JP2000342300A - Method for detecting bacterium and device for detecting bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the sensitivity of detection, when a bacterium is dyed with a fluorescent substance or the like by the use of bacteriophage and then detected. SOLUTION: This method for detecting a bacterium comprises multiply dyeing the detection target bacterium with two or more kinds of dyeing substances having optically different detection wavelengths and then detecting the dyed bacterium with the detection wavelengths of the multiply dyed dyeing substances. The bacterium detected with the detection wavelengths, respectively, is discriminated to be the detection target bacterium. Therein, at least one of the dyeing substances is a substance obtained by labeling the nucleic acid of a bacteriophage specifically adsorbed to the detection target bacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting bacteria such as E. coli.

[0002]

2. Description of the Related Art Bacteria and other microorganisms exist in various environments and maintain a close relationship with human life. However, some microorganisms cause disease in humans or livestock, and attention has been paid to human or livestock infection as a pathogenic microorganism. Among pathogenic microorganisms, bacteria are particularly fast-growing, less susceptible to environmental conditions such as low temperature, high temperature, and dryness. Increase and cause infection. From these facts, it is used not only for foods and drinking water (including tap water) that are directly ingested into the human body, but also for washing water and dishes used in food manufacturing processes. Such as water, rivers, lakes, marshes, seas, pools, baths, and other amenities for the use of scenic water, such as water for human contact and sewage discharge water.
For water that has a chance to come into contact with humans, proper management of bacteria is desired.

On the premise of the above, tap water used as food and drink is based on the criterion that "Escherichia coli group should not be detected". The following values are set. The target values are set such that the water for use in the scenic landscape is preferably "10 or less per 1 ml", and the general wastewater is preferably "3000 or less per 1 ml". As described above, the management criteria for items to check for the presence of enterobacteria, especially Escherichia coli and Escherichia coli, are set for each of the above-mentioned various types of water used (food, drinking water, or water for amenities at hydrophilic facilities, etc.). This is reasonable given that bacterial infections are most frequently caused by bacteria of human or animal origin, and the damage is likely to spread.

[0004] By the way, for example, the detection of bacteria necessary for controlling food, drinking water (tap water), pool water, water for landscaping and the like as described above has conventionally been generally carried out by a culture method. I have. In this culture method, for example, in the case of Escherichia coli or Escherichia coli group, for example, it is necessary to devise a method of detecting this in distinction from other general bacteria. Then, the culture is performed using a medium whose color tone is changed or a special color is generated in the color of the colony. However, this method requires a long time for the bacteria to grow by culturing, and in addition, the method for determining whether the grown bacteria are Escherichia coli or Escherichia coli is complicated, so that it takes more than several days until the final determination. There is a problem that it takes. In addition, in the case of using a method of obtaining from a general maximum dilution factor, there is a problem that only an approximate quantitative estimation value is obtained, and the accuracy of quantitative performance is poor.

[0005] Apart from such a simple culture method, a specific enzyme substrate medium method is also known as a simplified detection method. In this method, for example, a sample is cultured in the presence of a substrate for an enzyme specifically contained in Escherichia coli or an Escherichia coli group, and if positive, a characteristic color or fluorescence is generated.

In addition to the above methods, a PCR method (polymerase chain reaction method), a method for measuring ATP,
Furthermore, a method of binding a bacteriophage to the surface of a bacteriophage using a radioisotope and an enzyme as a label proposed in JP-A-8-154700,
A method of labeling NA by genetic recombination is also known.

However, in the conventional methods described above, problems that are extremely difficult to solve from various points such as quickness of processing, simplicity of processing operation, high detection accuracy, and the like are considered as follows from the viewpoint of actual implementation. There is like.

For example, the qualitative test for Escherichia coli and Escherichia coli is more difficult in the specific enzyme substrate medium method than in the simple culture method.
Although there is an advantage that it can be performed in about a time, it is not possible to detect all Escherichia coli and coliforms, and if a quantitative test is to be performed, it must be performed in the form of obtaining the positive value at the maximum dilution factor, and the measurement method There is a limit to the accuracy of quantitative detection derived from

Although the PCR method has a high sensitivity, it detects dead and actually harmless bacteria, so that there is a problem that dead bacteria and live bacteria cannot be distinguished from each other, and quantitativeness cannot be expected. .

[0010] The ATP method is excellent in that it is easy to operate and can specifically detect only live bacteria, but has a problem that its detection sensitivity is low (it cannot be detected unless there are more than 1,000 bacteria). In addition, there is a fatal problem in some applications that bacteria cannot be identified and detected.

[0011] Among the methods described in the above-mentioned JP-A-8-154700, a labeling method using a radioisotope (isotope) or an enzyme on the phage surface by labeling the phage surface is as follows:
There is a problem in processing operability and detection accuracy. That is, in the method using a radioisotope, the signal derived from the radioisotope label of the phage adsorbed on the bacteria cannot be distinguished from the signal derived from the label of the non-adsorbed phage. Extra operation to physically separate the phage is required. In addition, there is a risk of fatal erroneous detection in which radioactive isotopes labeled on phages non-specifically adsorbed to bacteria (or separation means such as a filtration membrane) are counted. In addition, the method of using an enzyme as a label has the problem of non-specific adsorption as in the case of using a radioisotope, and also uses a small amount of enzyme activity. There is a problem that it is not always easy to accurately detect a minute amount of an enzyme due to a phenomenon appearing on a substrate to be subjected to the reaction, and that a new technology development for improving the quantitative detection accuracy is required.

Further, a method using a reporter gene is as follows:
It is necessary to prepare a transgenic reporter gene for each phage specific to each bacterium, and it is necessary to perform replication, transcription, and translation of the phage nucleic acid containing this reporter gene, and to perform an operation to detect the produced protein. Therefore, versatility can hardly be expected.

