JPWO2010001841A1 - Method for aggregating and / or precipitating microorganisms with MDP1 - Google Patents

Abstract

[Problem] The present invention provides a method for easily collecting bacteria without applying stress by inducing aggregation and / or precipitation in the pretreatment stage of mycobacterial test, and further, specific to acid-fast bacteria. It is an object of the present invention to provide a very simple test method for identifying mycobacteria by a simple operation of agglutinating and / or precipitating and detecting this. [Solution] The present invention provides a separation method characterized in that a substance or microorganism having a mycolic acid-containing glycolipid is aggregated and / or precipitated using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid. It is.

Description

  The present invention relates to a method for aggregating and / or precipitating microorganisms by MDP1. More specifically, the present invention relates to a method for aggregating and / or precipitating the substance or the microorganism with MDP1 while minimizing stress on the substance or microorganism having the mycolic acid-containing glycolipid.

  About one third of humans are infected with tuberculosis, and 8 million people worldwide are affected by tuberculosis and 2 million people die worldwide. Most adult pulmonary tuberculosis is caused by regrowth of latent infections.

  In the examination of mycobacterial infection to which Mycobacterium tuberculosis belongs, detection and identification of the bacteria are conventionally performed by methods such as smear examination, culture examination, and genetic examination.

  The smear test is a method of staining bacteria and detecting this by microscopic observation, and can be performed in a relatively short time, but requires equipment such as a microscope and experienced staff. The culture test is a highly accurate method, but it takes a long time because the growth rate of acid-fast bacteria is very slow. Genetic testing is a method for amplifying and detecting nucleic acids eluted from bacteria, and although it has recently become possible to carry out relatively easily, a device for nucleic acid amplification can be used while reducing the cost of reagents. The point is the bottleneck.

  In any of the above-described examination methods, it is essential to concentrate or purify target bacteria from clinical specimens as so-called pretreatment. This process is generally performed by a centrifugal separation operation, but it is necessary to apply a strong centrifugal force in order to achieve a high bacterial collection rate. For this reason, a large-scale device (centrifugal separator) is required, but stress on the bacteria given by the centrifugal force cannot be ignored. For example, there is a problem of false negatives due to an increase in dead bacteria, which greatly increases the test accuracy. affect.

  As described above, although there are merits in each of the current inspection methods, there is no one that satisfies the point of point of care testing (POCT) that “it can be done easily anytime, anywhere”. In particular, the centrifugation operation that is unavoidable in any inspection is a major obstacle.

  Under such circumstances, various methods for inducing aggregation of cells and bacteria have been developed. For example, Patent Document 1 discloses cell aggregation using antibody-supported latex particles, Patent Document 2 uses antibodies, electricity, ultrasound, attractants, and Patent Document 4 uses cationic polymers to aggregate cells. A method of guiding is disclosed. Patent Document 3 describes a method for electrically inducing aggregation, as in Patent Document 2.

  However, the method using electricity or ultrasonic waves requires a relatively large apparatus and cannot specifically aggregate target cells or bacteria. On the other hand, the specificity is exhibited if an antibody is used, but regarding acid-fast bacteria, a technique for specifically agglutinating using an antibody is not disclosed.

  On the other hand, a polypeptide (MDP1) having immunogenicity against pathogenic mycobacteria and its use for vaccines or therapeutic agents have been proposed (Patent Document 5).

JP 2004-191332 A International Publication No. 2001/67084 Pamphlet JP 2003-223 A Japanese translation of PCT publication No. 2003-507032 JP 2000-219699 A

  The present invention has been made in view of such circumstances, and a method for easily collecting bacteria without applying stress by inducing aggregation and / or precipitation in the pretreatment stage of the acid-fast bacterium test, Another object of the present invention is to provide a very simple test method for identifying acid-fast bacteria by a simple operation of specifically agglutinating and / or precipitating acid-fast bacteria and detecting this.

  As a result of intensive studies to solve the above problems, the present inventors have found that substances or microorganisms having a mycolic acid-containing glycolipid easily aggregate and / or precipitate by MDP1, and thus complete the present invention. It came.

  That is, the separation method of the present invention is characterized in that a substance or microorganism having a mycolic acid-containing glycolipid is aggregated and / or precipitated using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid. It is.

  The protein and the polypeptide are MDP1 or a recombinant protein having an amino acid sequence in which one or several amino acids of MDP1 are deleted, substituted or added, and the recombinant protein is acid-fast bacilli It is preferable to have immunogenicity against.

  The agglomeration and / or precipitation can be promoted by flash centrifugation or spin-down, and the signal can be enhanced to a visible extent by a solid support that interacts with the mycobacterial surface. .

  The microorganism having the mycolic acid-containing glycolipid is preferably an acid-fast bacterium.

  The solid support is preferably a particle on which an antibody against acid-fast bacteria is immobilized.

  The acid-fast bacterium is preferably a pathogenic acid-fast bacterium, and the pathogenic acid-fast bacterium is preferably a tuberculosis bacterium.

