JP2006170646A - Prodegradent for microbe separation by fine tube electrophoresis and analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method for separating and detecting various microbes quickly with high sensitivity, and an analyzer used for the method. <P>SOLUTION: In the analysis method of microbes, a sample including the microbes is subjected to fine tube electrophoresis performed by using a migration buffer solution including an anionic polymer other than alginates. In the analysis method of microbes, the sample including the microbes is treated by a fluorescent antibody to be bonded to the microbes, and subjected to the fine tube electrophoresis, to thereby perform fluorescence detection of the microbes labeled by the fluorescent antibody. This analyzer for analyzing the microbes by using the fine tube electrophoresis is equipped with (1) a water tank storing the migration buffer solution including the anionic polymer other than alginates, (2) a fine tube for separating the microbes injected into the migration buffer solution, and (3) a detection means for detecting the separated microbes. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and an analyzer for analyzing microorganisms present in a specimen sample using microtubule electrophoresis. The analysis method and analysis apparatus of the present invention are useful for accurately or simultaneously analyzing microorganisms mixed in a sample sample in the environment, food, medical field and the like.

  In the present invention, analysis includes separation, detection (qualitative analysis), identification, quantification, screening, and life / death discrimination of a desired microorganism contained in a sample.

  In general, in order to investigate whether or not microorganisms are present in a sample, and to examine the type and amount of microorganisms contained in a sample, the purpose is to inoculate these samples once in a suitable medium and culture. It is necessary to grow the microorganisms. For this reason, conventionally, analysis of microorganisms requires a long time of at least several hours for culturing, and there has been a problem that results cannot be obtained quickly. In addition, since bacterial culture is basically performed aseptically, special culture techniques and equipment are required.

  From these facts, there is a demand for a method that can rapidly and easily detect microorganisms that can be contained in a sample without requiring culture of microorganisms and advanced techniques and devices.

  For the detection of microorganisms classified into many types, conventionally, after culturing in selective separation culture, it is necessary to catch suspicious colonies and determine with confirmation medium, identification kit or antiserum. It took a lot of experience and skill to identify. Moreover, when time for culture | cultivation etc. was put together, the long-term of 3-5 days was required for the determination result.

  For this reason, in the environment / food and medical fields, establishment of a quick and safe method for specifically detecting microorganisms, particularly microorganisms classified into many types, for each species / strain is required. It is a fact.

  In recent years, as a rapid method for separating and detecting microorganisms, there is an electrophoresis technique in which microorganisms are migrated in a microtube and reacted with an antibody (for example, Patent Document 1). The electrophoresis technique in this method is a moving means for reacting a microorganism with an antibody, and does not separate the microorganism cell itself electrophoretically. On the other hand, when migrating and separating microorganisms in a microtubule, the interaction between microbial cells and the inner wall of the tubule or between cells and cells is large. It is effective to contain an alginate (for example, Patent Document 2).

JP 2002-345451 A JP 2002-181781 A

  In order to promptly take appropriate measures against environmental water contamination, food contamination, and infectious diseases, or to quickly stop the contamination source, the microorganisms that can be the source of the contamination A detection method is required. Therefore, an object of the present invention is to provide an analysis method for separating and detecting various microorganisms widely and quickly and with high sensitivity, and an analyzer used in the method.

  In the above-described method using alginic acid, alginates as an additive for improving the separation efficiency of microorganisms are effective for the separation of Salmonella microorganisms, but function similarly for all microorganisms. It wasn't. Therefore, introduction of more types of separation efficiency improving agents capable of dealing with a wide variety of microbial species has been expected. In addition, it is unclear what mechanism alginic acid is effective in improving the separation efficiency of microorganisms, and it was completely unpredictable what other compounds could be useful in improving the separation efficiency of microorganisms.

  As a result of intensive studies to solve the above problems, the present inventors have used a microtubule electrophoresis method using a electrophoresis buffer containing an anionic sugar polymer other than an alginate. It has been found that microorganisms contained in various types can be separated with high resolving power and high reproducibility. In addition, the present inventors use a fluorescence detector as a detection means in microtubule electrophoresis, and in particular by subjecting a target microorganism to fluorescent labeling with a fluorescent antibody in advance, even a minute amount of microorganism can be cultured. It has been found that it can be detected with high sensitivity without undergoing growth treatment, and that this makes it possible to detect a desired microorganism with high accuracy and even higher resolution. Furthermore, the present inventors have confirmed that according to the method of the present invention, microorganisms mixed in a sample can be detected not only simply but also quantitatively measured. The present invention has been completed based on such findings.

That is, the aspect of this invention is as follows.
[1] A method for separating and analyzing microorganisms, which comprises subjecting a sample containing microorganisms to microtubule electrophoresis using an electrophoresis buffer containing an anionic polymer other than alginate.
[2] A method for analyzing a microorganism, comprising: treating a sample containing a microorganism with a fluorescent antibody that binds to the microorganism; and subjecting the sample to microtubule electrophoresis to detect the fluorescence of the microorganism labeled with the fluorescent antibody. .

[3] The method for analyzing a microorganism according to [2], wherein the microtubule electrophoresis is performed using an electrophoresis buffer containing an anionic polymer other than alginate.
[4] The method for separating and analyzing microorganisms according to [1], wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.
[5] The method for separating and analyzing microorganisms according to [4], wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.