As described above, all of the conventionally known methods have problems, and there are still many problems to be solved particularly in terms of speed, simplicity of operation, detection accuracy, detection sensitivity, and the like.

Therefore, the present inventor has solved the above-mentioned problems of the conventional method, and has made it possible to detect and judge a specific bacterium by specifically labeling the bacterium itself. Bacteriophage nucleic acid (DNA) is a method that can shorten the time, increase the efficiency, and increase the sensitivity.
A method using a bacteriophage (hereinafter referred to as “phage”) labeled with a dye or a fluorescent substance (hereinafter referred to as “phage DNA”) has already been proposed (JP-A-10-323).
178).

The invention according to this proposal has the following features.
That is, a phage that is a bacterial virus specifically binds to a host bacterium, injects phage DNA into living cells only depending on the membrane potential of the bacterium, and injects the phage DNA into the cells. If the phage DNA is labeled with a dye or a fluorescent substance, the whole host bacterium is stained to exhibit a large bright spot, because the phage DNA has a property of spreading (developing) in a spatially wide cell. Bacterial fluorescence exhibits a large, bright state that can be distinguished from tiny phage particles. Therefore, a bacterium (ie, a specific living bacterium) into which phage DNA labeled with a fluorescent substance has been injected and stained by phage infection can be optically easily detected in a short time by using the fluorescent substance. This advantage can be obtained not only for the fluorescent substance but also when a dye is used as the labeling substance.

According to the proposed invention, a special phage DNA is prepared by a DNA recombination operation for each phage (that is, for each phage having a specific relationship with a bacterium), as in the above-described gene recombination method for the reporter gene. This is advantageous in that phage DNA can be labeled using a common, identical and single universal operation.

[0017]

By the way, the detection of the target bacterium by the method of the above-mentioned Japanese Patent Application Laid-Open No. 10-323178 is as follows.
For example, a certain amount of phage labeled with a nucleic acid (DNA) by a staining substance is added to a sample solution to be measured, and the phage is brought into contact with a host bacterium to be stained, introduced into a detector, and a signal (color, Fluorescence), but the present inventor has found through further studies that the following problems are further encountered in order to carry out this method on an actual industrial level.

That is, depending on the properties of the sample liquid to be measured, for example, if the sample liquid contains a detergent or finely divided paper waste, these often contain impurities that emit color or fluorescence. Substances may be included from the beginning, and the signals emitted by these contaminants are indistinguishable from the signals of the host bacteria stained by the labeled phage during detection, and are erroneously detected as the target bacteria,
It can greatly affect detection accuracy and sensitivity. Therefore, when implementing the subject technology of the present invention on an industrial level, its solution is important. Furthermore, if the size of the contaminants is different from the target bacteria, it can be physically removed by a method such as membrane filtration, but those with similar sizes can be removed. There is a problem of difficulty.

The present inventor has studied the above problems, and has conducted further research and development from the viewpoint that in actual practice at the industrial stage, it is necessary to devise a more concrete implementation stage. It came to be.

[0020]

In order to achieve the above-mentioned object, the present invention provides the invention described in each of the above-mentioned claims.
The invention of the method for detecting bacteria according to the present invention is directed to a method for subjecting a bacterium to be detected to multiple staining with two or more kinds of staining substances having different optical detection wavelengths, and wherein the staining is detected at each detection wavelength of the multiple stained staining substance. In which at least one of the staining substances is labeled with a nucleic acid of a bacteriophage that specifically adsorbs to the bacteria to be detected.

Examples of combinations of bacteria and phages specific thereto include Escherichia coli and T-type phages, λ phages, etc., Salmonella and P-type phages, Pseudomonas and P-type phages, Klebsiara and phages of Stanford University 60 and 92. , Clostridium and each phage of 70, 71, 72, Shigella and φ80, Stanford University 37, D
20, Corynebacterium and C-type phage, Micrococcus and N-type phage, ML53-40 phage, and the like.

According to the present invention, since the host bacterium to be detected is multi-stained, it can be effectively distinguished from the color or fluorescence emitted by the contaminant, and the detection accuracy and sensitivity can be increased.

The inventions of claims 2 to 8 of the present application show specific combinations of the adsorbing substances for multiple staining of the above-mentioned bacteria. Nucleic acids are multiplexly labeled with two or more types of staining substances having different fluorescence detection wavelengths, and a bacteriophage that specifically adsorbs to the host bacterium to be detected is used. 4. The invention of the method for detecting bacteria according to claim 3, wherein the bacteria are multi-stained by injecting them into the host bacteria, and the bacteria to be detected are discriminated and measured by optically detecting the multi-stained bacteria. Is
Nucleic acids are labeled with staining substances having different color or fluorescence detection wavelengths, and at least two or more phages specifically adsorbed to the same host bacterium for the same detection purpose are used, and these phages are contacted with the host bacterium to label. The method is characterized in that bacteria are multi-stained by injecting nucleic acid into host bacteria, and the multi-stained bacteria are optically detected to discriminate and measure the bacteria to be detected. The phages used in this claim 3 may be the same phage or different phages.

Further, the invention of claim 4 is a method for specifically adsorbing at least one kind of phage whose nucleic acid is labeled with a staining substance capable of detecting color or fluorescence to a bacterium to be detected and placing the labeled nucleic acid in the bacterium. Injected and stained, the nucleic acid-labeled staining substance is another staining substance having a different color or fluorescence detection wavelength, and is capable of directly or indirectly staining at least the same bacteria for detection. In this method, the bacteria to be detected are multiply stained by staining the bacteria, and the bacteria to be detected are discriminated and measured by optically detecting the bacteria that have been multiply stained.

