JPH09131174A - Plate for detecting microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject plate, capable of detecting a microorganism and simultaneously confirming the presence or absence of the deterioration in a reagent and judging the effectiveness of the judgment by forming plural wells for filling the reagent for detecting ingredients derived from the microorganism and wells for positive control and negative control as one set. SOLUTION: This plate 4 for detecting a microorganism is obtained by preparing each of wells 1-1 to 1-3 for measuring a specimen comprising one or more reagent sheets 5, used for detection and located at the bottom thereof such as an endotoxin, a (1→3)β-D-glucan and a peptidoglucan as ingredients derived from a microorganism, each of wells 2-1 to 2-3 for positive control comprising the reagent sheets 5, used for detecting the ingredients derived from the microorganism and located at the bottom thereof and each of wells 3-1 to 3-3 for negative control comprising regent sheets 5, used for detecting the ingredients, derived from the microorganism and located at the bottom thereof as one set and forming the one set or plural sets for each kind of the ingredients derived from the microorganism on the plate 4. The resultant plate is capable of visually judging the presence or absence of the microorganism.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microorganism detection plate for visually detecting microorganisms such as Gram-negative bacteria, Gram-positive bacteria and fungi. The present invention relates to a microorganism detection plate capable of confirming the presence or absence of the effect or the influence of the sample diluent, and simultaneously determining the effectiveness of the determination.

[0002]

2. Description of the Related Art Endotoxin, (1 → 3) β-D-glucan (hereinafter abbreviated as β-glucan), peptidoglycan and the like are known as cell wall constituents of various microorganisms. It is conventionally known that the presence or absence of microbial species such as fungi, Gram-negative bacteria, and Gram-positive bacteria in a sample can be determined to some extent by examining the presence or absence of these components in the cell wall of the microorganism.

Conventionally, as a method for determining the presence or absence of these microbial species in a sample, a method using an endotoxin detection test paper (see Japanese Patent Application Laid-Open No. 6-341993) has been known. However, detection of components derived from microorganisms other than endotoxin such as β-glucan and peptidoglycan has not been actually performed or known.

[0004]

The conventional test paper for detecting endotoxin can detect the presence or absence of Gram-negative bacteria, but cannot detect the presence or absence of microbial species such as Gram-positive bacteria and fungi. Not only that, the test paper may not develop color due to the deterioration of the detection reagent contained in the test paper or the influence of the sample diluent, or the color may be erroneously developed. It was not very satisfying in terms.

The present invention detects the presence of microorganisms such as Gram-negative bacteria, Gram-positive bacteria and fungi in a sample, and at the same time, confirms whether or not the detection reagent is deteriorated and whether or not there is an influence of the sample diluent, and the effectiveness of the determination is determined. It is an object of the present invention to provide a plate for detecting a microorganism capable of discriminating.

[0006]

In order to achieve the above object, the present invention provides a sample measuring well in which a reagent for detecting a microorganism-derived component is located at the bottom, and a reagent for detecting the microorganism-derived component at the bottom. The positive control well and / or the negative control well with the reagent for detecting the microorganism-derived component located at the bottom are set as one set, and one set is formed on the plate or the microorganism-derived component is set. It is characterized in that a plurality of sets are formed for each type.

The fact that the reagent for detecting a microorganism-derived component is located at the bottom means that the reagent for detecting a microorganism-derived component or the reagent-containing material is preferably in the form of a film or a sheet and is held at the bottom, Alternatively, it is simply placed on the bottom and the well opening is covered with a film to prevent the film-like or sheet-like material from protruding from the opening.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described with reference to the drawings. As shown in FIG. 1, three circular wells 1, 2 and 3 are formed on a rectangular plate plate 4. Incidentally, 1 is a sample measuring well in which a reagent sheet 5 for detecting a microorganism-derived component is held at the bottom, and 2
Is a well for positive control in which the sheet 5 is held at the bottom, and 3 is a well for negative control in which the sheet 5 is held at the bottom.

