacterial antibody is attached to the polystyrene-coated surface acoustic wave element. A method for detecting food poisoning bacteria characterized by: 3. A sensor for detecting food poisoning bacteria, characterized in that the surface acoustic wave device is coated with polystyrene and an anti-food poisoning bacteria antibody is attached to the polystyrene.