JPH0956399A - Selective condensation of microorganism and microorganism inspection using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To selectively condense a microorganism for effective inspection of the microorganism by dipping a carrier having immobilized antibody against the objective microorganism in a sample solution, capturing the microorganism and suspending in a dispersion in a smaller amount than the sample solution and not containing a microorganism. SOLUTION: A polymer material (e.g.; nylon-6 stick) immobilizing an antibody specifically bonding with the objective microorganism (e.g.; Vibrio cholerae) is directly dipped in a sample solution or a suspended sample solution of human blood, human body fluid, urine, dejection, tissue, etc., and the objective microorganism existing in the sample solution is selectively captured, then the objective microorganism captured in the polymer material is suspended in a dispersion in a smaller amount than the sample solution and not containing a microorganism to condense the objective microorganism. A DNA from the microorganism condensed in the method is taken out, the gene is amplified by a PCR (polymerase chain reaction) method and the objective microorganism can be inspected according to the gene information.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for selectively concentrating microorganisms in a sample used in the fields of clinical testing and food testing, and a method for detecting microorganisms using the method.
More specifically, the target is selected from samples of human blood, body fluid, urine, feces, tissues, etc. directly or suspended, or plural kinds or a small amount of microorganisms contained in samples of food directly or suspended. Method for selectively capturing and concentrating microorganisms, and taking out DNA from the microorganisms to
The present invention relates to a method for inspecting microorganisms in a sample solution by the R method.

[0002]

2. Description of the Related Art In clinical tests and food tests, it is necessary to accurately and quickly test microorganisms in a sample. Culture testing methods that are commonly used in microbiological testing require skill in testing, and require 10 to 16 hours of enrichment culture and 18 to 24 hours of separate culture, so the test results after specimen acquisition are known. It takes a huge amount of time, and there are cases where the sample contains almost no viable bacteria due to the spread of chemotherapy. In Japan, it was desired to develop a highly sensitive and rapid test method.

In recent years, as means for solving the problems of these culture test methods, molecular genetic test methods such as DNA probe method, ELISA method, reverse passive latex agglutination reaction method (RP).
Immunological test methods represented by LA method) have been developed and researched. All of these methods are excellent in terms of specificity, rapidity, simplicity, etc., and have the possibility of being directly detected from the sample, and are therefore expected to be capable of extremely rapid diagnosis. , There are some that have been put to practical use.

[0004]

On the other hand, regarding the molecular genetic testing method, recently, PCR (Polymerase chain reaction) for synthesizing and amplifying a specific gene of interest has been recently conducted.
Laws have been developed and are rapidly spreading. This method uses a primer that specifically reacts with the nucleotide sequence of a target gene and a DNA synthase to amplify only the target gene, and the amplified gene is agarose gel electrophoresis or DNA probe method. Can be detected by PC
The R method can theoretically detect if the target gene is one or more in the sample, and if the specific nucleotide sequence of the gene of the target microorganism is determined, it is a highly sensitive and rapid detection method. Can be. However, when detecting microorganisms in a sample using this PCR method, there is no problem if the number of target microorganisms present in the sample is large, but conversely, if it is extremely small, a part of the sample is When the sample is subjected to the PCR reaction, there is a high possibility that the target microorganism is not present in the sample taken, and the target gene is often not detected even if the cycle of the amplification reaction is increased. Therefore, it is necessary to sample a plurality of samples from one sample, and as a result, there is a problem that the probability must be dependent. In addition, when a microorganism other than the target microorganism is mixed in the sample, there is a problem that a false positive is likely to occur because the genes of the microorganism are simultaneously amplified.

Because of these problems, amplification and detection of a gene contained in a target microorganism directly from a sample by the PCR method is limited to the application to the detection of a part of the microorganism contained in a relatively large amount in the sample. However, detection of most of the microorganisms still required an enrichment culture procedure.

It is an object of the present invention to provide a method for selectively concentrating only a desired microorganism in a sample solution, and an efficient method for inspecting a microorganism using the method.

[0007]

Means for Solving the Problems As a result of intensive investigations by the present inventors in order to solve the above problems, as a result, a polymeric material on which an antibody that specifically binds to a target microorganism is immobilized is used as a sample solution. It is possible to selectively capture only the target microorganisms by immersing it in water, and by suspending the polymeric material that has captured the target microorganisms in a smaller amount of dispersion liquid than the sample solution, only the target microorganisms are suspended. Found that it is possible to efficiently amplify the gene of the target microorganism by subjecting this concentrated microorganism to the PCR method, and found that it becomes possible to test the microorganism, The present invention has been reached.

