JP2002181823A - Detection method for microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rapid detection method for a target microorganism in a sample. SOLUTION: This microorganism detection method includes a process of bringing the sample containing the microorganism into contact with a bead and a process of detecting the microorganism bound to the bead by flow cytophotometry.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting microorganisms. More specifically, the present invention relates to a method for detecting microorganisms with high sensitivity using beads and flow cytometry. INDUSTRIAL APPLICABILITY The method for detecting microorganisms of the present invention is useful in the field of microbiological testing of foods, microbiological tests in feces at health centers, hospitals, and clinical testing centers.

[0002]

2. Description of the Related Art Detection of microorganisms present in a target sample is a very important task in a wide technical field.
For example, there are various tests of persons infected with foods caused by pathogenic microorganisms and infectious diseases, and investigations of toxic plankton causing red tide. Conventional detection and identification of microorganisms is performed by pre-culturing a specimen, separating and culturing from the pre-cultured solution in various selective media, and conducting a physiological test, genetic analysis, and immunological test of the finally separated microorganism. Is being done.

[0003] Preculture of a specimen is a process that must be performed whenever the number of microorganisms in a target sample is small. In this preculture, the primary purpose is to increase the amount of microorganisms contained in a sample, unlike the selective culture in which only a certain target microorganism is selectively grown. This pre-culture depends on the amount of the sample itself and the amount of microorganisms contained in the sample, but is usually performed for about 1 to 2 days. This pre-culture increases the absolute amount of the microorganisms contained in the sample, thereby facilitating the next step of selective culture.

[0004] The selective culture is a culture method used when a plurality of microorganisms are mixed in a sample, and is usually detected (separated, separated, etc.).
Use a medium that allows only the microorganism to be identified) to grow. In this selective cultivation, depending on the growth rate of the microorganism to be detected, cultivation for about 2 to 3 days
The appearance of colonies is observed. At that time, if it is possible to separate as a single colony, pick up the colony and separately stock-cultivate it for detailed analysis, conduct microscopic observation, other physiological tests, and identify microorganisms in the sample. Become. On the other hand, when a single colony cannot be separated by selective culturing, separation is performed using another selective medium, or the concentration of the sample is changed in the same medium, and the procedure is repeated until a single colony is obtained.

[0005] The operation of detecting (separating and identifying) microorganisms by culturing a selective medium has been conventionally performed.
With the advancement of biotechnology in recent years, approaches using genetic manipulation techniques and approaches using immunological reactions have also been put to practical use in the detection, isolation, and identification of microorganisms, and simple microbial detection methods have been used in each kit. ing.

For example, as an application example of the gene manipulation technique, a method of detecting a target microorganism using PCR and a method of detecting a toxin or the like derived from a microorganism have been actually put to practical use. In immunological reactions, ELISA kits and agglutination tests utilizing antigen-antibody reactions have been put to practical use for identification of microorganisms, detection of toxins, and the like. However, the genetic manipulation technique and the technique using immunological reaction are more accurate than conventional physiological tests and microscopic observations for confirming the detection and identification of microorganisms. When only microorganisms are present, preculture is essential, and in some cases, selective culture is also required, which is no different from the conventional method.

[0007] Accordingly, although a gene analysis method using biotechnology and an antigen-antibody reaction are being used as a final method for detecting and identifying microorganisms, the time required for detecting or identifying microorganisms hardly changes. Currently, several days to one day after the start of sample processing
It currently takes a week. In tests performed at public health centers, hospitals, test centers, and the like, such as the outbreak of food poisoning caused by Escherichia coli O-157 in Sakai City, Osaka in 1996, it is required that the tests be completed as soon as possible.

However, in the existing method, if the number of microorganisms to be detected in a specimen is small, it is impossible to detect the microorganisms. Cannot be separated, it is necessary to take one day for separation and culture in a selective medium, which hinders rapid inspection. That is, in immunological techniques such as ELISA, detection was not possible unless 1 × 10 ⁴ or more target microorganisms were contained in 1 ml of the sample. In E. coli O-157, 1
It is said that the presence of 000 Escherichia coli O-157 causes the disease, and development of a highly sensitive detection method is desired.

As one of the development approaches, even when the number of microorganisms to be detected in a sample is small, the microorganisms can be selectively cultivated without pre-culturing or by adding a special reagent to the culture solution. The development of a special medium that allows selective cultivation while separating is under development. If the absolute amount of microorganisms contained in the sample is insufficient, bind the antibody that reacts with the target microorganism to the beads adsorbed on the magnet, put the magnetic beads in the sample, and remove the microorganisms contained in the sample. A method of concentrating the beads and then culturing the beads with a selective medium is also being developed. However, even if these new methods are used, preculture or selective culture can be shortened to some extent, and the test period is several days after obtaining the specimen.