In the above description, “indirectly staining” means, as described later, a second substance that binds to the first substance by intermediating (bridging) the first substance adsorbed on the bacterial surface. Refers to labeling a substance with a staining substance and staining the substance.

The above-mentioned inventions have a common feature that at least one of the two types of labeling with a staining substance is always a phage nucleic acid. On the other hand, other staining substances other than those described above may be those that label the nucleic acid of the same phage multiple times as in the invention of claim 2 or the same host bacteria as in the invention of claim 3. The nucleic acid of another specific phage may be labeled. Examples of dyes for labeling phage nucleic acids include propidium iodide (hereinafter, referred to as “PI”) and ethidium bromide. Examples of fluorescent substances include acridine orange (C ₁₇ H ₂₀ ClN ₃ : hereinafter “ACO”).
R ": (excitation wavelength: nm) ex488, (fluorescence wavelength: nm) em530), 4,6-diamidino-2
-Phenylindole hydrochloride (DAPIex358, em461
), PI, ethidium homodimer and the like. In a preferred embodiment, the staining with the other staining substance may include either a case of directly staining a bacterium to be detected as in the invention of claim 4 or a case of indirectly staining the bacterium. Like the invention of 5,
By labeling the adsorbed substance adsorbed on the bacteria, the bacteria can be stained indirectly. Substances that adsorb to bacterial cell surfaces include specific sugar chains on the cell surface, antibodies that bind to proteins, and lectins that bind to specific sugar chains, and label the adsorbed substances that adsorb to bacterial cell surface antigens. Fluorescein (ex480, em530)
), Rhodamine (ex507, em530), Texas Red (ex590, em615), Quantum Red (Quantum Red)
: Hereinafter referred to as “QR”: ex488, em670) and the like. As commonly used in general immunoassays, the substance adsorbed on the cell surface of the cell may be pre-labeled with a staining substance such as fluorescein, rhodamine, Texas Red, QR, or directly adsorbed on the bacterial surface. May be indirectly labeled with a labeled second binding substance that specifically binds (adsorbs) to the first substance. An example of this is a general immunoassay in which bacteria are labeled with a primary antibody against a specific bacterium and then indirectly stained with a secondary antibody labeled with a stain that specifically binds to the primary antibody. Can be implemented in compliance. In addition, the labeled substance can be indirectly labeled by bridging and binding with a third substance. As such an example, for example, a method generally known as an avidin-biotin method can be exemplified. That is, this is a method in which a primary antibody is bound to avidin which binds to biotin, and a labeling substance bound to biotin is further bound to avidin which has been bound earlier. In this example, the primary binding substance and the final labeling substance are bound by using the specific binding ability between avidin and biotin as a bridge. Specific adsorbing substances other than phage include, for example, antibodies against bacterial surface antigens (antibodies that recognize and adsorb specific proteins and sugars on bacterial surfaces as antigens), as in the invention of claim 6. be able to. When an antibody is used, for example, if an antibody specific to only pathogenic E. coli O-157 is used,
It is useful because even more limited specific bacteria can be detected among Escherichia coli. That is, for example, a stain capable of specifying a strain-specific element such as an antibody specific to the surface antigen of O-157, and if this can be used, a phage can be used to specify a bacterial species, By using multiple staining in combination with staining that can identify strains using specific antigens on the surface of bacteria without using phage, strains in specific strains that were conventionally considered almost difficult were identified. This is because rapid bacterial detection can be realized.

In each of these inventions, the reason why at least one of the staining substances is always labeled with a phage nucleic acid is to surely detect only living bacteria into which phage nucleic acid injection occurs. Dyes and fluorescent substances can be given as staining substances to be labeled.
Alternatively, both may be used in combination.

Further, as in the invention according to claim 7, substances that stain cell membranes (for example, “FM1-43”, “4-Di-1-ASP” (both manufactured by Molecular Robe)) and bacterial cells A staining substance that adsorbs to the internal nucleic acid can also be used, and ethidium bromide, ethidium homodimer, LDS 751, and the like used for labeling the phage nucleic acid can be used as the labeling substance for staining the bacterial nucleic acid.

Further, as another staining substance for staining bacteria, a substance which produces color or fluorescence by a chemical change can be used. For example, it binds to a substance that produces a dye or a fluorescent substance only after its chemical structure is changed by reduction or decomposition by enzymes present in bacterial cells, or ions such as potassium, calcium, sodium, magnesium, and chlorine present in cells. A dye or a fluorescent substance is produced by forming a complex, or a substance that changes the color tone or the fluorescence wavelength by sensing the hydrogen ion concentration in cells can be used. Including. As the chemical substance that changes its chemical structure by the activity of the enzyme present in the bacterial cell to emit fluorescence, for example, it is oxidized by the activity of the enzyme (dehydrogenase) to change to ethidium, and binds to nucleic acid, emit red fluorescence by excitation dihydrochloride ethidium _{(C 21 H 21 N 3:} 5-ethyl-5,6-dihydro-6-phenyl-3,8-p
henanthridinediamine; W. = 315.4). This dihydroethidium is permeable to bacterial cell membranes. In addition, enzymes (esterases)
And fluorescein diacetate, which emits a fluorescent color when oxidized by the activity of the enzyme, and 4-methylscein diacetate, which emits a fluorescent color when oxidized by the activity of an enzyme (β-galactosidase).