That is, first, the present invention selects a target microorganism present in the sample solution by immersing a polymer material on which an antibody that specifically binds to the target microorganism is immobilized in the sample solution. The method for selectively concentrating microorganisms is characterized by suspending the microorganisms that are trapped in a polymer material and then trapped in a polymer material in a dispersion liquid containing a smaller amount than the sample solution and containing no microorganisms. Secondly, a gist of a microorganism inspection method characterized by extracting DNA from a microorganism concentrated by the above method, amplifying a gene by PCR method, and then inspecting the microorganism based on the gene information. To do.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The microorganism in the present invention, for example, tuberculosis, staphylococcus, streptococcus, gonorrhea, syphilis, pertussis, tetanus,
Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Shigella, Diphtheria, Salmonella typhi, Paratyphi, Bacillus cereus, Yersinia,
Campylobacter, Clostridium perfringens, Difficile, Salmonella, Vibrio parahaemolyticus, Cholera, Clostridium botulinum,
Helicobacter, Plesiomonas, Aeromonas, Legionella, Chlamydia, bacteria such as spirochete, Candida, yeast, Aspergillus, fungi such as mold, pneumonia virus, hepatitis virus, AIDS virus, rotavirus, herpes virus, RS virus, polio virus, In addition to pathogenic microorganisms such as influenza virus, measles virus, cytomegalovirus, dengue virus, etc., Bacillus, actinomycetes, photosynthetic bacteria, archaea, nitric acid bacteria, etc. that exist in general environment Microbes, algae, etc. are also included.

The sample solution containing microorganisms in the present invention means (1) a solution obtained by suspending human blood, body fluid, sputum, urine, feces, etc. as they are or in distilled water, buffer solution, physiological saline, etc.,
(2) Human tissue suspended in distilled water, buffer, physiological saline, etc., (3) Distilled foods such as fish, shellfish, meat, grains, vegetables and fruits, or processed foods thereof. Water, buffer solution, solution suspended in physiological saline, etc., (4) seawater, river water, industrial water, drinking water, waste water, water system existing in natural environment such as sewage,
(5) Distilled water, such as soil, a buffer solution, a solution in which physiological saline is suspended, (6) A colony or the like of the above-mentioned microorganism is a culture solution or distilled water,
Examples include buffer solutions, solutions suspended in physiological saline and the like.

In the present invention, the polymeric material for capturing the target microorganisms includes, for example, ethylene-vinyl acetate copolymer, polyvinyl chloride, polyurethane, polyethylene, polystyrene, polyamide such as nylon, polyester, polycarbonate, Examples thereof include polymeric materials such as polyacrylic acid, cellulose, natural rubber and glass, and derivatives thereof, regardless of whether they are organic polymeric materials or inorganic polymeric materials. Further, other materials may be materials obtained by coating these materials.

Examples of the shape of these polymer materials include a film, a sheet, a tube, a fiber, a stick, a rod, and the like, and an appropriate handle may be provided so that they can be easily operated by hand.

In the present invention, in order to selectively capture the microorganisms in the sample, an antibody capable of specifically binding to the desired microorganisms is immobilized on the polymer material. Any antibody may be used as long as it has a component characteristic of the structure of the target microorganism surface, for example, against the microorganism itself or glycoproteins, lipids, flagella, pili, and colonization factors present on the cell surface. An antibody etc. are mentioned.

Examples of the method for immobilizing an antibody on a polymer material for capturing microorganisms in the present invention include a physical adsorption method, an ionic bond method, a covalent bond method and the like. The covalent bond method is preferably used because it can be immobilized.

To immobilize an antibody on the surface of a polymeric material by covalent bonding, a carboxyl group, a formyl group, an amino group, an azido group, an isocyanate group, a chloroformyl group, an acid anhydride group and an epoxy group can be attached to the surface of the polymeric material. It is necessary to introduce a reactive functional group such as Materials having these functional groups existing in advance can be used as they are, but polymeric materials having these functional groups can also be used by introducing these functional groups.

As a method for introducing a reactive functional group onto the surface of a polymer material, a carboxyl group is introduced by, for example, carboxymethylating a hydroxyl group of cellulose or a saponified ethylene vinyl acetate copolymer. To be done. The carboxyl group is derived from an acid group via a hydrazide group. Further, the carboxyl group can be converted to a chloroformyl group by chlorination with thionyl chloride, acetyl chloride or the like. The amino group can be introduced by aminoacetalization in a polymer material having a hydroxyl group introduced therein, and can be introduced by reacting with 3-aminopropyltriethoxysilane in activated glass.