In the method for detecting microorganisms without performing pretreatment such as culture on the target sample, the target sample contains at least 1 × 10 ⁴ microorganisms / ml to be detected in the target sample; Unless the amount of contaminants is reduced as much as possible, detection of microorganism genes and detection of microorganisms by antigen-antibody reaction cannot be performed. Was not practically practical.

[0011] Flow cytometry is a reaction of an antibody conjugated with a fluorescent dye specific to a cell surface molecule,
It is characterized in that cells can be separated and identified. However, at present, most of the detection by flow cytometry detects cells derived from animals such as human peripheral blood mononuclear cells, and is hardly used for detecting microorganisms. The reason is that the size of the specimen needs to be 1 μm or more for detection by flow cytometry, but the size of microorganisms such as E. coli O-157 is almost 1 μm or less. In addition, in the detection of microorganisms in stool and food, if there are contaminants of the same size as the microorganisms in size, these may cause a decrease in detection sensitivity, and if the target microorganisms are 1% or less, high sensitivity. It could not be measured. That is, when the number of the target microorganisms was 1 × 10 ⁶ or less in 1 ml of a sample such as stool, it could not be detected by flow cytometry. For the above reasons, flow cytometry has been used for detection of microorganisms for a long time, but at present, it is used only for laboratory research purposes.

As described above, there is a demand for a method for rapidly detecting microorganisms present in a target sample. However, there is a need for selective culture of microorganisms in a special selective medium, a method for concentrating a sample using magnetic beads, and a method for genetic manipulation. Even if biotechnology technology is combined, only a few days can be achieved, and there is still a need to establish a method for rapidly identifying microorganisms.

[0013]

That is, the present invention provides:
An object of the present invention is to provide a method for rapidly detecting a target microorganism in a sample. More specifically, an object of the present invention is to provide a rapid microorganism detection method capable of directly detecting a microorganism from a specimen sample without performing pre-culture. Furthermore, the present invention provides a method for measuring a microorganism concentration of 1 ×
An object of the present invention is to provide a method for detecting a microorganism which can be detected even at 10 ³ cells / ml. Further, the present invention provides
An object of the present invention is to provide a method for detecting microorganisms, which can end the process from the start of processing of a sample sample to the detection of microorganisms within several hours.

[0014]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, bound an antibody that specifically reacts with a target microorganism to a magnetic bead, and put the magnetic bead in a sample. By adding only the target microorganisms to the magnetic beads from the sample containing many impurities by adding
By reacting the magnetic bead-microorganism conjugate with an antibody labeled with a fluorescent dye and detecting the magnetic bead-microorganism-fluorescent-labeled antibody complex by flow cytometry, the sample can be obtained within a few hours after obtaining the sample. It has been found that the target microorganism can be detected with high sensitivity. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a method for detecting microorganisms, comprising the steps of contacting a sample containing microorganisms with beads and detecting the microorganisms bound to the beads by flow cytometry.

According to the first aspect of the present invention, a step of contacting a sample containing a microorganism with magnetic beads to which a first substance having an affinity for the microorganism has been bonded, the method comprising: Binding a second substance having an affinity to the microorganism to which the substance has been bound;
A method for detecting a microorganism is provided, comprising a step of detecting a complex of the microorganism and a fluorescent labeling substance by flow cytometry.

According to a second aspect of the present invention, a step of contacting a sample containing a microorganism with magnetic beads to which a first substance having an affinity for the microorganism has been contacted, A step of binding a third substance having an affinity to a substance present in the cells of the microorganism to which the labeling substance has been bound, and a step of detecting a complex of beads / microorganism / fluorescent labeling substance by flow cytometry. ,
A method for detecting a microorganism is provided.

According to the third aspect of the present invention, a step of contacting a sample containing a microorganism with magnetic beads to which a first substance having an affinity for the microorganism is bonded, A second substance having an affinity for the microorganism to which the labeling substance is bound, and a third substance having an affinity for a substance present in the cells of the microorganism having the fluorescent labeling substance bound thereto.
And a method for detecting a complex of beads / microorganisms / fluorescent labeling substance by flow cytometry.

Preferably, a first material having an affinity for a microorganism is used.
Is an antibody against the microorganism, more preferably the antibody is a polyclonal antibody. Preferably, the substance to which the fluorescent label is bound is an antibody labeled with a fluorescent dye, and more preferably, the antibody labeled with a fluorescent dye is a monoclonal antibody.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and an embodiment of the present invention will be described in detail. The method for detecting a microorganism according to the present invention includes a step of bringing a sample containing the microorganism into contact with the beads, and a step of detecting the microorganisms bound to the beads by flow cytometry. The type of the sample containing the microorganism is not particularly limited. Bacteria need to be detected in cases such as identification of bacteria that cause bacterial infections such as food poisoning and hospital-acquired hospital infections, and identification of contaminating bacteria such as water used in food factories. No. More specifically, for example, there is a case where bacteria whose presence is to be avoided are detected in environmental water and air, food and water and sewage, hospitals, precision equipment manufacturing factories, and the like where detection of bacteria is required. Furthermore, pathogenic microorganisms (particularly, O-cells) in a sample (eg, stool, urine, blood, etc.) derived from a patient such as an infectious disease infected person.
157). The type of microorganism to be detected in the present invention is not particularly limited, and bacteria such as Escherichia coli, Staphylococcus aureus, Streptococcus, Klebsiella pneumoniae, Pseudomonas, Bacillus, Clostridium, and yeasts, molds, and actinomycetes are generally used. Applicable.