The reasons for adopting the configurations of the above inventions are as follows.
In some cases, it may not be possible to distinguish from signals generated by impurities that may be contained in the original sample only by detecting signals of various types of staining substances, and detection is performed by adding signals derived from these impurities and the like. Although it is possible, the bacterium to be detected is further labeled with a labeling substance having a different wavelength (for example, a labeling substance that emits red fluorescence with respect to green fluorescence), and all of these are detected to generate both signals. This is because, if only such bacteria are discriminated as bacteria to be detected, detection accuracy and sensitivity can be significantly improved. In particular, environmental water such as river water and lake water, sewage, and the like hardly have particles emitting both green and red fluorescence. Therefore, by cross-checking these two colors of fluorescence, one type of labeled phage can be obtained. And the problem of the dyeing method using other specific adsorbing substances can be simultaneously solved.

By the way, as described in Reference Example 2 described later, after removing a large impurity from a certain river water,
When water was passed through a filtration membrane having a pore size of 0.2 μm to capture the fluorescent contaminant particles present in the water and counted with a fluorescence microscope, the particles that emitted green fluorescence with a wavelength of 510 to 560 nm among the trapped contaminants were: 210 / ml, wavelength 590nm
There were 285 particles / ml emitting the above red fluorescence, but 13 particles / ml emitting both the green and red fluorescence.
There was only. In the same way, 5 for rivers, reservoirs, etc.
According to the investigation on the types of water, there were several hundred to 1,000 particles / ml emitting green fluorescence and several tens to several hundreds / ml of particles emitting red fluorescence. Were approximately 10 particles / ml.
From these facts, it can be understood that staining and cross-checking the bacteria in two colors (or more) of green and red is effective in reducing measurement errors and improving accuracy.

In the meaning of cross-check, as the number of stained substances with different detection wavelengths for multiple staining of one bacterium increases, the wavelength that can be detected from one contaminant and the number of multiple stained bacteria are Since the probability that the wavelengths emitted by multiple substances overlap can be reduced, it can be said that, from a stochastic point of view, the greater the number of staining substances to be multi-stained, the higher the accuracy will be. In general, the number of preparations is about two or three, because the labor of preparation increases and optical means for detecting a plurality of wavelengths is required to increase the complexity of the apparatus. Often preferred.

When a substance such as dihydroethidium, which changes its chemical structure and emits fluorescence due to the activity of the enzyme present in the bacterial cells, is used as a label, particles having the activity of the enzyme are used. That is, only live bacteria are stained to emit red fluorescence. This point is technically extremely useful in consideration of the fact that stained substances such as ethidium bromide, ethidium homodimer and the like which are adsorbed to nucleic acids may be fluorescently stained even in the case of dead bacteria, and are variously useful in water management. This is understood from the fact that it is generally important to measure the number of viable bacteria in controlling the degree of bacterial contamination, for example.

Specific combinations of staining substances for multiple staining can be variously adopted as described above. In the case where nucleic acid of one phage specific to a host bacterium is multiple-labeled, a common combination is used. When using two or more phages in which nucleic acid of a phage specific to a host bacterium to be labeled with a different staining substance is used, at least one phage labeled with the nucleic acid with the staining substance and a detection wavelength different from this staining substance And at least one of the staining substances capable of directly or indirectly staining the same bacterium for the same detection purpose.
The case of a combination of species can be exemplified.

Since the wavelength spectrum of the coloring and fluorescence emitted from the substance has a width of several tens to several hundreds nm across the peak, if the detection wavelengths of a plurality of staining substances adopted in the present invention are close, There is a high possibility that one contaminant will overlap its color or fluorescence wavelength with the color or fluorescence wavelength of a plurality of selected staining substances. Therefore, in order to more reliably eliminate the influence of the signal derived from the contaminant, a plurality of staining substances to be used are more preferably a combination having a large difference in coloring fluorescence wavelength. In addition, when there is a noise substance that emits fluorescence in a plurality of wavelength ranges, at least one of the plurality of staining substances that stains the bacteria in a multi-color manner has fluorescence having a wavelength outside the fluorescence wavelength range emitted by the noise substance. Is preferably selected so as to be a combination that emits

The adsorbing substance of bacteria labeled with a staining substance different from the staining substance labeled with the phage nucleic acid includes a substance that specifically adsorbs to the bacteria to be detected and a substance that is specific to the bacteria to which the bacteria to be detected belong. Either a substance that specifically adsorbs or a substance that specifically adsorbs to bacteria without distinguishing species can be used.

Next, the invention of a bacterium detection apparatus will be described. The invention of claim 9 relates to a bacterium which has been multistained by injection of a phage nucleic acid labeled with two or more kinds of staining substances having different color or fluorescence detection wavelengths. Means for preparing a sample comprising:
An optical detection means for detecting at each detection wavelength a plurality of stained substances which multiply stain the bacteria in the sample, and discriminating the detection target bacteria in the detected multistained state and other substances which are not multistained. The invention according to claim 10 is characterized by comprising a discriminating means, wherein one phage nucleic acid is labeled with two or more kinds of staining substances having different color or fluorescence detection wavelengths in the invention according to claim 9. In place of the above, two or more phage nucleic acids labeled with one staining substance are used. Further, the invention of the apparatus for detecting bacteria according to claim 11 is characterized in that the phage nucleic acid labeled with a staining substance capable of detecting color or fluorescence is stained by injection, and the nucleic acid-labeled staining substance is a color or fluorescence detection wavelength. Means for preparing a sample containing bacteria multiply stained by, for example, staining the bacterial surface with a bacterial stain labeled with a different stain, and a plurality of stains that multiple stain the bacteria in the sample It is characterized by comprising optical detecting means for detecting a substance at each detection wavelength, and discriminating means for discriminating a detection target bacterium in a detected multi-stained state and another substance not multi-stained.