Examples of the reagent for detecting a microorganism-derived component located at the bottom of the well include an endotoxin detection reagent, a β-glucan detection reagent and a peptidoglycan detection reagent. A positive control solution is added to the well 2 for positive control. The positive control liquid is a solution of the microorganism-derived component, for example, an aqueous solution of a purified product of the microorganism-derived component. Therefore, if the positive control liquid is added, it should naturally be colored, and if it is not colored, it is judged that the microorganism-derived component detection reagent is deteriorated. In addition, the purified product referred to here is
It refers to a component that does not contain a component that reacts with a reagent for detecting a microorganism-derived component in a well, other than the desired microorganism-derived component.
Further, the purification method is not particularly limited, and any method usually used in this field can be used.

A negative control solution is added to the well 3 for the negative control. As the negative control solution, water containing no microorganism-derived component such as water for injection or a sample diluent containing no microorganism-derived component is usually used. Therefore, even if the negative control liquid is added, it should not be colored.
It is determined that the microorganism-derived component detection reagent has deteriorated. When the sample diluent is used as the negative control solution and colored, the reagent for detecting a microorganism-derived component is deteriorated, or the sample diluent contains impurities for coloring the detection reagent. Is judged.

Either one of the well 2 for positive control and the well 3 for negative control may be used, but it is preferable to provide both in order to ensure sufficient reliability of determination. Reagents for detecting microorganism-derived components for positive control and negative control are
Use the same reagent for detecting the microorganism-derived component used for measuring the corresponding sample. For example, when an endotoxin detection reagent is held in a sample measuring well, the endotoxin detection reagent is also held in positive control well 2 and negative control well 3 corresponding thereto.

In the above embodiment, the wells 1, 2 and 3 are formed in a circular shape, but this is not necessarily the case and other shapes may be used. FIG. 2 shows another embodiment of the present invention in which a square plate-shaped plate 4 is provided with endotoxin detection wells 1-1 and 2-.
1, 3-1, β-glucan detection wells 1-2, 2-
2, 3-2 and peptidoglycan detection wells 1-3,
A total of 9 circular concave wells 2-3, 3-3 are formed. In addition, 1-1, 1-2, 1-3 are wells for specimen, 2-1, 2-2, 2-3 are positive control.
Wells 3-1, 3-2, and 3-3 for negative control are wells for negative control.

Endotoxin detection wells 1-1 and 2-
At the bottom of 1, 3-1 is a reagent sheet for endotoxin detection.
Well 1-2 for holding β-glucan,
At the bottom of 2-2 and 3-2, the reagent sheet 5b for detecting β-glucan is retained, and the well 1 for detecting peptidoglycan is used.
A reagent sheet 5c for detecting peptidoglycan is held at the bottom of -3, 2-3, 3-3.

In the above embodiment, the sample wells 1-1, 1
-2, 1-3, well for positive control 2-
The wells 1, 2-2, 2-3 and the negative control wells 3-1, 3-2, 3-3 are formed so as to be in the same row for each application.
-1, 2-1, 3-1, β-glucan detection well 1-
2, 2-2, 3-2 and peptidoglycan detection wells 1-3, 2-3, 3-3 are formed in the same row for each microorganism-derived component. By forming in this way, the experimental operation and its determination can be easily performed without mistake.

The plate-shaped plate 4 can be formed of a suitable material such as plastic that does not react with the detection reagent. The plate-shaped plate 4 is not particularly limited as long as it can form a well on its upper surface.
In addition, it is preferable to use a white plate or a plate in which the bottom surface of the transparent plate is white in view of ease of determination. In the above-mentioned Example, nine wells were formed in one plate-shaped plate 4, but each plate-forming plate was formed in a separate plate-shaped plate for each endotoxin, β-glucan and peptidoglycan detection well. The plates may be used together or separately.

The method for holding the predetermined detection reagent at the bottom of the well is not particularly limited as long as these reagents can be held at the bottom of the well so that they can react when a sample is added. For example, a method in which these detection reagents are supported on a sheet-like absorbent carrier and held at the bottom of the well, a polymer which is water-soluble and has a film-forming ability is used. A preferred method is to prepare a film-like (sheet-like) product using a compound, and hold the film at the bottom of the well.