  The cell isolation method of the present invention is characterized in that a target cell is isolated from a biological specimen, culture solution or cell-containing solution using the above method.

  The nucleic acid extraction method of the present invention includes a step of isolating a target cell by using the above-described cell isolation method, and a step of extracting a nucleic acid from the isolated cell.

  The step of extracting the nucleic acid is preferably performed by performing heat treatment, ultrasonic irradiation treatment or chemical reagent treatment.

  The cell detection method of the present invention is characterized by detecting the presence or absence of a target cell from a biological specimen, culture solution or cell-containing solution by aggregation and / or precipitation using the separation method. is there.

  The cell is preferably a tuberculosis bacterium.

  The kit of the present invention is characterized by being used in any of the separation method, the cell isolation method, the nucleic acid extraction method and the cell detection method.

  The present invention detects microorganism aggregation caused by the addition of MDP1, preferably by using a solid support to detect microorganisms that have been enhanced to the extent that this aggregation is visible as a signal. A method of identifying can be provided.

  Furthermore, although the present invention can suitably utilize a gentle centrifugal force field such as flash centrifugation or spin down, it does not require a large-scale apparatus, and is collected in a state where stress on bacteria is minimized. A method of sterilizing can be provided.

  In addition, the present invention can provide a safe and low cross-contamination method of bacteria collection that does not use high-speed centrifugation that is feared to generate aerosol.

FIG. 1 is an Eppendorf tube showing the results of Example 2 and Comparative Example 2. The tube at the left end is the same as in Example 2, except that 10 μg of anti-MDP1 antibody is added instead of rMDP1, and centrifuged at 800 × g for 1 second. After separation (flush), the second tube from the left represents that after flushing without adding rMDP1 in Example 2, and the third to rightmost tubes from the left represent rMDP1 in Example 1. This represents the state after adding 0.8 μg, 2.4 μg, and 8.0 μg, respectively, and flushing.

  The present invention comprises the following methods (1) to (4) and the following kit (5).

  (1) A separation method comprising aggregating and / or precipitating a substance or microorganism having a mycolic acid-containing glycolipid using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid.

  (2) A cell isolation method comprising isolating cells from a biological specimen, culture solution or cell-containing solution using the method (1).

  (3) A nucleic acid extraction method comprising the steps of: isolating cells by using the above method (2); and extracting nucleic acids from the isolated cells.

  (4) A cell detection method characterized by using the method (1) to detect the presence or absence of cells from an organism-derived specimen, culture solution or cell-containing solution by aggregation and / or precipitation.

  (5) A kit which is used in any one of the above methods (1) to (4).

  Hereinafter, the present invention will be specifically described.

<Method (1)>
The method (1) of the present invention is characterized in that a substance or microorganism having a mycolic acid-containing glycolipid is aggregated and / or precipitated using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid. Is the method.

(Mycolic acid-containing glycolipid)
In the present invention, “mycolic acid-containing glycolipid” refers to mycolic acid, a sugar ester of monosaccharide and mycolic acid, a sugar ester of disaccharide and mycolic acid, and a disaccharide, mycolic acid, and a fatty acid other than mycolic acid. It means at least one fatty acid selected from the group consisting of sugar esters.

  Examples of the “monosaccharide” include glucose, fructose, ribose, xylose, mannose, galactose, talose and the like.

  Examples of the “disaccharide” include sucrose, maltose, lactose, cellobiose, trehalose and the like.

  Examples of “fatty acids other than mycolic acid” include octadecanoic acid, nonadecanoic acid, icosanoic acid, docosanoic acid, tetradocosanoic acid, hexadocosanoic acid, octadocosanoic acid and the like.

  Examples of the “mycolic acid-containing glycolipid” include abimycolic acid I, hominomycolic acid I, mycolic acid IIa, mycolic acid IIb, mycolic acid IIIa, mycolic acid IIIb, corinomycolic acid, trehalose-6-monomycolate, trehalose-6,6 Examples include '-dimycolate and meromicolic acid. Mycolic acid-containing glycolipids are contained in large amounts in the cell walls of acid-fast bacteria.

(Substance / Microorganism)
The “substance having a mycolic acid-containing glycolipid” is not limited to an organism, an inanimate object, or an inanimate object as long as it has the “mycolic acid-containing glycolipid”.

  “Microorganisms having mycolic acid-containing glycolipids” include not only prokaryotes (eubacteria, archaea) but also eukaryotes (algae, protozoa, fungi, slime molds), viruses, and worms. Small animals are also included. It should be noted that even if fungi and the like form macroscopic colonies, those whose structural units are microscopically sized, such as mold, are treated as “microorganisms having mycolic acid-containing glycolipids”. . In addition, unicellular organisms as well as individual cells constituting multicellular organisms are also included in “microorganisms having mycolic acid-containing glycolipids”.