In the above configuration, the means for preparing the sample containing the multi-stained bacteria is simply prepared in advance with respect to the original sample solution (test sample such as river water) containing the bacteria to be detected. The nucleic acid is a phage labeled with a staining substance, or another staining substance having a different color or fluorescence detection wavelength from the staining substance labeled with the nucleic acid such as a bacterial adsorbing substance labeled with the staining substance, and at least It can be constituted as a means for adding a staining solution containing a staining substance capable of directly or indirectly staining the same bacteria for detection. Further, when the concentration of bacteria in the original sample solution is low, a filtration membrane (microfiltration membrane (microfilter: M
F))) and other concentrating means for capturing bacteria before or after multiple staining can also be used. Furthermore,
The multi-stained bacteria captured by the filtration membrane can be separated and recovered into a recovery solution such as physiological saline, and used as a sample.

As the optical detecting means in the above configuration, there can be exemplified a means having a light projecting system and a light receiving system for illuminating and receiving a stained substance of a bacterium which is multiply stained. In the present invention, whether or not a bacterium to be detected is identified based on the spectrum of light emitted by the bacterium.In a preferred embodiment, a configuration capable of more strictly limiting the observation wavelength band for detailed identification. Is desirable. For example, if the staining substance is a fluorescent substance, a laser is used as an excitation light source, and by using an optical filter with a narrow transmission wavelength band in the light receiving system,
More information can be extracted from the emission spectrum. As a detection device, a flow system detection device comprising a means for flowing a sample containing multi-stained bacteria into a flow cell and detecting light emitted from the passing bacteria at a plurality of predetermined wavelengths, as in the invention of claim 12 An apparatus can be used. In this case, only a target that simultaneously detects luminescence of a plurality of spectral patterns derived from multiple staining in the sample solution can be determined as a detected bacterium. The bacteria count can be easily realized by counting the signal from the light receiving element with an oscilloscope, a pulse counter, or the like. Further, a configuration in which the filtration membrane capturing the multi-stained bacteria is observed with a CCD camera or the like and the image obtained thereby is analyzed can be adopted. In this case, a bright spot having a plurality of colors derived from multiple staining is determined as a bacterium to be detected by the image processing, and the number of bacteria can be obtained as a total thereof.

In the case of using the above-described flow cell detection device, an object (bright spot) in which the detected fluorescence wavelengths of, for example, two kinds of fluorescent substances in the sample flowing through the flow cell are detected simultaneously.
Can be detected to distinguish bacteria.

In order to discriminate an object which is subjected to multi-staining and the detection fluorescence wavelengths of, for example, two kinds of fluorescent substances are detected simultaneously, a computer in which an image processing program is incorporated is generally used.

[0042]

Next, an embodiment of the present invention will be described. The following example illustrates the use of the fluorescent material ACOR
The case where Escherichia coli is detected using phage labeled with, and avidin (adsorbed substance) labeled with QR, which is a fluorescent substance, will be described. As described above, avidin is known to have an extremely high affinity for biotin.

FIG. 1 is a plan view showing an outline of a sheath flow type bacterial detection apparatus using flow cytometry. In FIG. 1, reference numeral 1 denotes a sheath flow cell, and a sample solution is supplied by a sample syringe pump 2. The liquid flows into the flow cell 1 from below in a direction perpendicular to the plane of the drawing. The sample solution is prepared by reacting ACOR-labeled phage with biotin-labeled anti-Escherichia coli antibody in advance with Escherichia coli, and then mixing QR-labeled avidin.

Around the sample liquid flux, the sheath liquid is supplied from the sheath syringe pump 3 and flows at a high speed, whereby the sample liquid flux is made thinner. The overlap of the fluorescent spots observed from the side of the sample solution is reduced, and the rise of the background due to the overlap of the fluorescence of the free phage can be suppressed,
In addition, the possibility of detection errors due to overlapping of bacteria that emit fluorescence is reduced.

Reference numeral 4 denotes an argon (Ar) laser light source.
The sheath flow cell 1 is irradiated with excitation light having an OR or QR excitation wavelength (488 nm).

The fluorescence emitted from the Escherichia coli upon excitation by the laser beam is converted into parallel light by the objective lens 6, and after scattered light is cut by the sharp cut filter 7, green light derived from ACOR and QR by the dichroic mirror 8. It is split into red light of origin. 510 green light
The red light passes through an interference filter 9 that transmits a wavelength of 630 nm or more, passes through second objective lenses 11 and 12, and pinholes 13 and 14, respectively. , 16 are converted into electronic signals. The electric signal from the photomultiplier tube is green,
After each of the red systems is amplified by the preamplifiers 17 and 18, only the case where both the green and red signals are recognized by the logic circuit 19 is regarded as the E. coli signal to be detected and is output as the TTL signal. By counting the TTL signal with the pulse counter 20, the number of bacteria in the sample solution can be obtained.

According to the above apparatus, the flow of the sample (sample) liquid becomes thinner due to the sheath flow, and the irradiation range of the excitation light becomes narrower by using the laser light source. As a result, the increase in background fluorescence can be suppressed as much as possible, thereby improving the detection sensitivity and the detection accuracy.

[0048]

EXAMPLES Examples of the present invention will be described below by taking as an example a case where Escherichia coli is detected by using the bacteria detection apparatus based on the sheath flow type flow cytometry described in the above embodiment. Reference examples are also given.

In Reference Example 1, Example 1 and Example 2, the above-mentioned ACOR, which is a green fluorescent substance as a nucleic acid labeling substance, is used.
(Acridine orange) -labeled T4 phage, and QR-labeled avidin or PI, in which a biotin-labeled anti-Escherichia coli antibody was previously reacted with Escherichia coli using the aforementioned “system using biotin-avidin complex”, was used.

REFERENCE EXAMPLE 1 River water A was filtered through a 30 μm pore size filtration membrane to remove large contaminants, and filtered water was filtered through a 0.45 μm pore size black membrane and captured on the membrane. Was observed with a fluorescence microscope. The observation was performed under conditions (detection wavelength: 500 to 600 nm) in which green fluorescence of Escherichia coli stained with ACOR-labeled phage could be observed. as a result,
Some green fluorescent contaminants were observed.