[6] The method for separating and analyzing a microorganism according to [5], wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin, and chondroitin sulfate.
[7] The method for separating and analyzing microorganisms according to [1], wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.
[8] The anionic polymer having a sulfonic acid group and / or a carboxylic acid group is selected from the group consisting of a polyglutamate salt, a polystyrene sulfonate salt, and a salt of poly (styrene sulfonic acid / maleic acid). ] Method for separating and analyzing microorganisms.

[9] The method for analyzing a microorganism according to [2] or [3], wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.
[10] The method for analyzing a microorganism according to [9], wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.
[11] The method for analyzing a microorganism according to [10], wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin, and chondroitin sulfate.

[12] The method for analyzing a microorganism according to [2] or [3], wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.
[13] The anionic polymer having a sulfonic acid group and / or a carboxylic acid group is selected from the group consisting of a polyglutamate salt, a polystyrene sulfonate salt, and a salt of poly (styrene sulfonic acid / maleic acid). ] Microbial analysis method.
[14] An apparatus for separating microorganisms using microtubule electrophoresis, wherein (i) a water tank containing an electrophoresis buffer containing an anionic polymer other than alginate, and (ii) injected into the electrophoresis buffer A microbe separation apparatus, comprising: a microtube for separating the microbe produced.

[15] The microorganism separation apparatus according to [14], wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.
[16] The microorganism separation apparatus according to [15], wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.
[17] The microorganism separation apparatus according to [16], wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin, and chondroitin sulfate.

[18] The microorganism separation apparatus according to [14], wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.
[19] The anionic polymer having a sulfonic acid group and / or a carboxylic acid group is selected from the group consisting of a polyglutamate salt, a polystyrene sulfonate salt, and a salt of poly (styrene sulfonic acid / maleic acid). ] Microbe separation device.

[20] An apparatus for analyzing microorganisms using microtubule electrophoresis, (i) a water tank containing an electrophoresis buffer containing an anionic polymer other than alginate, and (ii) injected into the electrophoresis buffer A microbe analysis apparatus comprising: a microtubule that separates the microbe; and (iii) a detection means for detecting the microbe that has been separated.
[21] The microorganism analysis apparatus according to [20], further comprising a fluorescence detector as a detection means.

[22] The microorganism analysis apparatus according to [20] or [21], wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.
[23] The microorganism analysis apparatus according to [22], wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.
[24] The microorganism analysis apparatus according to [23], wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin, and chondroitin sulfate.

[25] The microorganism analysis apparatus according to [20] or [21], wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.
[26] The anionic polymer having a sulfonic acid group and / or a carboxylic acid group is selected from the group consisting of a polyglutamate salt, a polystyrene sulfonate salt, and a salt of poly (styrene sulfonic acid / maleic acid). ] Microbe analysis equipment.
[27] A separation promoting agent for separating microorganisms in microtubule electrophoresis for separating microorganisms, comprising an anionic polymer other than alginate and a buffer solution.

[28] The microorganism separation promoter according to [27], wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.
[29] The microorganism separation promoter according to [28], wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.
[30] The microorganism separation promoter according to [29], wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin, and chondroitin sulfate.

[31] The microorganism separation promoter according to [27], wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.
[32] The anionic polymer having a sulfonic acid group and / or a carboxylic acid group is selected from the group consisting of a polyglutamate salt, a polystyrene sulfonate salt, and a salt of poly (styrene sulfonic acid / maleic acid). ] Microbial separation promoter.

  ADVANTAGE OF THE INVENTION According to this invention, the method and apparatus which can analyze (separate and detect) the microorganisms in a sample sample accurately in a short time can be provided. According to the microtubule electrophoresis using the electrophoresis buffer containing the anionic sugar polymer other than the alginate according to the first aspect, the microorganisms can be separated with high performance and high reproducibility. According to such a method, it is possible to isolate the microorganism species and strains individually. According to the microtubule electrophoresis using the fluorescent antibody according to claim 2, the microorganisms contained in the specimen sample can be easily and accurately accurately in a short time without requiring a special technique and without a culture operation. Therefore, it is possible to establish a highly accurate microbiological examination and monitoring system. According to the analysis method of claim 3, microorganisms contained in a sample can be separated with high reproducibility and high separation ability, and further, a desired microorganism can be obtained by using a laser-induced fluorescence detector as a fluorescence detection means. Since it can be detected with high sensitivity and specificity, a minute amount of microorganisms can be accurately separated and detected without subjecting the sample to a growth treatment such as culture. Since these methods of the present invention have quantitative properties, it is possible to discriminate and measure (quantitative analysis) the number of bacterial cells present in a sample.

  Further, according to the method and apparatus of the present invention, it is possible not only to detect microorganisms in a sample, but also to identify bacterial species by using specific detection reagents (for example, antibodies) specific to various microorganisms. Is also possible. In particular, since the method of the present invention is excellent in the ability to separate microorganisms, it is possible to simultaneously analyze and identify several types (subspecies, serotypes) of microorganisms present in a sample.

  Thus, since the method and apparatus of the present invention are suitable for the analysis of microorganisms (qualitative analysis, quantitative analysis), it is possible to eliminate foods and foods contaminated by pathogenic microorganisms at an early stage and is useful for preventing the occurrence of food poisoning. is there. In addition, since early diagnosis of food poisoning patients due to pathogenic microorganisms becomes possible, it helps to prevent the spread of food poisoning and early treatment. Furthermore, the quality control period of food can be shortened, and the net period can be extended due to the strictness of hygiene management, which can greatly contribute to the economics of food distribution.