The type of the beads used in the present invention is not particularly limited as long as they can specifically adsorb the microorganism to be detected. As the beads used in the present invention, it is preferable to use beads obtained by binding a substance having an affinity to a microorganism to be detected (for example, an antibody against the microorganism to be detected) to ordinary beads. The antibody that binds to the beads may be a monoclonal antibody or a polyclonal antibody as long as it is specific to the target microorganism to be detected.However, in this process, the antibody must be a polyclonal antibody because the microorganism must be strongly adsorbed. Is preferred. The method for binding an antibody to beads is known to those skilled in the art, and can be appropriately selected depending on the type of beads. For example,
Activate the beads by an appropriate method (such as tosyl activation),
A method of binding this to the amino group of the antibody can be used.

The type of beads used in the present invention may be plastic beads or magnetic beads adsorbed on a magnet, but from the viewpoint of ease of separation operation and detection sensitivity, it is preferable to use magnetic beads in the present invention. .

The size of the beads used in the present invention is particularly large as long as the target microorganism to be detected can be isolated while avoiding contamination with foreign substances, and the size allows measurement with high sensitivity. Not limited. From this perspective,
The size of the beads used in the present invention is preferably 1 μm to 10 μm, more preferably 2.8 μm to 6 μm.
m. When the size of the beads is 1 μm or less, the noise level is almost equal to the noise level. When the impurities cannot be completely removed, it is difficult to distinguish between the impurities and the noise. On the other hand, in the case of using 10 μm beads, contamination of contaminants does not pose a problem, but the size of the beads themselves varies, so that the amount of obtained fluorescence also varies greatly, and as a result, measurement with high sensitivity becomes impossible. Impossible.

In a preferred embodiment of the method for detecting a microorganism according to the present invention, a step of contacting a sample containing the microorganism with magnetic beads to which a first substance having an affinity for the microorganism has been bound; A step of binding a second substance having an affinity to the microorganism to which the fluorescent label is bound, and a step of detecting a complex of beads / microorganism / fluorescent label by flow cytometry. A second substance having an affinity for the microorganism to which the fluorescent label is bound
As the substance, an antibody labeled with a fluorescent dye is preferable.
Such a fluorescently labeled antibody may be a monoclonal antibody or a polyclonal antibody as long as it is an antibody specific to the microorganism to be detected. In this process, it is necessary to avoid non-specific binding to microorganisms, so that a monoclonal antibody is preferred as the fluorescently labeled antibody used here.

That is, according to a particularly preferred embodiment of the present invention, a sample containing a microorganism is contacted with magnetic beads to which a polyclonal antibody against the microorganism has been bound, and then a fluorescent labeling substance is bound to the microorganism adsorbed on the beads. And a monoclonal antibody against the microorganism. The magnetic beads are required to capture only the target microorganism from the sample, but if this is relaxed, the target microorganism will not be missed, but other microorganisms may also be captured by the magnetic beads. On the other hand, if this is made strict, the capture rate of other microorganisms can be reduced as much as possible, but there is a risk that the capture of the target microorganism will fail. Therefore, in the present invention, it is preferable in the present invention to use a monoclonal antibody having a strict specificity as a fluorescent-labeled antibody, in which the microorganism is adsorbed with a polyclonal antibody somewhat broadly in the microorganism adsorption process.

The type of the fluorescent labeling substance used in the present invention is not particularly limited as long as it can be detected by flow cytometry. For example, phycoerythrin (PE), FITC
In addition to these, ethidium bromide (Ethidium bromide), propidium iodide (Pr
opidium iodide), Hoechst 33258/33342,
DAPI, Acridine orange, Chromomycin, Mithramyci
n), Olivomycin, Pyronin
n) Y, Thiazole orange, Rhodamine 101 isothiocynate
anate), BCECF, BCECF-AM, C.SNARF-1, C.SNARF-1-AM
A, Aequorin, Indo-1, Indo-1-AM, Fluo
-3, Fluo-3-AM, Fura-2, Fura-2-AM, oxonol (O
xonol), Rhodamine 123, 10-N-nony-acridine orange, Fluorescein, Fluorescein diacetate (Flu
orescein diacetate), carboxyfluorescein (Car
boxyfluorescein) (CF), carboxyfluorescein diacetate (CFDA), carboxydichlorofluorescein (Carboxydichlorofluore)
scein) (CDCF), carboxydichlorofluorescein diacetate (CDC
FDA). The fluorescent-labeled antibody used for detection by flow cytometry may be such that the above fluorescent substance is directly bound to the antibody, or biotin is labeled on the antibody, and avidin is bound to the fluorescent substance. These labeling protocols are described in the protocol collection "Cu
rrent Protocols in Cytometry '' (ISAC (The Internatio
nal Society for Analytical Cytology)) 4.2. chapter
Conjugation of fluorochromes to monoclonal antibod
Described in y. The same contents can be viewed on the Internet, and the address (HYPERLI
NK "http://www.drmr.com/abcon/" http://www.drmr.co
m / abcon /) Conjugation of monoclonal antibodies
Can be labeled according to this method.