Next, the E. coli solution cultured in the LB liquid medium, diluted with the river water A, was added to T-labeled with ACOR.
4 phages (hereinafter referred to as ACOR phage) in 5 × 1
Escherichia coli was stained by adding the cells at a concentration of 0 ¹⁰ cells / ml.

First, the sample was detected at a detection wavelength of 5
When observing the vicinity of 00 to 600 nm with a fluorescence microscope,
Bacteria that emitted green fluorescence and fluorescence that could be attributed to other contaminants could be observed.

Therefore, the bacterial count of this sample was measured by the above-mentioned flow system detection device. As a control, a sample in which Escherichia coli solution was diluted with river water A, to which no labeled phage was added, and a sample in which Escherichia coli solution was diluted with physiological saline (the dilution ratio was the same as that of river water), to which labeled phages were added For, the number of bacteria was measured in the same manner. The operation method is described below. <Pretreatment for background reduction> 5 ml of the above sample was filtered from the top of a UF membrane (FP030 / 2 manufactured by Schleicher & Schuell) having a pore size of 0.45 μm. ↓ 10 ml of physiological saline was filtered from the top of the filter as a washing solution. ↓ 5 ml of physiological saline was filtered from the opposite side of the filter as a backwash, and the permeate was collected. ↓ Bacteria count is measured with a flow detection device.

As a result, the number of samples obtained by diluting the E. coli solution with physiological saline and adding the labeled phage was 1,270 cells / ml.
Samples diluted with river water A and added with labeled phage were 2
The number of samples without labeled phage diluted with 300 cells / ml and river water A was 1,000 cells / ml. From these results, the number of signals of the river water A contains signals other than the stained bacteria, that is, the signals of the fluorescence generated by the contaminants contained in the river water A in advance, and the number of bacteria cannot be accurately measured. You can see that

Example 1 A biotin-labeled anti-Escherichia coli antibody and QR-labeled avidin were prepared. First, a cultured Escherichia coli was reacted with a biotin-labeled antibody, and then reacted with QR-labeled avidin.
Observation of the vicinity of a detection wavelength of 650 to 670 nm with a fluorescence microscope confirmed that the bacteria emitted red fluorescence.

Next, the sample reacted with the biotin-labeled antibody and the QR-labeled avidin was subjected to the AC reaction shown in the comparative example.
OR-labeled phage was added and double staining was performed. When this was observed near each detection wavelength with a fluorescence microscope, it was confirmed that one germ emitted red fluorescence by QR and green by ACOR.

Then, these were measured by the above-mentioned flow system detecting apparatus. The target sample was the same as that in Reference Example 1 described above. First, Escherichia coli in the sample was stained with a labeled antibody, and then stained with a labeled phage and double-stained to reduce the background described in Reference Example 1. Pretreatment was performed.

Next, this was introduced into the above-mentioned flow detection device capable of simultaneously detecting the fluorescence of ACOR and the fluorescence of QR, and the number of signals emitting both fluorescences was confirmed. As a result, the sample obtained by diluting the E. coli solution with physiological saline and adding the labeled antibody and the labeled phage was 1,
The sample to which the labeled antibody and the labeled phage were added after dilution with 170 cells / ml and river water was 1,200 cells / ml, and the sample not stained was undetected. That is, by detecting both signals, the signal of the contaminant was eliminated, and only Escherichia coli could be detected.

Next, another embodiment 2 of the present invention will be explained. In this embodiment, a phage in which nucleic acid is labeled with ACOR which is a fluorescent substance and a dye which directly stains bacterial nucleic acid and PI which is also a fluorescent substance are used. This is an example in which Escherichia coli was used to detect E. coli.

Example 2 A T4 phage labeled with ACOR and a solution prepared by dissolving PI powder in physiological saline to a concentration of 1 mg / ml (hereinafter referred to as “PI solution”) were prepared. After adding and labeling phage, 10 ml of the bacterial solution was directly stained with bacterial nucleic acid by adding 100 μl of PI solution, and double-stained.

When this was observed with a fluorescence microscope, it was confirmed that one bacterium emitted red by PI and green by ACOR.

Therefore, these were measured by the above-mentioned flow system detecting device. The target sample was the same as that in Reference Example 1. First, Escherichia coli in the sample was stained with ACOR-labeled phage, and then directly stained with PI, and the pretreatment for reducing the background shown in Reference Example 1 was performed. Was.
Next, this is detected simultaneously with the detection of ACOR fluorescence,
Was introduced into a flow-type detection device capable of detecting the fluorescence of both, and the number of signals emitting both fluorescences was confirmed. As a result, those which produced both signals were those obtained by diluting Escherichia coli with physiological saline and performing double staining, 1,200 cells / ml, and those diluting with river water A and performing double staining. Was 1,250 / ml, and the unstained sample was undetected.

Thus, it was confirmed that by detecting both signals of the double staining, the signal of the contaminant was eliminated and only Escherichia coli could be detected.

Separately from Examples 1 and 2 and Reference Example 1 above, bacterial cells were not distinguished as nucleic acid stains using the above-described sheath flow type flow cytometry bacteria detection apparatus, instead of QA-labeled adipine. Using dihydroethidium, which is a red fluorescent dye to be stained with, and Reference Examples 2, 3 to confirm that the discrimination with fluorescent contaminant particles other than Escherichia coli contained in environmental water such as river water is improved. The test of Example 3 was performed.

Reference Example 2 In a certain river water B (water of the same river as in Reference Example 1, but containing different particles due to different sampling time and location, is basically completely different test water). The contained fluorescent contaminant particles were examined.