(1) Microbial Analysis Method The microorganism analysis method of the present invention first uses a microtubule electrophoresis method using a migration buffer containing an anionic polymer other than alginate for the separation of microorganisms. It is characterized by. In the microorganism analysis method of the present invention, secondly, the microorganism is separated by microtubule electrophoresis and fluorescently detected. Specifically, the target microorganism is fluorescently detected by labeling with a fluorescent antibody. It is characterized by this.

  The microtubule electrophoresis method used here is not particularly limited except that a buffer solution containing an anionic polymer other than alginate is used as the electrophoresis buffer, and any conventionally known method is arbitrarily used. be able to. In addition, as long as a buffer solution containing an anionic polymer other than alginate is used as an electrophoresis buffer, a microtube electrophoresis method developed in the future including a microchip electrophoresis technique can be used without limitation.

  The ratio of the anionic polymer other than alginate contained in the electrophoresis buffer is not particularly limited, but is usually 0.001 to 1% by weight, preferably 0.001 to 0.1% by weight, more preferably 0.001 to 0.01% by weight. it can. Anionic polymers other than alginate include anionic sugar polymers other than alginate and anionic polymers that do not contain sugar. Examples of anionic sugar polymers other than alginate include anionic sugar polymers containing a sulfonic acid group, such as dextran sulfate, chondroitin sulfate, and heparin. Examples of the anionic polymer containing no sugar include anionic polymers having a sulfonic acid group and / or a carboxylic acid group. For example, polyglutamate such as poly-γ-glutamate, polystyrene sulfonate, and the like. And salts of poly (styrene sulfonic acid / maleic acid). Furthermore, as the anionic polymer other than the alginate of the present invention, specifically, collate, polygalacturonate, pectate, chondroitin sulfate (chondroitin sulfate A to E or chondroitin sulfate K salt), hyaluron Examples thereof include acid salts, heparin, fucan sulfate, dextran sulfate, heparan sulfate, keratan sulfate, carrageenan, fucoidan and the like. Specific examples of salts of anionic polymers other than alginates include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, and ammonium salts, preferably sodium salts. It is.

  The average molecular weight of the anionic polymer other than the alginate used in the method of the present invention is not limited, but is preferably 5000 or more, more preferably 20000 or more, further preferably 100000 or more, particularly preferably 150000 or 20000 or more.

  In addition, the electrophoresis buffer may be any buffer having a buffer capacity in the range of pH 3 to 10, preferably pH 7 to 9, and specifically, Tris buffer, phosphate buffer, oxalate buffer, phosphate Examples thereof include a buffer solution, a borate buffer solution, or a mixed solution of these buffers. Moreover, as a density | concentration of these electrophoresis buffers, the range of 1-1000 mM is preferable, Preferably it is 2-200 mM.

  FIG. 1 shows an example of a basic configuration of a microtubule electrophoresis system that can be used in the present invention. Here, both ends of the hollow microtube 10 are immersed in the water tanks 11 and 12 containing the migration buffer solution, respectively, and the inside of the microtube is filled with the migration buffer solution. Electrodes (13, 14) are attached to the water tanks 11 and 12, respectively, and these are connected via a power source 15 like a high voltage supply.

  A sample containing microorganisms to be analyzed is injected from one end (inlet side, 10a) of the microtube, and then the power supply 15 is activated to generate a potential gradient in the microtube in the migration region. When surface charges are present on the inner wall of the microtube, this potential gradient generates an electroosmotic flow that is the flow of the entire liquid in the microtube, and usually it is a flow in the cathode direction. In addition, the microorganisms injected into the microtubule move toward the electrode having a polarity opposite to the surface charge of the microorganisms in response to the generated potential gradient. As a result, the migration direction and velocity of the microorganism in the microtubule are determined by the electroosmotic flow velocity and the mobility of the microorganism, and are detected by the detector 18 provided near the opposite end (exit side, 10b) of the microtubule. Detected. When an optical detection method is used as the detection means, the microtube itself becomes the flow cell 16 and is directly optically detected through the microtube wall.

  The method for injecting the sample into the microtube is not particularly limited, and any of conventionally used gravity method, pressurization method, decompression method, and electrophoresis injection method can be used. Although the amount of injection is not particularly limited, it is usually 0.1 to 100 nL, preferably 1 to 100 nL, more preferably 1 to 10 nL.

  Note that the pair of electrodes (13, 14) and the power source 15 exemplified above have a potential gradient of a strength necessary for the microorganisms injected into the microtube to migrate in the electrophoresis buffer in the microtube. Any means (potential gradient can be applied) without any limitation to these (power supply and a pair of electrodes) as long as it can achieve the purpose. Means for applying) can be used.

  When the power source and the electrode are used as the application means as described above, the electrode is inserted in the counter water tank filled with the respective electrophoresis buffer, and when the power is turned on, the anode (anode) and the cathode (cathode) A potential gradient is formed between them, and the microorganisms injected into the microtubule move toward the appropriate electrode due to the balance between the charge and the electroosmotic flow velocity it has. In general, since the surface charge of microorganisms is often negative, microorganisms have the property of moving in the anode direction, but the electroosmotic flow velocity flowing in the cathode direction is often greater than the microorganism migration rate. Microorganisms move to the cathode as a result.