A preferred embodiment of the present invention will be described below, taking as an example the case where the microorganism to be detected is Escherichia coli O-157. First, an antibody that specifically reacts with E. coli O-157 is bound to the magnetic beads. Next, the antibody-bound magnetic beads are put into a target sample, mixed well, and Escherichia coli O-157 is adsorbed on the antibody-bound magnetic beads, and the remaining sample is discarded while adsorbing only the magnetic beads by a magnet. Then, after contaminants (microorganisms other than Escherichia coli O-157) non-specifically adsorbed to the antibody-bound magnetic beads with a phosphate buffer or the like, phycoerythrin (PE)
The phycoerythrin-labeled antibody-E. Coli O-157
Detecting the complex of magnetic beads by flow cytometry.

Flow cytometry is a method in which isolated cells are made into a suspension, passed through a flow cell at high speed, and the cells are measured by an optical method. In flow cytometry, it is necessary to stain microorganisms with a fluorescent dye, and the fluorescence emitted during the process of a sample passing through a microtube is measured by a high-sensitivity detector. That is, in flow cytometry, usually, the test liquid is irradiated with excitation light while flowing the test liquid through a fine tubular flow cell having an inner diameter of about several tens μm to several hundreds μm, and the fluorescence emitted in the flowing test liquid is emitted. Receive and detect.

Further, according to the present invention, a step of contacting a sample containing a microorganism with a magnetic bead to which a first substance having an affinity for the microorganism is bound, and binding a fluorescent labeling substance to the microorganism adsorbed on the bead. A step of binding a third substance having an affinity to a substance present in the cells of the microorganism thus caused, and a step of detecting a complex of beads / microorganism / fluorescent labeling substance by flow cytometry. A method is provided for detecting a substance present in a cell.

[0030] Among the microorganisms, there are microorganisms that produce special toxins. Some of these toxins are not released outside the cells after production, but are stored in the cells. If such toxins in the cells could be detected, it would be possible to directly detect microorganisms that produce special toxins. In addition, there may be bacteria that produce a toxin and bacteria that do not produce a toxin even if they are the same kind of microorganism. In such a case, even if it is determined to be positive by the above-described method for detecting a microorganism according to the present invention, bacteria that do not produce a toxin may be detected. Therefore, direct detection of toxins in microorganisms such as bacteria is extremely important for clinical diagnosis.

As the third substance used in the present invention, which has an affinity for the substance present in the cells of the microorganism to which the fluorescent labeling substance is bound, an antibody labeled with a fluorescent dye is preferable. As such a fluorescently labeled antibody, a monoclonal antibody or a polyclonal antibody may be used as long as it is an antibody specific to an intracellular substance to be detected, but a monoclonal antibody is preferred. The kind of the fluorescent labeling substance mentioned here is not particularly limited as long as it can be detected by flow cytometry, and specific examples include those described above in this specification.

As an example of a combination of such a bacterium and a toxin produced thereby, Escherichia coli O-157 and a verotoxin produced thereby can be mentioned. E. coli O-1
The method of the present invention will be specifically described below, taking as an example the case of detecting verotoxin produced by 57. First,
An antibody that specifically reacts with Escherichia coli O-157 is bound to the magnetic beads, and the antibody-bound magnetic beads are placed in a target sample that has been previously subjected to a heat treatment to form holes in the cell membrane of a microorganism such as Escherichia coli. After mixing, the E. coli O-157 is adsorbed on the antibody-bound magnetic beads, and the remaining sample is discarded as it is while only the magnetic beads are adsorbed by the magnet. Next, impurities (specifically, microorganisms other than Escherichia coli O-157) adsorbed nonspecifically to the antibody-bound magnetic beads with a phosphate buffer or the like.
After washing away, a specific antibody is reacted with phycoerythrin (PE) -labeled verotoxin, and the complex of phycoerythrin-labeled anti-verotoxin antibody-E. Coli O-157-magnetic beads is subjected to flow cytometry. Detect with.