Examples of the absorptive carrier for carrying the above detection reagent include plastics such as polypropylene, polyethylene, polyvinylidene fluoride, polyacrylonitrile, nylon, polyethylene, polyvinyl acetate, polytetrafluoroethylene, polyurethane, glass fiber, Cellulose, such as methylcellulose, acetate cellulose,
Absorbent carriers made of cellulose derivatives such as nitrocellulose and the like (more specifically, for example, woven cloth, nonwoven cloth,
Filter paper, sponge, etc.).

The polymer compound which is water-soluble and has the ability to form a film is, for example, polyvinyl alcohol, for example, a cellulose derivative such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose or hydroxyethyl cellulose. Examples thereof include polycarboxylic acids such as polyacrylic acid and polymethacrylic acid.

The detection reagent may be supported on the absorptive carrier by a known method such as impregnating the absorptive carrier with the detection reagent solution and then drying. As a method for holding the absorptive carrier or film supporting each detection reagent at the bottom of the well,
For example, a method of cutting them out according to the shape of the well and adhering them to the bottom of the well or holding them by press-fitting is performed. Examples include a method of sandwiching and adhering.

Examples of the endotoxin detection reagent carried on the bottom of the well of the microorganism detection plate of the present invention include, for example, genus Limulus and Tachypleu genus.
s), or a reagent containing a blood cell component of horseshoe crab belonging to the genus Calcinoscorpius, a so-called Limulus reagent, which has a property of not reacting with β-glucan. In addition, in the present invention, since endotoxin is visually detected, such a reagent is preferably a reagent for a synthetic substrate method which is colored by the presence of endotoxin. In addition, commercially available reagents satisfying such conditions, such as Biowita
hittaker Inc.), Endosafe. Inc., Seikagaku Kogyo Co., Ltd., Wako Pure Chemical Industries Ltd., etc., and reagents specific to endotoxin are, of course, However, it can be used for the purposes of the present invention.

As the β-glucan detection reagent, for example, a reagent specifically prepared from β-glucan prepared from an insect body fluid component, for example, Limulus genus (Limulus), Tachypleus genus (Tachypleus), or Carsino Scorpius (Ca
β containing blood cell components of horseshoe crab belonging to lcinoscorpius)
-A reagent that has a property of reacting with glucan but not with endotoxin and capable of visually confirming the presence of β-glucan (for example, a reagent using a synthetic substrate) can be mentioned. As these reagents, commercially available products may be used as appropriate, or those produced by a known method may be used.

Examples of peptidoglycan detection reagents include reagents specific to peptidoglycan prepared from insect body fluid components, and commercially available reagents may be used as appropriate, or known methods are available. It is also possible to use a self-made product.

As a method for preparing a β-glucan detection reagent or a peptidoglycan detection reagent from an insect body fluid component, for example, as for the β-glucan detection reagent, JP-A-63-141598, Peptidoglycan detection reagent is known. For example, the method described in JP-A-63-141599 can be used.

Next, an example in which the presence or absence of a microorganism is determined by using the plate for detecting a microorganism of the present invention will be shown. Regarding the samples (1) to (6), the presence or absence of microorganisms and their types were determined. The plate for detecting microorganisms of the present invention used had the form shown in FIG. 2, and the determination was performed by the following operation.

That is, in the wells 1-1, 1-2, 1-3 of the plate for detecting microorganisms, a predetermined sample is
Wells for positive control 2-1, 2-2, 2-3
And wells 3-1, 3-2, 3 for negative control
-3, a predetermined control liquid is dropped in a fixed amount,
After a lapse of a predetermined time from the start of the reaction, the state of color development in each well was visually evaluated to determine the presence or absence of microorganisms and their types.

Judgment based on the color development status in each well is as follows.
Except when the positive control was negative and when the negative control was positive, the procedure was as follows. If the positive control is negative and the negative control is positive, it is determined to be invalid. -If all of endotoxin, β-glucan and peptidoglycan are negative: it is determined that no microorganism is present. -If only β-glucan is positive: it is determined that a fungus is present. -If only peptidoglycan is positive: It is determined that Gram-positive bacteria are present.