  When the application means includes electrodes, the voltage applied between the electrodes is generally about 1 kV / m to about 500 kV / m, preferably about 2 kV / m to about 100 kV / m, more preferably about the microtubule length. 5 kV / m to about 20 kV / m.

  Examples of the fine tube include a hollow tube having an inner diameter of 1 to 150 μm and a length of 0.1 to 100 cm, preferably an inner diameter of 20 to 100 μm and a length of 1 to 50 cm. The material is not particularly limited, and glass, Teflon, etc. can be arbitrarily exemplified, but preferably it is made of fused silica.

  Any of spectroscopic, electrochemical, and gravimetric methods can be used as means for detecting microorganisms separated in the microtubule, but preferably UV-visible detection, fluorescence detection, luminescence detection, and light scattering detection. An optical detection method such as Among them, a fluorescence detection method excellent in detection specificity and sensitivity is preferable, and a laser-induced fluorescence detection method capable of detecting with higher sensitivity is particularly preferable. These fluorescence-based detections can be made more specific and sensitive by appropriately combining reactions such as immune reactions (antigen-antibody reactions), enzyme reactions, and nucleic acid complementary strand formation reactions depending on the target microorganism. Can be detected. Among these methods, the immune reaction is one of the preferred methods because it does not require any special pretreatment on the microorganism sample and is excellent in specificity. Specific examples of such an immune reaction include a method of labeling a target microorganism with fluorescence, an enzyme, or the like using a fluorescent dye-labeled antibody (also simply referred to as a fluorescent antibody), an enzyme-labeled antibody (also simply referred to as an enzyme antibody) or the like. be able to.

  For example, the fluorescent reagents used for labeling microorganisms are not particularly limited and widely known ones that can be conventionally known or known in the future. Specifically, BODIPY FL (4,4-difluoro -5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic Acid, Succinimidyl Ester (excitation wavelength 503 nm, fluorescence wavelength 512 nm, manufactured by Molecular Probe), SYTO 9 (excitation Wavelength 480 nm, fluorescence wavelength 500 nm, manufactured by Molecular Probe), FITC (Fluorescein Isothiocyanate) (excitation wavelength 490 nm, fluorescence wavelength 520 nm, manufactured by Dojin), TexasRed (excitation wavelength 596 nm, fluorescence wavelength 620 nm, Molecular Probe TRITC (Tetramethylrhodamine Isothiocyanate) (excitation wavelength 541 nm, fluorescence wavelength 572 nm, manufactured by Research Organics), Cy3 (N, N'-diethyl-indodicarbocyanine-5,5'-disulfonic acid) (excitation wavelength 552 nm) , Fluorescence wavelength 565 nm, manufactured by Amersham Pharmacia Biotech), Cy5 (N, N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid) Wavelength 650 nm, fluorescence wavelength 667 nm, manufactured by Amersham Pharmacia Biotech), Acridine Orange (excitation wavelength 490 nm, fluorescence wavelength 530 nm, 640 nm, manufactured by Wako), Ethidum Bromide (excitation wavelength 545 nm, fluorescence wavelength 605 nm, Dojin), Propidum iodide (excitation wavelength 530 nm, fluorescence wavelength 615 nm, Dojin), Calcein (excitation wavelength 495 nm, fluorescence wavelength 520 nm, Dojin), BCECF-AM (2 ', 7'- Bis- (2-carboxyethyl) -5- (and-6) -carboxyfluorescein Acetoxymethyl Ester (excitation wavelength 500 nm, fluorescence wavelength 530 nm, manufactured by Molecular Probe), Evans blue (excitation wavelength 550 nm, fluorescence wavelength 610 nm, Wako), Lucifer Yellow CH (excitation wavelength 430 nm, fluorescence wavelength 535 nm, ICN Pharmaceutical), Dil (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate) (excitation wavelength) 550 nm, fluorescence wavelength 565 nm, manufactured by Molecular Probe), Dio (excitation wavelength 484 nm, fluorescence wavelength 501 nm, manufactured by Molecular Probe), DioC6 (3) (3,3'-Dihexyloxacarbocyanine Iodide) (excitation wavelength 480 nm Fluorescence wavelength 501 nm, manufactured by Lambda Probe & Diagnostics), Rhodamine123 (excitation wavelength 500 nm, fluorescence wavelength 540 nm, manufactured by Molecular Probe), Fluo-3 (excitation wavelength 506 nm, fluorescence wavelength 526 nm, manufactured by Dojin), Examples thereof include Calcium Green (excitation wavelength 506 nm, fluorescence wavelength 526 nm, manufactured by Molecular Probe) and the like.

  Microorganisms targeted by the method of the present invention include bacteria, fungi, chlamydia, viruses and rickettsia. Bacteria are preferable. Examples of the bacteria include staphylococci, octococci, spirillum, streptococci, staphylococci, gonococci, spirochetes, quasicoccus, comma type cocci and actinomycetes. Specific examples include Rhodospirillaceae, Chromatiaceae, Chlorobiaceae, Myxococcaceae, Archangiaceae, Cysttobacteraceae, Polyangiaceae C, ophiaceae (Beggiatoaceae), Simonsiellaceae, Leucotrichaceae, Achromatiaceae, Pelonemataceae, Spirochaetaceae, Spirichatobi (A), Pseudomonadaacter (A) Methylomonadaceae, Halobacteriaceae, Enterobacteriaceae, Vibrionaceae, Bacteroidaceae, Neise Neisseriaceae, Veillonellaceae, Organisms oxidizing ammonia or nitrite, Organisms metabolizing sulfer and sulfer compounds, Iron oxide and / or Manganisms depositing iron and / or manganese oxides), Siderocapsaceae, Methanobacteriaceae, aerobic and / or facultatively anaerobic Micrococcaceae, Streptococcaceae, anaerobic Peptococcus ae ), Bacillaceae, Lactobacillaceae, Acetobacter, Coryneform group of bacteria, Propionibacteriaceae, Actinomycetaceae, Mycobacterium bacteriaceae, Frankiaceae, Actinoplanaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae, RickettsiaceBar, Tone And microorganisms belonging to Anaplasmataceae, Chlamydiaceae, Mycoplasmataceae, and Acholeplasmataceae.