Further, in the present invention, together with a second substance having an affinity for the microorganism to which the fluorescent label is bound, the substance having an affinity for the substance present in the cells of the microorganism to which the fluorescent label is bound is attached. By using together with the third substance having the above, it is also possible to detect the substance in the cells of the microorganism simultaneously with the detection of the microorganism. In this case, a fluorescent label of the second substance having an affinity for the microorganism,
Although it is different from the fluorescent label of the third substance having an affinity for the substance present in the cell to the toxin or the like, it is preferable that it can be directly and simultaneously detected by a laser having the same wavelength.

[0034]

The present invention will be described in more detail with reference to the following examples. In this example, Escherichia coli O-157 was detected, but E. coli O-157 was selected by selecting an antibody.
Detection of other bacteria is also possible. That is, the materials, reagents, ratios, operations, and the like shown in the following examples can be appropriately changed without departing from the spirit of the present invention, and the scope of the present invention is not limited to the specific examples shown below. .

Example 1: Preliminary study (1) Examination of optimal size of beads The optimal size of beads that can be measured by flow cytometry was examined. That is, 1 μm, 2.8 μm, 3 μm,
6 μm, 10 μm polystyrene beads and magnetic beads were measured by flow cytometry, and FSC (forward scattered light: correlated with cell and bead size), SSC (side scattered light: reflected cell and bead shape) Was measured to determine the size of beads suitable for flow cytometry measurement.

The apparatus for flow cytometry used in the examples was FACScan (registered trademark) (Becton Dic
Kinson). Therefore, the excitation wavelength 48
This system is excited by an 8 m air-cooled argon laser (output 15 mW) and detected by PMT. Measurement conditions are FLUID
Control was set to Low, and the measurement was completed when 10,000 samples were taken in. The settings of fluorescence (FL1 to FL3) -scattered light (FSC, SSC) were as follows.

FIG. 1 shows the results. As can be seen from the results in FIG. 1, 2.8 μm, 3 μm, and 6 μm correspond to the C of the beads.
V was also found to be good and could be detected as a coherent population.

(2) Examination of Types of Beads As beads for capturing microorganisms, plastic beads,
Various types of beads such as magnetic beads that are attracted to magnets
There are, however, these beads are the target among many contaminants.
We examined whether microorganisms could be detected with high sensitivity. <Method using plastic beads> Sample (10
% Suspension) with anti-O-157 antibody fixed 3μm bead
And then centrifugally washed, and FITC anti-O-157 antibody
(PoAb) and centrifugally washed beads into PBS
Suspended. The sample was E. coli O-157: H7 (1 × 1
0 ⁷, 1 × 10⁶, 1 × 10^Five, 1 × 10^Four/ Ml)
And the mixed bacteria are Salmonella typhimrium (1 × 10⁹Pieces / m
l).

FIG. 2 shows the outline of the method and the results. With plastic beads such as polystyrene, it is possible to distinguish between beads and other contaminants such as microorganisms in the sample. After that, data had to be captured. In this method, detection was not possible unless 1 × 10 ⁵ or more target microorganisms were present in the sample (FIG. 2).

<Method Using Magnetic Beads> Sample (1
0% suspension), magnetic beads with anti-O-157 antibody were added thereto, adsorbed by a magnet, FITC anti-O-157 antibody (PoAb) was added, and the beads adsorbed by the magnet were added to PBS.
Suspended in water. The sample was E. coli O-157: H7 (1 ×
10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ cells / ml), and the mixed bacteria are Salmonella typhimrium (1 × 10 ⁹ cells / ml).

The outline of the method and the results are shown in FIG. After reacting the sample with the magnetic beads conjugated with the anti-O-157 antibody, the magnetic beads are adsorbed by a magnet, so that 1 × 10 ⁴ or more E. coli O-157 can be detected in the sample. (FIG. 3). However, even when magnetic beads were used, it was clarified that setting a gate for fractionation of beads was a condition for increasing sensitivity (FIG. 3).

Example 2: E. coli O- using magnetic beads
Detection of 157 (1) Binding of antibody to magnetic beads In order to capture E. coli O-157 in a sample, it is necessary to immobilize an antibody that specifically reacts with E. coli O-157 on the magnetic beads. As an antibody used for
It was examined whether a polyclonal antibody or a monoclonal antibody was suitable. That is, a polyclonal antibody (anti-O-157 antibody) obtained by binding an antibody of KPL to magnetic beads (tosyl-activated magnetic beads: 2.8 μm, Dynabeads) and a monoclonal antibody (anti-O-157 antibody)
Antibody 57 (ATCC HB-10452) was bound to magnetic beads (tosyl-activated magnetic beads: 2.8 μm, Dynabeads), and the extent to which Escherichia coli O-157 could be captured was examined. As an immobilization method, the amino group of the antibody was bound to the tosyl-activated magnetic beads. The measurement conditions were E. coli O-157: H7 (1 × 10 ⁶ , 1 ×
10 ⁵ , 1 × 10 ⁴ , 1 × 10 ³ beads / ml) and the bead concentration is 5 × 10 ⁵ beads / ml.