That is, after passing the river water B through a filtration membrane having a pore diameter of 20 μm to remove large contaminants, the water diameter of the river water B was reduced to 0.1 μm.
Water was passed through a 2 μm filtration membrane (Whatman anodisc) to capture impurities. Wavelength of 5 among the impurities trapped in the filtration membrane
When particles emitting green fluorescence of 10 nm to 560 nm were counted by a fluorescence microscope, there were 219 particles / ml (green fluorescence of a wavelength of 510 nm to 560 nm is a fluorescence wavelength emitted by acridine orange, which is used to label bacteria using phage. ).

The number of particles emitting red fluorescence having a wavelength of 590 nm or more was 285 particles / ml.

However, among these particles, only 13 particles / ml emitting both green and red fluorescence were present.

Reference Example 3 A 1 ml of a pure culture of Escherichia coli (1 × 10 ⁸ cells / ml)
0.1 ml of COR-labeled T4 phage solution (1 × 10 ¹² cells / ml) was added (additional concentration: 1 × 10 ¹¹ cells / ml) to give 5
The mixture was reacted for minutes, and an E. coli solution in which E. coli was stained with ACOR was prepared.

0.1 μl of this Escherichia coli solution was added to 5 ml of 0.8% saline containing no fluorescent contaminants to obtain a measurement sample 1, and 5 ml of the above-mentioned river water B was added to the measurement sample 1. And 2.

For these measurement samples 1 and 2, measurement was performed using the above-mentioned flow system detection apparatus, and the number of green fluorescence detection signals was counted. As a result, the number of detected signals is
In the case of measurement sample 1 (without contaminants), it was 1760 / ml, but in the case of measurement sample 2 (with contaminants of river water B), 3
It was 372 / ml.

It can be seen from the measurement results that the results of the number of detected signals of the measurement sample 1 are all derived from Escherichia coli, and thus correspond to the true number of Escherichia coli. Although the number of Escherichia coli is almost the same as that of the measurement sample 1, the number of Escherichia coli is increased by about twice, and it is considered that a large number of detection signals caused by impurities in the river water are generated. Therefore, the ACOR indicator T
It can be seen that it is difficult to obtain high measurement accuracy with single-color staining of 4 phages or the like.

Example 3 In addition to the staining of Escherichia coli with a green fluorescent substance using ACOR using T4 phage and the double staining of Escherichia coli with a red fluorescent substance using dihydroethidium, the T4 phage was subjected to the measurement operation of Reference Example 3 described above. Was performed to prepare a double-stained measurement sample 3, and the measurement was performed using the above-described sheath flow type flow cytometry bacteria detection device.

The staining of Escherichia coli for preparing the measurement sample 3 was performed as follows.

First, 25 mg of dihydroethidium, which is a solid powder, was added to an organic solvent dimethyl sulfoxide (DMS).
O) It was dissolved in 2.5 ml to prepare a 10 mg / ml dihydroethidium solution. This is because dihydroethidium has a low solubility in water and is not suitable to be directly added to sample water, so that it is once dissolved in an organic solvent and then added to sample water.

Then, 5 μl of the above dihydroethidium solution (additional concentration: 50 μg / ml) was added to 1 ml of the pure culture solution (1 × 10 ⁸ cells / ml) of Escherichia coli of Reference Example 3 and allowed to react for 20 minutes. Thereafter, 0.1 ml of an ACOR-labeled T4 phage solution (1 × 10 ¹² cells / ml) was added (additional concentration: 1 × 10 ¹¹ cells / ml) and reacted for 5 minutes to prepare an E. coli solution.

0.1 μl of this Escherichia coli solution was added to 5 ml of 0.8% saline containing no fluorescent contaminants to obtain a measurement sample 3, and 5 ml of the river water B was added to obtain a measurement sample. And 4.

For these measurement samples 3 and 4, measurement was performed using the above-mentioned flow system detection apparatus, and the number of green and red fluorescence detection signals was counted. As a result, the number of detection signals was 1770 / ml for the measurement sample 3 (with no contaminants) and 1816 / ml for the measurement sample 4 (having contaminants in the river water B), and almost the same number of counts were obtained. Was done.

From the results of Reference Examples 2, 3 and Example 3, it can be seen that dihydroethidium and ACOR using ACOR-labeled T4 phage stained in two colors, green and red, and the detection of each of these colors was performed. According to the method performed,
For example, it was confirmed that E. coli contained in water to be measured such as river water can be detected with high accuracy.

[0080]

According to the present invention, it is possible to detect and identify a specific bacterium, and further, only a viable bacterium by a quick and simple operation.
For example, trace bacteria that are repelled by water and air in environmental water, environmental air, food and water, hospitals, precision machinery factories, and food manufacturing and processing factories are detected with higher detection accuracy and sensitivity to increase sensitivity. In addition to the effect of being able to do so, the following effects are also achieved in addition to this.

According to the second or third aspect of the present invention, bacteria can be stained multiple times using one or more kinds of phages, and highly accurate and highly sensitive bacterial detection can be realized.

According to the invention of claims 4 to 8, at least one kind of phage and highly accurate and sensitive detection of bacteria can be realized by using a bacteria-adsorbing substance and the like, and the phages can be realized by using a bacteria-adsorbing substance and the like. Bacteria can be detected using specificity different from the specificity of, and the advantage that the identification of the bacterium to be detected can be more detailed can be obtained.

According to the ninth to eleventh aspects of the present invention, it is possible to provide an apparatus for detecting bacteria suitable for realizing the above method.