  As the antibodies used for labeling the various microorganisms to be detected with fluorescence or enzymes, antibodies that specifically bind to the microorganisms (bacteria, fungi, chlamydia, virus, rickettcher) (hereinafter, Examples of antibodies include monoclonal antibodies and polyclonal antibodies). For example, conventionally known antibodies against bacteria include Mycobacterium tuberculosis antibody, anti-Mycobacterium avium antibody, anti-Streptococcus pneumoniae antibody, anti-Streptococcus A group antibody, anti-Streptococcus B group antibody, anti-Streptococcus D group antibody, anti-Streptococcus mutans antibody, anti-Staphylococcus aureuse antibody, anti-Staphylococcus epidermidis antibody, anti-Esherichia coli antibody, anti-intestinal tract Hemorrhagic E. coli O-157: H7 antibody, anti-Salmonella sp. Antibody, anti-Shigella sp. Antibody, anti-Campylobacter sp. Antibody, anti-Vibrio cholerae, paraheamoliticase ) Antibody, anti-Klebsiella sp. Antibody, anti-Brotheus antibody, anti-Legionella pneumophila antibody, Helicobacter pylori antibody, Pseudomonas aeruginosa antibody, Hemophilisinflenzae, Heamophilis sp. Antibody, Pasteurella sp. Antibody, Neisseria meningitidis group A, B , C, X, Y, Z, W, E) antibodies, anti-Neisseria gonorrhea antibodies, anti-Listeria sp. Antibodies, Clostridium botulium antibodies, Clostridium difficile Examples include antibodies. Examples of conventionally known antibodies against fungi include anti-Candida (Candida albicanse, Candida tropicals, Candida stllatoidea) antibodies. In addition, antibodies against antigens common to all bacterial species can be used, and antibacterial cell wall peptide glycan (Bacterial peptidoglycan) antibodies, anti-gram positive bacterial cell wall lipotic acid (Bacterial Lipoteichoicacid) antibodies, and the like are known. Antibacterial bacteria (Enterobacteriaceae α, β) antibodies are useful antibodies for separating bacteria inhabiting the intestinal tract, and anti-mannans, anti-galactomannans, and 1-3β-D glucans are common antibodies to fungi. Etc. are known.

  The sample to be analyzed by the method of the present invention is a sample suspected of containing microorganisms. In the medical field, blood, urine, feces, sputum, cerebrospinal fluid, pus, collected from various (suspected) infected patients Biological samples such as saliva, raw materials and various processed products such as meat, eggs, vegetables, fish, and drinking water in the food field, and tap water, factory effluent, bath water, sewer water, activated sludge in the environmental sanitation field Water can be exemplified.

(2) Microorganism separation and analysis device The microorganism separation and analysis device of the present invention is based on the principle of microtubule electrophoresis. Specifically, (i) an electrophoresis buffer containing an ionic polymer other than alginate is used. A pair of water tanks, (ii) a microtube that separates the microorganisms injected into the electrophoresis buffer, and (iii) means for applying a potential gradient to the electrophoresis buffer in the microtube. The microorganism separation / analysis apparatus of the present invention is further characterized by comprising (iv) a detection means for detecting the microorganisms separated in the microtubule.

  FIG. 1 is an example showing a schematic configuration of an apparatus for analyzing microorganisms by microtubule electrophoresis according to the present invention. The analyzer according to the present invention will be described in more detail with reference to the drawings. The analyzer 1 according to the present invention includes a pair of water tanks (anode-side water tank (11) containing an electrophoresis buffer containing an anionic polymer other than alginate. ), Cathode side water tank (12)), microtube 10 for separating injected microorganisms, a pair of electrodes (anode side (13), cathode as means for applying a potential gradient to the electrophoresis buffer in the microtube) Side (14)) and a power source 15, and a light source 17 such as a laser light source and a detector 18 for detecting fluorescence generated by the laser irradiation as detection means for detecting microorganisms separated in the microtubule. It is configured. Both ends of the microtube 10 are respectively immersed in water tanks 11 and 12 containing an electrophoresis buffer, and the inside of the microtube is filled with the electrophoresis buffer. Electrodes 13 and 14 are attached to the water tanks 11 and 12, respectively, and a power source 15 is connected to them through a conducting wire. The detecting means is provided near the outlet side (10b) of the microtube. The microtube portion is optically non-absorbing in the corresponding wavelength region, and the microtube itself is formed as a detection cell (flow cell) 16 so that it can be directly optically detected through the microtube. The water tank may be provided with a cooling means on its outer periphery in order to prevent the water temperature of the aqueous solution in the capillary from rising due to heat generated when voltage is applied (not shown).