FIG. 4 shows the obtained results. As shown in FIG. 4, the use of a polyclonal antibody to capture Escherichia coli O-157 from the sample was approximately 1000 times higher and could be detected.

(2) Examination of Fluorescent Dye and Antibody Species of Fluorescent Labeled Antibody for Detection In order to surely capture Escherichia coli O-157 from a sample, it is more sensitive to use magnetic beads to which a polyclonal antibody is bound. Was found, but finally E. coli O-1
Preferred antibodies for detecting magnetic beads capturing 57 were examined. First, whether a monoclonal antibody or a polyclonal antibody was suitable as an antibody for detection was examined. In addition, FI is used as a fluorescent dye.
TC and phycoerythrin (PE) were also examined.
Antibodies for examination include polyclonal antibodies (anti-O-15
7 antibody) and the monoclonal antibody (anti-O-157 antibody) was ATCC HB
-10452 was used. The measurement conditions were E. coli O-15.
7: H7 (1 × 10 ⁷ , 1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 1
0 ^4, 1 × a 10 ³ / ml), bead concentration is 5 × 1
0 is ^five / ml.

FIG. 5 shows the obtained results. As shown in FIG. 5, it was found that the polyclonal antibody was more sensitive than the detection antibody, and that PE was a highly sensitive fluorescent dye. However, using a polyclonal antibody for capturing Escherichia coli O-157 and using a polyclonal antibody for detection may lead to a determination of negative as positive. When a monoclonal antibody is used for detection, the sensitivity is lower than the polyclonal antibody, but the specificity is high. If PE is used as the fluorescent dye, a monoclonal antibody can be detected with high sensitivity. Therefore, a PE-labeled monoclonal antibody (ATCC: H
B-10452) was found to be preferable.

Example 3 Examination of Various Conditions for Detection Reaction (1) Examination of Sample Amount Depending on the type of specimen, a small amount or a sufficient amount of the sample may be obtained. Therefore, E. coli O-
We examined how many samples were needed to detect 157. Escherichia coli O-157 was added to a 10% suspension (a rabbit stool was diluted to 10% with PBS (phosphate buffer solution) and then filtered with a 100 μm filter) at a concentration of 1 × 10 ³ / ml to 1 × 10 ^7. / Ml, and reacted with magnetic beads to which a polyclonal antibody was bound and a PE-labeled monoclonal antibody. The sample liquid volume is 0.25 ml, 0.5 ml, 1.0 ml,
2.0 ml and 4.0 ml. FIG. 6 shows the obtained results. As shown in FIG. 6, it can be seen that if the sample amount is 250 μl, there is no difference from the case where there is a 4 ml sample, and it can be said that this method is also effective when a test must be performed based on a small amount of sample.

(2) Examination of Reaction Time The time required to capture Escherichia coli O-157 from the specimen and the reaction time of the fluorescently labeled antibody were examined. Sample is E. coli O
-157: H7 (1 × 10 ⁶ / ml), the beads were anti-O-157 antibody-bound magnetic beads (2.5 × 10 ⁴ / sample), and the antibody for detection was FITC-labeled anti-O -157 antibody (PoAb) with a reaction time of 0.5
Minutes to 90 minutes. FIG. 7 shows the obtained results. The time required for the E. coli O-157 in the sample to bind to the antibody-bound magnetic beads is 30 minutes as shown in FIG. 7, and the reaction time of the fluorescently labeled antibody for detection is as shown in FIG. It has been found that more than 20 minutes is sufficient.

(3) Examination of Stirring When capturing Escherichia coli O-157 from a specimen using magnetic beads, the specific gravity of the magnetic beads is large (1.45 g / c).
m ³ ), the magnetic beads immediately precipitate if left still during the reaction. Therefore, a comparative study was conducted between a rotating method (a method of stirring a sample while rotating it), a method of performing pipetting for sucking / discharging a liquid with a pipette at intervals of 5 minutes, and a stationary method. The sample was E. coli O-157: H7 (1 ×
10 ⁷ , 1 × 10 ⁵ , 1 × 10 ³ / ml), and the beads were anti-O-157 antibody-bound magnetic beads (5 × 10 ⁵ / ml).
ml), and the antibody for detection was FITC-labeled anti-O-1.
57 antibody (PoAb) and the reaction volume is 4 ml. The results obtained are shown in FIG. As shown in FIG. 9, the detection sensitivity is deteriorated when left standing, but it is found that the rotation and pipetting have good detection sensitivity, and it is clear that detection can be performed with high sensitivity by stirring with either method. I made it.