The epoxy group is introduced by reacting a polymer material having a hydroxyl group introduced therein with epichlorohydrin, diethylene glycol diglycidyl ether or the like.
Isocyanate group is hexamethylene diisocyanate,
It is introduced by reacting with tolylene diisocyanate or the like. The formyl group is introduced by reacting a polymer material having a hydroxyl group introduced therein with glutaraldehyde, dialdehyde starch or the like. The acid anhydride group is introduced by reacting an amino group-introduced polymer material with ethylene-maleic anhydride copolymer, methyl vinyl ether-maleic anhydride copolymer, styrene-maleic anhydride copolymer or the like. It In addition, as a method of introducing a reactive functional group to the material surface, for example, Japanese Patent Application Laid-Open No. 4-1730
Physically introducing a reactive functional group, for example, by graft-polymerizing a copolymer of maleic anhydride with polyurethane or polystyrene disclosed in Japanese Patent Publication No. 90 by γ ray or electron beam to introduce an acid anhydride group. You can also

There is also a method of introducing a reactive functional group onto the surface of a polymer material by coating a compound having a reactive functional group. Further, even in the case of a polymeric material having a reactive functional group in advance, for example, nylon is acid-treated in order to introduce a large amount of amino groups onto the surface of the nylon, and the surface is hydrolyzed to expose the carboxyl group, followed by polyethylene. A large amount of reactive functional groups can be introduced by performing a treatment such as a reaction with a polyamine such as imine.

Many methods have been developed and are known as covalent bonding methods between a reactive functional group and an antibody.
A method of diazoniumizing a polymer material having an amino group introduced with dilute hydrochloric acid and sodium nitrite to diazo couple with an antibody, or a method of condensing a carboxyl group or an amino group into a polymer material such as a carbodiimide reagent to form an antibody protein and an amide A method of binding, a method of reacting a polymeric material introduced with a carboxyl group with an amino group of an antibody protein as a derivative of azide, chloride, isocyanate, etc., an amino acid of an antibody protein added to a polymeric material introduced with a reactive functional group such as halogen. Group, phenolic hydroxyl group, thiol group, etc. can be alkylated (“immobilized biocatalyst” edited by Ichiro Chibata, Kodansha). In addition, JP-A-5
There is also a method disclosed in JP-A-249116 in which an amino group or a thiol group of an antibody protein is immobilized on a polymer material into which an acid anhydride group has been introduced, under relatively mild conditions.

A desired reactive functional group such as an amino group can be introduced into a polymer compound having no reactive functional group by an ammonia-plasma treatment or a radiation treatment such as γ-ray or electron beam. As a position for immobilizing the antibody of the polymer material, the antibody-immobilized polymer material is immersed in the sample solution to capture the target microorganisms, and then suspended in the PCR reaction dispersion liquid in the PCR reaction. It suffices that the antibody is immobilized on the portion immersed in the dispersion liquid for use. For example, when a certain amount of a PCR reaction dispersion is placed in a PCR reaction tube and immersed in a polymeric material on which the antibody is immobilized to suspend it, the portion on which the antibody is immobilized is dispersed for PCR reaction. It need only be completely immersed in the liquid.

In the present invention, a polymeric material on which an antibody against a target microorganism is immobilized is immersed in a sample solution to capture the microorganism. The temperature in the sample solution at the time of capturing is as follows.
It is 0 ° C. to room temperature, preferably room temperature. The immersion time is several seconds to 24 hours, preferably 1 minute to 1 hour, more preferably 1 minute to 10 minutes. At this time, the polymer material on which the antibody is immobilized may be immersed in a static state, but if it is appropriately moved up and down, left and right, or rotated in the axial direction, the microorganisms can be captured more efficiently. . Also, for example,
The antibody-immobilized polymer material processed into a shaft may be rotated in a sample solution using a machine such as a motor to capture the microorganism.

The polymer material in which the microorganisms in the sample solution are captured can be selectively concentrated by suspending it in a dispersion liquid for PCR reaction or the like. Examples of the dispersion liquid used in the present invention include a culture medium for microorganisms, distilled water, a buffer solution, a solution suspended in physiological saline and the like. The temperature of the dispersion liquid at the time of suspension is 0 ° C. to room temperature,
It is preferably room temperature. The suspension time is a few seconds to 1 hour, preferably a few seconds to 10 minutes, more preferably 30 seconds to 5
It's a minute. At this time, the polymer material that has captured the microorganisms can be dipped and suspended in a static state, but by appropriately moving it up and down, left and right, or rotating it axially, the microorganisms can be dispersed more efficiently. Can be suspended in Also,
For example, the material processed into the shape of a shaft may be rotated in a dispersion using a machine such as a motor to suspend the microorganisms.