When both endotoxin and peptidoglycan are positive: It is determined that only Gram-negative bacteria or both Gram-negative bacteria and Gram-positive bacteria are present. -If both β-glucan and peptidoglycan are positive: It is determined that both Gram-positive bacteria and fungi are present. -If the endotoxin, β-glucan, and peptidoglycan are positive, it is determined that both Gram-negative bacteria and fungi are present, or that Gram-negative bacteria, Gram-positive bacteria, and fungi are present.

The following Tables 1 and 2 show the judgment results from the color development status of each well of the specimens (1) to (6). In addition, in Tables 1 and 2, + indicates that a color is developed, and − indicates that a color is not developed. The abbreviations have the following meanings. S: Sample PC: Positive control NC: Negative control ET: Endotoxin GL: β-glucan PG: Peptidoglycan

[0029]

[Table 1]

[0030]

[Table 2]

Next, Table 3 below shows an example of the case where the detection result of microorganisms using the plate of the present invention should be judged invalid.
Each symbol in Table 3 has the same meaning as in Tables 1 and 2.

[0032]

[Table 3]

When the detection results shown in Table 3 are obtained, the results are not reliable, and it is necessary to reexamine the sample.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, the presence and type of microorganisms such as fungi, Gram-negative bacteria, Gram-positive bacteria and the like in a sample are qualitatively determined by primary screening in a laboratory or the like. At the same time as determining whether the sample is positive or negative, it is possible to confirm the deterioration of the detection reagent or the influence of the sample diluent on the detection reaction and to confirm the validity of the determination. The accuracy of can be significantly increased.

Further, the experimental operation is simple because a predetermined solution is added to each well in a fixed amount, and since each well is separated by a partition wall, the solution added to other wells may be used. It can be measured accurately without being contaminated.

[0036]

[Brief description of the drawings]

FIG. 1 is a perspective view showing an embodiment of the present invention.

FIG. 2 is a perspective view showing another embodiment of the present invention.

[Explanation of symbols]

1,1-1,1-2,1-3 Sample measurement wells 2,2-1,2-2,2-3 Positive control wells 3,3-1,3-2,3-3 Negative Control wells 4 Plate-shaped plates 5, 5a, 5b, 5C Reagent sheets for detecting microorganism-derived components

Claims (9)

[Claims] 1. A sample measuring well in which a microorganism-derived component detection reagent is located at the bottom, a positive control well in which the microorganism-derived component detection reagent is located in the bottom, and / or the microorganism-derived component detection. A set of wells for negative control with reagents for the bottom positioned,
A plate for detecting microorganisms, which comprises visually determining the presence or absence of microorganisms, characterized in that one set or a plurality of sets are formed on the plate for each type of the microorganism-derived component. 2. The plate according to claim 1, wherein the reagent for detecting a microorganism-derived component is held at the bottom. 3. The pre-treatment according to claim 1 or 2, wherein the type of the microorganism-derived component is one or more of endotoxin, (1 → 3) β-D-glucan and peptidoglycan.
G. 4. The plate according to claim 3, wherein the set of wells is formed separately for each endotoxin, (1 → 3) β-D-glucan or peptidoglycan as the microorganism-derived component. 5. The specimen measuring well and the positive control well and / or the negative control well are formed in the same row for each microorganism-derived component, and the specimen measurement well is used. 5. The plate according to claim 4, wherein the plate is formed in the same row for each use for positive control and / or for negative control. 6. The method according to claim 1, wherein the positive control well is a well to which a positive control liquid is added, and the positive control liquid is a solution containing the microorganism-derived component. Plate. 7. The negative control well is a well to which a negative control solution is added, and the negative control solution is water or a sample diluent which does not contain the microorganism-derived component. Or the plate according to item 5. 8. A sheet-like absorbent carrier carrying the reagent for detecting a microorganism-derived component or a sheet-like product of a water-soluble polymer compound containing the reagent for detecting a microorganism-derived component,
The plate according to any one of claims 2 to 7, which is held at the bottom of each well. 9. The plate according to claim 8, wherein the absorbent carrier is a woven fabric, a non-woven fabric, a filter paper or a sponge-like polymer compound.