(4) Examination of Washing Solution After magnetic beads were added to the sample and allowed to react, only the magnetic beads were adsorbed by a magnet, and the other sample solutions were discarded (aspirated and removed with a pipette), and then a fluorescent label for detection was used. An antibody will be added. The study was conducted on the necessity of washing before adding the fluorescently labeled antibody, with what kind of washing solution it should be washed, and on how many washings it was necessary to wash. The sample was E. coli O-15
7: H7 (1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ pieces / m
l), the beads are anti-O-157 antibody-bound magnetic beads (2.5 × 10 ⁴ / sample), and the antibody for detection is FITC-labeled anti-O-157 antibody (PoAb). In the examination of the number of washings, the number of washings was 0 using PBS.
Once, twice or once. In the examination of the type of washing solution, PBS, 0.05% Tween 20-PBS,
0.1% BSA-PBS was used.

The results obtained are shown in FIGS. 10 and 11.
As shown in FIGS. 10 and 11, cleaning is necessary, and a detergent having a detergency (such as Tween 20) is used as a cleaning liquid.
It was clarified that the solution to which the protein and the protein (BSA) were added had a detergency and that the number of times of washing was sufficient.

Example 4 Detection Protocol A protocol for detecting Escherichia coli O-157 with high sensitivity based on the results of Examples 1 to 3 is shown below. (1) Dispense a sample. If the sample volume is 250 μl or more, detection is possible. (2) Add antibody-labeled magnetic beads. A polyclonal antibody-bound antibody is used at a concentration of 2 × 10 ⁴ cells / sample. (3) Perform the reaction. Stirring can be performed by either rotation or pipetting, and the reaction time is 30 minutes or more. (4) Wash (0.01% BSA or 0.05%
Wash with PBS containing Tween 20 at least once. (5) Suspend the sample in PBS (250 μl to 1
ml). (6) Add a fluorescently labeled antibody. (7) Perform the reaction. Stirring can be performed by either rotation or pipetting, and the reaction time is at least 20 minutes. (8) Wash. 0.01% BSA or 0.05%
Wash with PBS containing Tween 20 at least once. (9) Suspend (200 μl) in PBS. (10) Measure by flow cytometry.

Example 5: Flow cytometric detection of bacterial endotoxin (verotoxin) A characteristic of E. coli O-157 is that it produces a toxin called verotoxin. It has also been reported that this verotoxin is not released one after another after being produced, but is once stored in the cells. If Vero toxin in the cells can be detected, direct detection of toxin-producing Escherichia coli O-157 becomes possible. Some of Escherichia coli O-157 and its distant bacteria do not produce toxins. Even if a positive result is obtained by the aforementioned method for detecting Escherichia coli O-157, the bacteria may not produce toxins. Therefore, direct detection of toxins in bacteria is extremely important for clinical diagnosis. Therefore, it was examined whether or not it is possible to detect the vero toxin in the cells by modifying the aforementioned method for detecting bacteria.

The antibody cannot react with the vero toxin in the cells without any treatment. However, if it is possible to make a hole through which the antibody penetrates into the cells while maintaining the shape of the cells, it is possible to detect the verotoxin in the cells with the antibody. Therefore, as a means for allowing the antibody to react with the verotoxin while maintaining the shape of the cells, detection was performed using a sample that had been subjected to heat treatment of the cells (95 ° C., 5 minutes). The detection protocol uses Escherichia coli heat-treated as a sample in advance, captures Escherichia coli O-157 with magnetic beads to which anti-Escherichia coli O-157 is bound, and then detects Verotoxin in Escherichia coli with a fluorescently labeled anti-verotoxin antibody. Considered by method.

As the measurement conditions, the sample was E. coli O-
157: H7 (1 × 10 ⁶ / ml), magnetic beads were used at 5 × 10 ⁵ / ml, and anti-VT was used as an antibody.
1,2 monoclonal antibody (clone 19) and FITC
A labeled anti-mouse Ig antibody (PharMingen) was used. FIG. 12 shows the obtained results. As shown in FIG. 12, E. coli O-157 producing verotoxin was detectable.

Example 6: Detection sensitivity of Escherichia coli O-157 Based on the results of the above examination, detection sensitivity was measured using 10% suspension of rabbit stool to which various amounts of Escherichia coli O-157 were added as samples. The sample was E. coli O-
157: H7 (in 10% suspension) (1 × 1
^{0 7, 1 × 10 6,} 1 × 10 5, 1 × 10 4, 1 × 10 3,
1 × 10 ² beads / ml), the magnetic beads were used at 5 × 10 ⁵ beads / ml, the reaction solution volume was 1 ml, and the reaction time was 60 minutes. FIG. 13 shows the obtained results. FIG.
As can be seen from FIG. 3, the concentration of E. coli O-157 was 1 × 1
0 was able to distinguish a ³ cells / ml, even positive.