The dispersion obtained by selectively concentrating the desired microorganism obtained by the above method can be used for the inspection of the microorganism by treating it with the PCR method. Here, the inspection of microorganisms is based on the gene sequence peculiar to the microorganisms, determining the type of microorganisms, the structure specific to the cell surface such as serotype, the production of pathogenic factors such as toxins, other enzyme proteins, sugars. It is to detect the quality of producing quality and lipids. The microorganism testing method of the present invention involves extracting DNA from cells in the dispersion liquid obtained by the above method, amplifying the gene using a DNA amplification method such as PCR, and then analyzing the gene sequence peculiar to the microorganism. It is to inspect microorganisms. As a method for extracting DNA from microbial cells, for example, 90 ° C
The method of destroying cells by the above heat treatment, alkali treatment, surfactant treatment, enzyme treatment or the like, or a combination of these methods can be mentioned.

The DNA extracted from the microorganism is subjected to PCR
It is amplified by the (polymerase chain reaction) method and used for the inspection of microorganisms. The principle of PCR method is double-stranded DNA
Is divided into single strands by heating above 90 ° C, and returns to the same double strands when the temperature is lowered. Basically, double stranded DNA is heated to 92-94 ℃ to make it single stranded by heat denaturation (Denature), temperature is 45-60 ℃
By annealing to a part of the single-stranded DNA that is complementary to the target gene sequence and a base sequence (primer) synthesized in advance based on the known base sequence of the DNA is annealed. To 72-75 ° C and reacting with DNA polymerase.
An extension (Extens
ion) operation, and by repeating this operation n times, the target gene sequence is amplified by (2 ⁿ -n-1) times.

As a method for inspecting a microorganism from the DNA amplified by the PCR method, a known method may be used, and examples thereof include agarose gel electrophoresis and a method for examining the nucleotide sequence by a labeled DNA probe method and the like. .

[0026]

EXAMPLES The present invention will be described in more detail below with reference to examples. Reference Example 1 (Preparation of Antibodies for Capturing Microorganisms) As an antibody against microorganisms, a polyclonal antibody against Vibrio cholerae was prepared by the following method. Vibrio cholerae O1 was used as the cholera bacterium. This microorganism is owned by the Research Institute for Microbial Diseases of Osaka University, but it is a "microorganism having a property that causes or has a risk of causing damage to health or the environment." It cannot be done.
Cholera was suspended in 10 mM phosphate buffer containing 0.9% sodium chloride (pH 7.0: abbreviated as PBS hereafter) at a concentration of 1 × 10 ⁷ cells / ml, and an equal volume of Freund's complete (Freund's) solution was added.
complete) Adjuvant (manufactured by Difco) was added to a rabbit having a body weight of about 2 kg for immunization. After 25 days, instead of Freund's complete adjuvant, Freund's incomplete adjuvant (manufactured by Difco) was added to immunize. One week after the second immunization, blood was collected to obtain antiserum. 50% ammonium sulfate was added to the obtained antiserum to cause precipitation. This operation was repeated twice, and the resulting precipitate was dissolved in 10 mM phosphate buffer (pH 7.0) and dialyzed against the same buffer, and the obtained immunoglobulin was used as a polyclonal antibody against Vibrio cholerae.

Reference Example 2 (Preparation of antibody-immobilized polymer material) [Immobilization of antibody to nylon stick by covalent bond]
(1)] The antibody obtained in Reference Example 1 was immobilized on nylon 6 via an acid anhydride group. Immobilization was performed by the following method. Ten nylon 6 sticks (diameter 1.0 mm, length 5 cm, manufactured by Unitika Ltd.) were immersed in 10 ml of 3N hydrochloric acid at 30 ° C. for 30 minutes and then washed with distilled water. After drying, 10 ml of 10
% (W / v) Polyethyleneimine aqueous solution and methanol in a 1: 5 mixture at room temperature for 30 minutes, then 20 ml of 5%
A solution of dicyclohexylcarbodiimide in methanol was added, and the mixture was then immersed at room temperature for 2 hours to introduce amino groups on the nylon surface. Next, after being washed with methanol and dried, it was immersed in 20 ml of a dehydrated acetone solution of a 2% (w / v) styrene-maleic anhydride copolymer at room temperature for 1 hour, washed with acetone, and then vacuum dried. . The 5 mm tip of the obtained stick was immersed in a 10 mM phosphate buffer containing 2 μg / ml of an antibody against V. cholerae at 4 ° C. for 16 hours to immobilize the antibody. After the immobilization reaction, the stick was washed with distilled water and then dried.