  According to such an analyzer, after injecting a sample containing microorganisms to be analyzed into one end (inlet side, 10a) of the microtube 10, the power source 15 is operated to apply a voltage between the electrode 11 and the electrode 12. As a result, the entire liquid in the migration region (in the microtube) generates a flow toward the cathode due to the electroosmosis phenomenon. On the other hand, the microorganism tries to move to the electrode side (water tank) having the opposite polarity to the charge that it has. For example, a microorganism having a negative surface charge tries to move to the anode side. In many cases, such movement is sufficiently small compared to the electroosmotic flow, and microorganisms separated in the microtubule from the migration process are UV-flowed in the detection section near the outlet of the microtube (10b) in the form of flowing into the electroosmotic flow. -It can be detected optically, such as by a visible or fluorescent detector. When the microorganism to be analyzed is fluorescently labeled with a fluorescent antibody or the like, the fluorescence generated by the microorganism is detected by laser irradiation from the light source 17 by using a laser-induced fluorescence detector as the detector 18. Can do. In FIG. 1, arrows drawn along the microtubule 10 indicate the direction in which cells migrate.

  Regarding the electrophoresis buffer containing an anionic polymer other than alginate used in the analyzer, a microtube, a means for applying a potential gradient to the electrophoresis buffer of the microtube, a microorganism to be analyzed, and a detection means A wide variety of the above-described methods for the microorganism analysis method of the present invention can be mentioned.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to the said Example at all.
Example 1
Lactobacillus deburueckii subsp. Bulgaricus B-5b ( L. deburueckii B-5b), Lactococcus lactis subsp . Lactis JCM-1158 ( Lc. Lactis JCM-1158), Streptococcus thermophilus 510 ( S. thermophilus 510) and Acetobacter aceti JCM-7641 One platinum loop was taken from each of the slant solid cultures, and these were separately inoculated into a 200 mL Erlenmeyer flask containing 50 mL of MRS medium prepared according to a conventional method, and then cultured with shaking at 30 ° C. for 48 hours. After shaking culture, transfer 500 μL of the culture solution to a 1.5 mL sample tube, and add 0.75 μL of SYTO9 solution A from LIVE / DEAD BacLight Bacterial Viability Kits (Molecular Probes) to the tube as it is. After being allowed to stand, it was subjected to capillary electrophoresis. The capillaries used were pre-washed with 1N and 0.1N sodium hydroxide at 20 psi for 1 minute, ultrapure water at 20 psi for 3 minutes, and electrophoresis buffer at 20 psi for 2 minutes before electrophoresis. did. The sample was injected into the capillary at 5 psi for 0.4 seconds, and electrophoresis and detection were performed under the following conditions.

<Capillary electrophoresis and detection conditions>
Equipment: Biofocus (Bio-rad)
Capillary: Fused silica tube (GL Sciences),
Inner diameter 50μm, total length 24cm, effective length 20cm
Electrophoretic conditions: 10 kV (4 μA), 3 min running buffer: For Lactobacillus deburueckii subsp. Bulgaricus B-5b ( L. deburueckii B-5b), 10 mM borate buffer (pH 9) was used as a control, and further 10 mM boric acid Use each buffer solution (pH 9) with each anionic sugar polymer (dextran sulfate sodium (average molecular weight 500,000), heparin sodium and chondroitin sulfate C sodium) added and dissolved to a concentration of 0.1% (w / v). It was. For Lactococcus lactis subsp. Lactis JCM-1158 ( Lc. Lactis JCM-1158) and Streptococcus thermophilus 510 ( S. thermophilus 510), 10 mM borate buffer (pH 9) was used as a control, and further 10 mM borate buffer (pH 9). ) And dextran sulfate sodium (average molecular weight 500,000) added and dissolved so that the concentration becomes 0.1% (w / v). For Acetobacter aceti JCM-7641, 10 mM borate buffer solution (pH 9) was used as a control, and sodium poly-γ-glutamate was added to 10 mM borate buffer solution (pH 9) to a 0.05% concentration (w / v). A solution in which was added and dissolved was used.
Detection conditions: Absorption detection at an ultraviolet wavelength of 210 nm, or detection at an excitation wavelength of 488 nm (argon ion laser module laser light) at a fluorescence wavelength of 530 nm Detector: UV-visible spectrophotometer, or laser-induced fluorescence (LIF) detector.

Lactobacillus deburueckii subsp. Bulgaricus B-5b ( L. deburueckii B-5b), Lactococcus lactis subsp . Lactis JCM-1158 ( Lc. Lactis JCM-1158), Streptococcus thermophilus 510 ( S. thermophilus 510) and Acetobacter aceti JCM Ferrograms of microtubule electrophoresis for -7641 are shown in FIGS. 2 to 5, respectively.

In FIG. 2, the analysis conditions (A) to (D) are as follows.
Electrophoresis solution: (A) 10 mM borate buffer (pH 9.0), (B) 10 mM borate buffer (pH 9.0) containing 0.10% (w / v) dextran sulfate sodium (average molecular weight 500,000), (C ) 10 mM borate buffer (pH 9.0) containing 0.10% (w / v) heparin sodium, (D) 10 mM borate buffer (pH 9.0) containing 0.10% (w / v) chondroitin sulfate C sodium, capillary : 24cm x 50μm (effective length 20cm), applied voltage: 10kV [(A) 2μA, (B) 4μA], sample: B-5b [(A) approx. 2,700, (B) approx. 1,600, (C) approx. 2,600, (D) about 2,500]

  3 and 4, the analysis conditions of (A) and (B) are as follows: (A) 10 mM borate buffer (pH 9.0), (B) 0.10% (w / v) dextran sodium sulfate (average molecular weight 500,000) 10 mM borate buffer solution (pH 9.0). In FIG. 5, the analysis conditions of (A) and (B) are (A) 10 mM borate buffer (pH 9.0), (B) 0.05% (w / v) sodium poly-γ-glutamate (average molecular weight 500,000) 10 mM borate buffer solution (pH 9.0).