In this regard, commercially available kits for the same sample are used.
(VIP: Gunze Sangyo, TECRA: Safety Limi)
Tid). VIP FOR E
When using HTC (E. coli O157) (Gunze Sangyo)
Means that the sample is E. coli O-157: H7 (10% suspension)
(Pending) (1 × 10 ⁸, 1 × 10⁷, 1 × 10⁶,
1 × 10^Five, 1 × 10^Four, 1 × 10^ThreePcs / ml),
The liquid volume is 0.1 ml and the reaction time is 10 minutes.
TECRA E.coli O157 VISUAL IMMUNOASSAY (Sefte
Sample), the sample should be colon
Bacteria O-157: H7 (in 10% suspension) (1 ×
10⁸, 1 × 10⁷, 1 × 10⁶, 1 × 10^Five, 1 × 1
0^Four, 1 × 10^Three, 1 × 10^Two/ Ml) and the liquid volume is
0.2 ml and the reaction time is 2 hours.

When VIP is used, 1 × 10⁸, 1 × 1
0⁷, 1 × 10⁶Positive in cells / ml, but less than that
Was not positive. In TECRA, 1 ×
10 ⁸~ 1 × 10^FiveDepends on the concentration of O-157 in cells / ml
Color development was recognized and determined to be positive, but 1 × 10^FourPieces
/ Ml was recognized as positive with the same coloration as the negative control
I couldn't.

[0058]

The method of the present invention has the following advantages. (1) Microorganisms can be directly detected from a specimen sample without performing pre-culture. That is, stool or food directly collected from the subject can be used as a sample, and the sample is pre-cultured in advance,
It is unnecessary to grow the target microorganism to be detected by selective culture. (2) Detection is possible even when the concentration of microorganisms in the sample is 1 × 10 ³ cells / ml. (3) The process from the start of sample processing to the detection of microorganisms can be completed within several hours.

[Brief description of the drawings]

FIG. 1 shows the results of studying the optimal size of beads used in flow cytometry.

FIG. 2 shows an outline of the method using plastic beads and the results thereof.

FIG. 3 shows an outline of a method using magnetic beads and the results thereof.

FIG. 4 shows the results of a study of an antibody for immobilizing beads.

FIG. 5 shows the results of examination of an antibody for detecting O-157.

FIG. 6 shows the results of a study on the amount of a sample liquid.

FIG. 7 shows the results of a study on the reaction time between a sample and magnetic beads.

FIG. 8 shows the results of a study on the reaction time of a fluorescently labeled antibody.

FIG. 9 shows the results of a study of the presence or absence of stirring and stirring conditions.

FIG. 10 shows the results of a study of cleaning conditions (the number of times of cleaning).

FIG. 11 shows the results of a study of cleaning conditions (the type of cleaning solution).

FIG. 12 shows the results of a study of the detection of verotoxin.

FIG. 13 shows the results of evaluation of the magnetic bead agglutination method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/569 G01N 33/569 Z

Claims (8)

[Claims] 1. A method for detecting microorganisms, comprising: contacting a bead with a sample containing the microorganism; and detecting the microorganisms bound to the beads by flow cytometry. 2. A step of contacting a sample containing a microorganism with a magnetic bead to which a first substance having an affinity for the microorganism has been bound, wherein the microorganism adsorbed on the bead and the microorganism having a fluorescent labeling substance bound thereto The method for detecting a microorganism according to claim 1, comprising a step of binding a second substance having affinity and a step of detecting a complex of beads / microorganism / fluorescent labeling substance by flow cytometry. 3. A step of contacting a sample containing a microorganism with magnetic beads to which a first substance having an affinity for the microorganism has been bound, wherein the microorganism adsorbed on the beads has a microorganism labeled with a fluorescent labeling substance. The method according to claim 1, further comprising: binding a third substance having an affinity to a substance present in a cell; and detecting a complex of beads / microorganism / fluorescent labeling substance by flow cytometry. Detection method. 4. A step of contacting a sample containing a microorganism with a magnetic bead to which a first substance having an affinity for the microorganism has been bound, wherein the microorganism adsorbed on the bead and the microorganism having a fluorescent labeling substance bound thereto A step of binding the substance present in the cells of the microorganism to which the second substance having the affinity and the fluorescent labeling substance are bound and the third substance having the affinity,
The method for detecting a microorganism according to claim 1, comprising a step of detecting the complex of beads / microorganism / fluorescent labeling substance by flow cytometry. 5. The first substance having an affinity for a microorganism,
The method for detecting a microorganism according to any one of claims 2 to 4, which is an antibody against the microorganism. 6. The method for detecting a microorganism according to claim 5, wherein the antibody against the microorganism is a polyclonal antibody. 7. The method for detecting a microorganism according to claim 2, wherein the substance to which the fluorescent labeling substance is bound is an antibody labeled with a fluorescent dye. 8. The method for detecting a microorganism according to claim 7, wherein the antibody labeled with a fluorescent dye is a monoclonal antibody.