Reference Example 3 (Preparation of antibody-immobilized polymer material) [Immobilization of antibody to nylon stick by covalent bond]
(2) The antibody obtained in Reference Example 1 was immobilized on nylon 6 using glutaraldehyde. Immobilization was performed by the following method. Nylon 6 Stick (Diameter 1.0mm, Length
5 cm, manufactured by Unitika Ltd.) 30 pieces in 10 ml of 3N hydrochloric acid
After soaking at ℃ for 30 minutes, it was washed with distilled water. After drying
After soaking in 10 ml of a 1: 5 mixture of 10% (w / v) polyethyleneimine aqueous solution and methanol at room temperature for 30 minutes,
An amino group was introduced into the nylon surface by adding ml of a 5% solution of dicyclohexylcarbodiimide in methanol and then immersing the solution in room temperature for 2 hours. Next, after washing with methanol and drying, soak in 10 ml of 0.02N NaOH,
Add 0.15 ml of 25% (v / v) glutaraldehyde and
It was treated at 30 ° C for 30 minutes. Further, it was immersed in 10 ml of 0.1% tannic acid solution at 30 ° C. for 15 minutes, washed, and dried. The 5 mm tip of the obtained stick was immersed in a 10 mM phosphate buffer containing 2 μg / ml of an antibody against V. cholerae at 4 ° C. for 16 hours to immobilize the antibody. After the immobilization reaction, the stick was washed with distilled water and then dried.

Reference Example 4 (Preparation of antibody-immobilized polymer material) [Immobilization of antibody to nylon stick by covalent bond]
(3)] The antibody obtained in Reference Example 1 was immobilized on nylon 6 by a condensation reaction. The antibody was immobilized by the following method. Nylon 6 stick (diameter 1.0mm, length 5cm,
Unitika 10) in 10 ml of 3N hydrochloric acid at 30 ℃, 30
After soaking for a minute, it was washed with distilled water. After drying, 10 ml
After soaking in a 1: 5 mixture of 10% (w / v) polyethyleneimine aqueous solution and methanol at room temperature for 30 minutes, 20 ml of 5
% Dicyclohexylcarbodiimide in methanol was added, and the mixture was then immersed at room temperature for 2 hours to introduce amino groups on the nylon surface. Next, after washing with methanol and drying, soak in 10 ml of 0.01N HCl for 10 minutes at room temperature, wash with distilled water, and then get the tip 5 of the stick.
mm portion is 10 ml of 0.1 M phosphate buffer: 0.9% NaCl =
It is immersed in a solution of 1: 1 (pH 5.0), and 0.2 ml of 0.1 mg / ml WSC [1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride] is added to the solution, and the mixture is kept on ice for 10 minutes. After allowing to stand for a minute, 0.2 ml of an antibody against 2 μg / ml of cholera was added, and the antibody was immobilized by immersing on ice for 16 hours. After the immobilization reaction, the stick was washed with distilled water and then dried.

Reference Example 5 (Preparation of Antibody-Immobilized Polymer Material) [Immobilization of Antibody on Polystyrene Stick by Physical Adsorption] The antibody obtained in Reference Example 1 was immobilized on polystyrene by a physical adsorption reaction. The antibody was immobilized by the following method. Polystyrene ELISA plate (Falcon) stick shape (length 5 cm, width 0.2 mm, thickness)
0.2 mm). Into a 1.5 ml microtube (Eppendorf), add 0.3 ml of 50 mM carbonate buffer (pH 9.6) containing 2 μg / ml of antibody against cholera, and put the 5 mm tip of the resulting stick at 4 ° C at 16 ° C. It was immersed for a period of time to carry out an antibody immobilization reaction. After the immobilization reaction, the stick was washed with distilled water and then dried.

Reference Example 6 (Preparation of antibody-immobilized polymer material) [Immobilization of antibody to glass stick by covalent bond]
The antibody obtained in Reference Example 1 was immobilized on an alkyl aminated glass. The antibody was immobilized by the following method. Porous glass (manufactured by Corning Incorporated) is used as a stick (vertical 5 c
m, width 0.2 mm, thickness 0.1 mm) and heated in 0.1 MHNO ₃ for 3 hours. After washing, vacuum dry at 90 ℃, and
Heat treatment was performed at 500 ° C. for 3 hours. This glass stick
The mixture was refluxed in a toluene solution of 10% (v / v) 3-aminopropyltriethoxysilane for 3 hours, washed with toluene and dried to obtain an alkylaminated glass stick. In a 1.5 ml microtube (Eppendorf), 0.3 ml of 2 μg / ml antibody against cholera and 2 μg of dicyclohexylcarbodiimide in 10 mM phosphate buffer (pH)
5.0) was added and the 5 mm tip of the obtained glass stick was immersed at 4 ° C. for 16 hours to carry out an antibody immobilization reaction.
After the immobilization reaction, the stick was washed with distilled water and then dried.