As can be seen from Fig. 2, it can be confirmed that L. deburueckii B-5b can be well separated and detected as sharp peaks by adding anionic sugar polymers (sodium dextran sulfate, sodium heparin, sodium C chondroitin sulfate). It was. As can be seen from FIGS. 3 and 4, Lc. Lactis JCM-1158 and S. thermophilus 510 can be well separated and detected as sharp peaks by adding an anionic sugar polymer (sodium dextran sulfate). It could be confirmed. Furthermore, as can be seen from FIG. 5, it was confirmed that Acetobacter aceti JCM-7641 could be well separated and detected as a sharp peak by adding an anionic polymer (poly-γ-glutamic acid).

Next, for L. deburueckii B-5b, the culture was sequentially diluted with 10 mM borate buffer, and each tube was subjected to microtubule electrophoresis under the above conditions (using dextran sulfate sodium as an additive). The ferrogram result obtained by measuring the absorbance is shown in FIG. FIG. 6 shows the relationship between the number of injected bacteria and the electropherogram obtained by UV-visible absorption detection, and the numerical value at the upper right of each pherogram indicates the number of injected bacteria. When the number of injected cells and the detected peak area were plotted, it was shown that the peak intensity was proportional to the number of bacteria as shown in FIG. From this, it was found that the microorganisms in the specimen can be quantitatively measured by the microtubule electrophoresis method according to the present invention. In addition, for the fluorescently labeled L. deburueckii B-5b, the culture solution was diluted with 10 mM borate buffer sequentially, and microtube electrophoresis was performed under the above conditions (using dextran sulfate sodium as an additive). FIG. 8 shows the ferrogram result obtained by performing fluorescence measurement. FIG. 8 shows the relationship between the number of injected bacteria and the electropherogram obtained by laser-excited fluorescence detection, and the numerical value at the upper right of each pherogram indicates the number of injected bacteria. When the number of injected cells and the fluorescence detection peak area were plotted, as shown in FIG. 9, it was shown that the peak intensity was proportional to the number of bacteria. From this, it was found that the microorganisms in the specimen can be quantitatively measured with higher sensitivity by the microtubule electrophoresis method according to the present invention.

Example 2
A 200 mL Erlenmeyer flask containing 50 mL of MRS medium prepared by taking one platinum loop from each slanted solid culture of L. deburueckii B-5b and Lactobacillus acidophilus L-54 ( L. acidophilus L-54). And inoculated separately at 30 ° C. for 48 hours. After shaking culture, mix 500 μL of each culture into a 1.5 mL sample tube, add 0.75 μL of SYTO9 solution A from LIVE / DEAD BacLight Bacterial Viability Kits (Molecular Probes), and add it to room temperature in the dark. After leaving for 15 minutes, it was subjected to capillary electrophoresis. Electrophoresis and detection conditions were the same as in Example 1.

The ferrogram is shown in FIG. L. acidophilus L-54 was separated in about 1.0 minutes by L. deburueckii B-5b in about 1.5 minutes, and both microorganisms were completely separated and detected by microtubule electrophoresis. It was the same thing. This revealed that two types of lactic acid bacteria were separated and detected in a short time.

It is a figure which shows schematic structure of the analysis apparatus of microorganisms by the microtube electrophoresis of this invention. In Example 1, it is a figure which shows the result of having carried out capillary electrophoresis, adding dextran sodium sulfate, heparin sodium, chondroitin sulfate C sodium to the electrophoresis solution about L. deburueckii B-5b. The vertical axis represents absorbance at 210 nm, and the horizontal axis represents time (minutes). In Example 1, Lc. Lactis JCM-1158 is a pherogram when capillary electrophoresis is performed by adding dextran sulfate sodium to the electrophoresis solution. In Example 1, it is a ferrogram figure at the time of performing a capillary electrophoresis for S. thermophilus 510, adding dextran sulfate sodium to the electrophoresis solution. In Example 1, it is a ferrogram figure at the time of performing a capillary electrophoresis by adding poly-g-sodium glutamate to the electrophoresis solution about Acetobacter aceti JCM-7641. In Example 1, it is a ferrogram figure at the time of performing capillary electrophoresis by adding sodium dextran sulfate to the electrophoresis solution about L. deburueckii B-5b. In Example 1, it is a figure which shows the ultraviolet visible absorption peak area with respect to the number of microbe used for electrophoresis at the time of performing a capillary electrophoresis by adding sodium dextran sulfate to the electrophoresis solution about L. deburueckii B-5b. In Example 1, it is a ferrogram figure at the time of performing a capillary electrophoresis by adding a sodium dextran sulfate to the electrophoresis solution about the fluorescent label L. deburueckii B-5b. In Example 1, it is a figure which shows the fluorescence peak area with respect to the number of microbes used for the electrophoresis at the time of performing a capillary electrophoresis by adding sodium dextran sulfate to the electrophoresis solution about the fluorescence label L. deburueckii B-5b. . In Example 2, it is a figure which shows the result of having isolate | separated and detected L. deburueckii B-5b and L. acidophilus L-54 by fluorescence microtube electrophoresis. The vertical axis represents fluorescence intensity, and the horizontal axis represents time (minutes). The peak that appears first is derived from the coumarin fluorescent dye added as an osmotic flow marker.