According to the twelfth aspect of the present invention, it is possible to continuously detect bacteria in a sample using a flow-type detection device, and it is advantageous in that the bacterium is quick, highly accurate and easy to operate. Can be

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of an outline of an apparatus using a sheath flow cell type flow system detection apparatus of Example 1 for detecting Escherichia coli.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12Q 1/68 G01N 21/77 D G01N 21/77 21/78 Z 21/78 33/53 D 33/53 SY 33 / 566 33/566 33/569 B 33/569 33/58 A 33/58 33/483 C // G01N 33/483 C12N 15/00 A (72) Inventor Takako Nogami 1-2-2 Shinsuna, Koto-ku, Tokyo No. 8 Organo Co., Ltd. (72) Inventor Haruki Haruka 1-2-8 Shinsuna, Koto-ku, Tokyo Organo Co., Ltd. (72) Inventor Yuichiro Toba 1-2-8 Shinsuna, Koto-ku, Tokyo Organo Fuji Electric Co., Ltd. Inside (72) Inventor Tokio Oto 1-1, Tanabe Nitta, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Inside Fuji Electric Co., Ltd. (72) Inventor Hiroshi Tada Ichikawa, Kawasaki, Kanagawa Prefecture Subdivision Tanabeshinden No. 1 No. 1 Fuji Electric Co., Ltd. in

Claims (12)

[Claims] 1. A bacterium to be detected is multi-stained with two or more kinds of dyes having different optical detection wavelengths, and an object whose staining is detected at each detection wavelength of the multi-stained dye is detected for the purpose of detection. A method for determining a bacterium, wherein at least one of the staining substances is labeled with a nucleic acid of a bacteriophage that specifically adsorbs to the bacterium to be detected. 2. A bacteriophage in which a nucleic acid is multiplexly labeled with two or more kinds of staining substances having different color or fluorescence detection wavelengths, and which specifically adsorbs to a host bacterium to be detected.
Contacting the bacteriophage with the host bacterium and injecting the labeled nucleic acid into the host bacterium causes multiple staining of the bacterium, and optically detecting the multistained bacterium to discriminate and measure the bacterium to be detected. A method for detecting bacteria, characterized in that: 3. The nucleic acid is labeled with a staining substance having a different color or fluorescence detection wavelength, and at least two or more kinds of bacteriophages that specifically adsorb to the same host bacterium for the purpose of detection are used. Bacteria characterized in that bacteria are multi-stained by contacting the host bacteria and labeled nucleic acid is injected into the host bacteria, and the multi-stained bacteria are optically detected to discriminate and measure the bacteria to be detected. Detection method. 4. At least one bacteriophage labeled with a phage nucleic acid with a staining substance capable of detecting color or fluorescence is specifically adsorbed to a bacterium to be detected, and the labeled nucleic acid is injected into the bacterium for staining. Together, the nucleic acid-labeled staining substance is another staining substance having a different color or fluorescence detection wavelength, and stains the bacterium with a staining substance capable of directly or indirectly staining at least the same bacteria for detection. A method for detecting bacteria, wherein the bacteria to be detected are multi-stained, and the multi-stained bacteria are optically detected to discriminate and measure the bacteria to be detected. 5. The method for detecting bacteria according to claim 4, wherein the other staining substance that stains bacteria is labeled with at least an adsorbing substance that adsorbs to the bacteria to be detected. 6. The method for detecting bacteria according to claim 5, wherein the other stain that stains bacteria is labeled with an antibody that specifically adsorbs to a membrane protein or sugar on the surface of bacteria. . 7. The method for detecting bacteria according to claim 4, wherein the other staining substance is a substance that stains bacterial cell membranes or a substance that stains bacterial nucleic acids. 8. At least one kind of bacteriophage whose nucleic acid is labeled with a staining substance capable of detecting color or fluorescence is specifically adsorbed to a bacterium to be detected, and the labeled nucleic acid is injected into the bacterium for staining. The color of the nucleic acid-labeled staining substance is different from that of the detection wavelength of fluorescence or fluorescence, and stained with another staining substance that stains the bacterium depending on the bacterial cytoplasmic conditions, thereby multiplex-staining the bacterium for the detection purpose. A method for detecting a bacterium, wherein a bacterium to be detected is discriminated and measured by optically detecting a stained bacterium. 9. A means for preparing a sample containing bacteria multiply stained by injecting bacteriophage nucleic acids labeled with two or more kinds of staining substances having different color or fluorescence detection wavelengths, and multiplexing the bacteria in the sample. An optical detection means for detecting a plurality of stained substances at each detection wavelength, and a discrimination means for discriminating a detection target bacterium in a detected multiple staining state and other substances that are not multistained, A bacterium detection device, characterized in that: 10. Includes a bacterium that is labeled with a staining substance having a different color or fluorescence detection wavelength and that has been stained multiple times by injection of the labeled nucleic acid into a bacteriophage that specifically adsorbs to the same host bacterium to be detected. Means for preparing a sample, optical detection means for detecting at each detection wavelength a plurality of stained substances which are multi-stained for the bacterium, and a detection target bacterium in a multi-stained state detected and other not multi-stained A device for detecting bacteria, comprising: a determination unit configured to determine a substance. 11. A dye which is stained by injection of a bacteriophage nucleic acid labeled with a staining substance capable of detecting color or fluorescence, and is stained with another staining substance having a different color or fluorescence detection wavelength from the nucleic acid-labeled staining substance. Means for preparing a sample containing bacteria that are multi-stained by being subjected to the detection, optical detection means for detecting, at each detection wavelength, a plurality of stained substances that multi-stain the bacteria in the sample, A bacterium detection device, comprising: a determination unit configured to determine a bacterium to be stained and another substance that is not multistained. 12. The bacterium according to claim 9, wherein the optical detection means for detecting a plurality of staining substances at each detection wavelength is performed on a sample flowing through a flow cell. Detection device.