Example 1 (Method for selectively capturing and concentrating microorganisms by antibody-immobilized polymer material) [Selective capture of cholera bacteria by antibody-immobilized stick]
Concentration] The antibody-immobilized nylon stick obtained in Reference Example 1 was used to selectively capture and concentrate microorganisms. Vibrio cholera 01 (Vibrio cholera 01 (Vibrio cholera)
e O1)] cholera toxin (CT) producing strain AQ-1001 was used. This microorganism is owned by the Institute for Microbial Diseases, Osaka University, but is a "microorganism having a property that causes or may cause harm to health or the environment." It is something that cannot be done. The above cholera fungus was inoculated on a heart infusion plate (manufactured by Difco) containing 3% salt,
It was cultured at 37 ° C for 16 hours. The obtained colonies were cultured in a liquid medium of Heart Infusion (manufactured by Difco) containing 3% sodium chloride, and the number of bacteria was adjusted to 1 × 10 ² from the absorbance at 610 nm. The obtained culture solution
1 ml in a 1.5 ml microtube (Eppendorf)
added.

The antibody-immobilized nylon stick obtained in Reference Example 2 was attached to the chuck portion of a stirring motor (Shinto Kagaku Co., Ltd., Three One Motor 300G), and 0.5 cm of the antibody-immobilized tip portion was cultured in the microtube. After immersing in the liquid and rotating it with a stirring motor at 60 rpm for 3 minutes, the stick was taken out from the culture liquid and the tip of the stick was washed with sterile distilled water. Next, in a 0.5 ml microtube (made by Eppendorf), 3% containing no bacterial cells
Heart infusion containing salt (Difco
20 μl of liquid medium was added, and the tip portion of the stick that had been subjected to the above operation was immersed in this liquid medium and rotated by a stirring motor at 180 rpm for 3 minutes to suspend the captured bacterial cells. After suspension, the same liquid medium was added to make the total volume 100 μl, and this was applied to a selective medium for cholera bacteria (TCBS medium, manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. for 16 hours. After culturing, the number of colonies obtained was examined. As a result of the above operation, almost all of the 1 × 10 ² cholera bacteria present in 1 ml of the culture solution were suspended in 20 μl of the liquid medium, It was confirmed that the cell culture solution was concentrated 50 times.

Examples 2 to 5 (Method for selectively capturing and concentrating microorganisms by antibody-immobilized polymer material) [Selective capture of cholera bacteria by antibody-immobilized stick]
Concentration] Example 1 except that the antibody-immobilized stick obtained in Reference Example 3, Reference Example 4, Reference Example 5, and Reference Example 6 was used in place of the antibody-immobilized nylon stick obtained in Reference Example 2.
The same operation as described above was performed. (Example 2 using the antibody-immobilized stick obtained in Reference Example 3 Example 2 using the antibody-immobilized stick obtained in Reference Example 4 Example 3 using the antibody-immobilized stick obtained in Reference Example 4 The one using the immobilized stick was used as Example 4 and the one using the antibody-immobilized stick obtained in Reference Example 6 was used as Example 5.) As a result of the above operation, any antibody-immobilized stick was used. Almost all 1 × 10 ² cholera bacteria present in 1 ml of the culture solution were suspended in 20 μl of liquid medium, and it was confirmed that the cell culture solution was concentrated 50 times. .