Explanation of symbols

1. Microorganism analyzer by microtubule electrophoresis
Ten. Fine tube
10a. Entrance side
10b. Exit side
11． Water tank (anode side)
12． Water tank (cathode side)
13. Electrode (anode side)
14. Electrode (cathode side)
15． Power supply
16． Detection cell (flow cell)
17． Light source (laser light source)
18． Detector (laser induced fluorescence detector)
19. Microorganisms isolated

Claims (32)

  A method for separating and analyzing microorganisms, comprising subjecting a sample containing microorganisms to microtubule electrophoresis using an electrophoresis buffer containing an anionic polymer other than alginate.   A method for analyzing a microorganism, comprising: treating a sample containing a microorganism with a fluorescent antibody that binds to the microorganism; and subjecting the sample to microtubule electrophoresis to detect fluorescence of the microorganism labeled with the fluorescent antibody.   3. The method for analyzing a microorganism according to claim 2, wherein the microtubule electrophoresis is performed using an electrophoresis buffer containing an anionic polymer other than alginate.   2. The method for separating and analyzing microorganisms according to claim 1, wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.   5. The method for separating and analyzing microorganisms according to claim 4, wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.   6. The method for separating and analyzing microorganisms according to claim 5, wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin and chondroitin sulfate.   The method for separating and analyzing microorganisms according to claim 1, wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.   The anionic polymer having sulfonic acid groups and / or carboxylic acid groups is selected from the group consisting of polyglutamate, polystyrene sulfonate and poly (styrene sulfonic acid / maleic acid) salts. A method for separating and analyzing microorganisms.   The method for analyzing a microorganism according to claim 2 or 3, wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.   The method for analyzing microorganisms according to claim 9, wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.   The method for analyzing a microorganism according to claim 10, wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin and chondroitin sulfate.   4. The method for analyzing microorganisms according to claim 2, wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.   13. The anionic polymer having sulfonic acid groups and / or carboxylic acid groups is selected from the group consisting of polyglutamate, polystyrene sulfonate and poly (styrene sulfonic acid / maleic acid) salts. Microbial analysis method.   An apparatus for separating microorganisms using microtubule electrophoresis, wherein (i) a water tank containing an electrophoresis buffer containing an anionic polymer other than alginate, and (ii) a microorganism injected into the electrophoresis buffer A microbe separation apparatus comprising: a microtubule for separating the microbe.   The apparatus for separating microorganisms according to claim 14, wherein the anionic polymer other than alginate is an anionic sugar polymer other than alginate.   The apparatus for separating a microorganism according to claim 15, wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.   The apparatus for separating a microorganism according to claim 16, wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin and chondroitin sulfate.   The apparatus for separating microorganisms according to claim 14, wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.   19. The anionic polymer having sulfonic acid groups and / or carboxylic acid groups is selected from the group consisting of polyglutamate, polystyrene sulfonate and poly (styrene sulfonic acid / maleic acid) salts. Microbe separation device.   An apparatus for analyzing microorganisms using microtubule electrophoresis, comprising: (i) a water bath containing an electrophoresis buffer containing an anionic polymer other than alginate; and (ii) a microorganism injected into the electrophoresis buffer. A microbe analysis apparatus comprising: a microtubule to be separated; and (iii) a detection means for detecting the separated microbe.   21. The microorganism analyzing apparatus according to claim 20, further comprising a fluorescence detector as the detecting means.   The microbial analyzer according to claim 20 or 21, wherein the anionic polymer other than an alginate is an anionic sugar polymer other than an alginate.   23. The microorganism analyzing apparatus according to claim 22, wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.   24. The microorganism analyzing apparatus according to claim 23, wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin and chondroitin sulfate.   22. The microorganism analyzing apparatus according to claim 20 or 21, wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.   26. The anionic polymer having sulfonic acid groups and / or carboxylic acid groups is selected from the group consisting of polyglutamate, polystyrene sulfonate and poly (styrene sulfonic acid / maleic acid) salts. Microbe analysis device.   A separation promoting agent for separating microorganisms in microtubule electrophoresis for separating microorganisms, comprising an anionic polymer other than alginate and a buffer.   28. The microorganism separation promoter according to claim 27, wherein the anionic polymer other than an alginate is an anionic sugar polymer other than an alginate.   29. The microorganism separation promoter according to claim 28, wherein the anionic sugar polymer other than alginate is an anionic sugar polymer having a sulfonic acid group.   30. The microorganism separation promoter according to claim 29, wherein the anionic sugar polymer having a sulfonic acid group is selected from the group consisting of dextran sulfate, heparin and chondroitin sulfate.   28. The microorganism separation promoter according to claim 27, wherein the anionic polymer other than alginate is an anionic polymer having a sulfonic acid group and / or a carboxylic acid group.   32. The anionic polymer having sulfonic acid groups and / or carboxylic acid groups is selected from the group consisting of polyglutamate, polystyrene sulfonate and poly (styrene sulfonic acid / maleic acid) salts. Microbial separation promoter.

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