Example 6 (P of selectively enriched microorganisms
Detection method by CR method) [Detection of cholera toxin gene from selectively concentrated cholera bacterium by PCR method] Detection of cholera toxin gene using the concentrated cell culture of cholera bacterium obtained in Example 1 Was performed according to the method of Miyagi et al. (Bacteriological Techniques Series 12, "Basic Techniques for Research on Bacterial Pathogenic Factors and Application to Clinical Examinations", Nane Shuppan 1993). × 10 Reaction buffer (Reactio
n buffer, manufactured by Toyobo) 5 μl, 2 mM deoxynucleotide triphosphate mixed solution (dNTP mixture, manufactured by Toyobo) 5
μl, 100 μM DNA primer sense
e, Cholera toxin, Wako Pure Chemical Industries, Ltd.) 1 μl, 100 μM DNA primer antisense (Cholera toxin, Wako Pure Chemical Industries, Ltd.) 1 μl, 2 μg / ml Thermus thermophilus HB8 (Tth) DNA polymerase (Toyobo Co., Ltd.) 45 μl of reaction solution consisting of 1 μl and 32 μl of sterile distilled water
Was prepared and dispensed into a 0.5 ml microtube (manufactured by Eppendorf). To this reaction solution, 5 μl of a cell culture solution that had been boiled for 10 minutes and lysed by heating was added, 1 drop of liquid paraffin was added, and the mixture was centrifuged. This was set in a PCR device and pre-denatured at 94 ° C for 1 minute,
℃, 25 seconds), annealing (55 ℃, 35 seconds), extension (75
The gene was amplified by carrying out 25 cycles of steps (° C, 20 seconds). After amplification of the gene by PCR, the cholera toxin gene was confirmed by 0.8% agarose gel electrophoresis (100 V, about 30 minutes).
The presence of the cholera toxin gene was confirmed by the presence of an amplified band of the cholera toxin gene at the position of 0 bp (base pair). From the above results, it is apparent that microorganisms can be detected by applying the cell culture solution obtained by the method for selectively capturing and concentrating microorganisms of the present invention to the PCR method.

Examples 7 to 10 (Detection method of selectively concentrated microorganisms by PCR method) [Detection of cholera toxin gene by PCR method from selectively concentrated cholera bacteria] Obtained in Example 1 A gene was amplified by the PCR method in the same manner as in Example 6 except that the concentrated cholera bacterial cell culture solution was used instead of the concentrated cholera bacterial cell culture solution. Detection of microorganisms was performed. (Examples using the concentrated microbial cell culture solution of V. cholerae obtained in Example 3 and Example 8 using the concentrated cell culture medium of V. cholerae obtained in Example 4) The one using the concentrated microbial cell culture solution of V. cholerae obtained in Example 5 was designated as Example 9, and the one using the concentrated cell culture medium of V. cholerae obtained in Example 6 was designated as Example 10.) After the amplification of the gene by PCR, the cholera toxin gene was confirmed by 0.8% agarose gel electrophoresis (100 V, about 30 minutes), and the band of the cholera toxin gene amplified at the position of 380 bp (base pairs) was confirmed. The presence of the cholera toxin gene was confirmed from the fact that From the above results, it is apparent that microorganisms can be detected by applying the cell culture solution obtained by the method for selectively capturing and concentrating microorganisms of the present invention to the PCR method.

Reference Example 7 [Immobilization of anti-Escherichia coli antibody on nylon stick by covalent bond] 10 mM containing 2 μg / ml of antibody against Vibrio cholerae
The antibody was immobilized in the same manner as in Reference Example 2 except that 10 mM phosphate buffer containing 2 μg / ml of anti-Escherichia coli monoclonal antibody (Chemicon) was used instead of the phosphate buffer.

Example 11 [Selective capture / concentration of Escherichia coli by antibody-immobilized stick] The antibody-immobilized nylon stick obtained in Reference Example 7 was used to selectively capture / concentrate microorganisms. Escherichia coli (Escherichia coli strain HB101, purchased from INVITROGEN) was inoculated on a heart infusion plate (manufactured by Difco) and cultured at 37 ° C for 16 hours. The obtained colonies were cultured in a liquid medium of Heart Infusion (manufactured by Difco), and the number of bacteria was adjusted to 1 × 10 ² from the absorbance at 610 nm. 1 ml of the obtained culture solution was added to a 1.5 ml microtube (manufactured by Eppendorf).

The antibody-immobilized nylon stick obtained in Reference Example 7 was attached to the chuck of a stirring motor (Shinto Kagaku Co., Ltd., Three One Motor 300G), and 0.5 cm of the antibody-immobilized tip portion was cultured in the microtube. After immersing in the liquid and rotating it with a stirring motor at 60 rpm for 3 minutes, the stick was taken out from the culture liquid and the tip of the stick was washed with sterile distilled water. Next, to a 0.5 ml microtube (Eppendorf), add 20 μl of heart-infusion [Difco] liquid medium containing no cells to the tip of the stick that was subjected to the above operation in this liquid medium. Immerse part and 180 with a stirring motor
The captured bacterial cells were suspended by rotating at rpm for 3 minutes. After suspension, the same liquid medium was added to make the total volume 100 μl, and this was applied to a selective medium for E. coli (DHL medium, Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. for 16 hours. After culturing, when the number of obtained colonies was examined, 1 ml was obtained by the above operation.
It was confirmed that almost all of the 1 × 10 ² Vibrio cholerae existing in the culture broth were suspended in 20 μl of the liquid medium, and the cell culture was concentrated 50 times.

Example 12 [Identification of Escherichia coli by PCR method from selectively concentrated culture solution] Instead of the concentrated microbial cell culture solution of V. cholerae obtained in Example 1, it was obtained in Example 11. Genes were amplified by the PCR method to detect and identify Escherichia coli in the same manner as in Example 6 except that the concentrated bacterial culture of E. coli was used.
After the amplification of the gene by PCR, the Escherichia coli gene was confirmed by 0.8% agarose gel electrophoresis (100 V, about 30 minutes), and the amplified gene band was observed. Was confirmed. From the above results, it is apparent that microorganisms can be detected by applying the cell culture solution obtained by the method for selectively capturing and concentrating microorganisms of the present invention to the PCR method.

Example 13 [Selective capture of Escherichia coli from human sample by antibody-immobilized stick] 1 ml of human watery diarrhea stool (sample) expected to contain Escherichia coli was collected, and 1.5 ml micro It was added to a tube (Eppendorf). The antibody-immobilized nylon stick obtained in Reference Example 7 was attached to the chuck of a stirring motor (Shinto Kagaku Co., Ltd., Three One Motor 300G), and 0.5 cm of the antibody-immobilized tip was immersed in the sample in the microtube. , With stirring motor 6
After rotating at 0 rpm for 3 minutes, the stick was taken out from the culture solution and the tip of the stick was washed with sterile distilled water.
Next, 0.5 ml microtube (Eppendorf)
Heart infusion [manufactured by Difco]
20 μl of liquid medium was added, and the tip portion of the stick that had been subjected to the above operation was immersed in this liquid medium and rotated by a stirring motor at 180 rpm for 3 minutes to suspend the captured bacterial cells. After suspending, add the same liquid medium to a total volume of 100 μ
l was applied to a heart infusion agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and incubated at 37 ° C. for 16 hours. The obtained colonies were transferred to a selective medium for E. coli (DHL agar medium, manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 37 ° C. for 16 hours. After the culture, the presence of E. coli colonies was confirmed on the selective medium. From the above results, it is clear that the method of the present invention can selectively capture and concentrate Escherichia coli from human watery diarrhea stool specimen.

Example 14, Comparative Example 1 [Identification of Escherichia coli by PCR method from selectively concentrated culture medium] Instead of the concentrated microbial cell culture medium of V. cholerae obtained in Example 1, Example 13 was used. A gene was amplified by the PCR method to detect and identify Escherichia coli in the same manner as in Example 6 except that the concentrated bacterial cell culture solution of Escherichia coli obtained in (1) was used (Example 14). For comparison, the human watery diarrhea stool used in Example 13 was subjected to the gene amplification by the PCR method in the same manner as in Example 6 without performing the concentration method of the present invention to detect and identify Escherichia coli. It carried out (Comparative example 1). After the amplification of the gene by PCR, the Escherichia coli gene was confirmed by 0.8% agarose gel electrophoresis (100 V, about 30 minutes). As a result, in Example 14, the band of the amplified gene was observed to be dark and Was confirmed. On the other hand, in Comparative Example 1, the gene band was recognized only very thinly. From the above results, the bacterial cell culture solution obtained by the method for selectively capturing and concentrating microorganisms of the present invention was PCR
It is clear that the gene can be efficiently amplified by the method and the microorganism can be detected from the amplified gene.

[0043]

According to the method for selectively concentrating microorganisms of the present invention, only the desired microorganism in the sample solution can be selectively concentrated. In addition, since the concentrated microorganisms can efficiently perform gene amplification by the PCR method, the microorganism inspection method of the present invention enables the inspection of microorganisms,
Particularly, it is useful for detecting a pathogenic factor existing in a pathogenic microorganism.

Claims (2)

[Claims] 1. A polymeric material, on which an antibody that specifically binds to a target microorganism is immobilized, is immersed in a sample solution to selectively capture the target microorganism present in the sample solution, and then to increase the concentration of the target microorganism. A method for selectively concentrating microorganisms, which comprises suspending a target microorganism trapped in a molecular material in a dispersion liquid containing a smaller amount of a microorganism than a sample liquid. 2. A method for testing microorganisms, which comprises extracting DNA from a microorganism concentrated by the method according to claim 1, amplifying the gene by PCR, and then inspecting the microorganism based on the gene information. .