  Of these microorganisms having a mycolic acid-containing glycolipid, an acid-fast bacterium that is a bacterium belonging to the genus Mycobacterium is preferable. The “acid-fast bacterium” is preferably a pathogenic acid-fast bacterium. Examples of “pathogenic mycobacteria” include Mycobacterium tuberculosis, nontuberculous mycobacteria, and leprosy. Among these, Mycobacterium tuberculosis is preferable.

  As "tuberculous bacteria", in addition to Mycobacterium tuberculosis which is a causative bacterium of human tuberculosis, M. bovis and Mycobacterium african having pathogenicity against humans (M. africanum). BCG is an attenuated version of M. bovis that has been subcultured for a long period of time, and is currently used as a vaccine for preventing tuberculosis (a vaccine for attenuated bacteria).

  Mycobacteria are slow-growing, that is, a group of late-growing bacteria that require a week or more to grow until the colonies can be discerned by the naked eye, a group of fast-growing rapidly growing bacteria, The tubercle bacillus belongs to the group of late-growing bacteria and requires 3 to 6 weeks for the separation culture.

(Protein / polypeptide)
“Protein” and “polypeptide” are MDP1 (Mycobacterial DNA-binding protein 1) or an amino acid sequence in which one or more, preferably one or several amino acids of MDP1 are deleted, substituted or added. It is desirable that the recombinant protein and the modified protein have immunogenicity against acid-fast bacteria.

  “MDP1” is a protein consisting of 205 amino acids and can be separated and purified from the BCG Tokyo strain. However, the MDP1 according to the present invention is not limited to this strain, and is a BCG strain other than the BCG Tokyo strain, It may be a protein obtained by separation and purification from Mycobacterium bacteria such as Mycobacterium tuberculosis.

  The range of “several” is not particularly limited, but means, for example, 2 to 10, preferably 2 to 5, more preferably about 2 or 3.

  “Having immunogenicity against acid-fast bacteria” means that the protein and the polypeptide are mixed with an adjuvant, lipopolysaccharide (LPS), bacterial cell walls or cells other than acid-fast bacteria, β-glucan, etc. It means having the ability to induce antibody production against acid-fast bacteria when administered to mammals in combination with polysaccharides.

  Examples of techniques for inducing mutations such as deletion, substitution or addition of one or several amino acids in the amino acid sequence of MDP1 include well-known techniques such as site-directed mutagenesis / PCR (SN Ho et al., Gene 77, p. 51 (1989); co-translated by Satoshi Saigo and Yuko Sano, Current protocols compact version, Molecular Biology Experiment Protocol I, June 1997, Maruzen).

  As the “recombinant protein” or “modified protein” (hereinafter also referred to as “rMDP1”), a recombinant protein embodiment such as a 6 × histidine fusion protein or a GST fusion protein is preferable. When producing such a recombinant protein, it is first necessary to obtain DNA encoding MDP1. Patent Document 5 describes the acquisition of DNA encoding MDP1, the base sequence of the DNA, and the like. This DNA can be expressed as a recombinant protein by incorporating it into an appropriate expression vector such as a plasmid and introducing it into Escherichia coli or yeast. The expressed recombinant protein can be separated and purified by affinity chromatography. Specifically, DNA encoding MDP1 is incorporated into a plasmid such as pQE30 or pGEX, and introduced into a cell such as Escherichia coli or BCG or a cell such as yeast, and cultured as a transformant, whereby rMDP1 Is produced (Patent Document 5).

(Interaction)
The “interaction” according to the method (1) of the present invention means that an intermolecular force acts between the mycolic acid-containing glycolipid and MDP1 or rMDP1, and these associate or bind specifically. However, the covalent bond is not included in the “interaction”.

(Aggregation / precipitation)
The term “aggregation” according to the method (1) of the present invention is used in the meaning normally used in the field, and the substance or the microorganisms are aggregated between surfaces for a certain period of time or for a long time. A phenomenon that forms. Aggregation differs from “concentration” in that the aggregated substances or microorganisms form a group of objects, but in enrichment, the concentrated substances or microorganisms are still separated from the individual substances or microorganisms independently. And are disjointed.

  The “aggregation and / or precipitation” can be facilitated by flash centrifugation or spinning down, or by a solid support that interacts with the mycobacterial surface. That is, the aggregation and / or precipitation can form “lumps” in a shorter time by performing such operations.

  As the “flash centrifugation or spin down”, it is desirable to apply a centrifugal force of preferably about 800 × g for 1 to several seconds using a small centrifuge such as a table centrifuge. Since the centrifugal operation is performed for such a short time, it is not particularly necessary to control the temperature, and therefore, the centrifuge need not be large.

  The “acid-fast bacterium surface” refers to at least one compound among compounds constituting the cell walls of acid-fast bacteria (eg, mycolic acid-containing glycolipid, murein, tycoic acid, etc.).

  The solid support that interacts with the mycobacterial surface is used as an alternative to indicate the presence of bacteria, and enhances the aggregation of bacteria to a visible level as an aggregation signal of the solid support. Can be visually recognized.

(Solid support)
“Solid support” means insoluble in water or aqueous solution that interacts with the surface of mycobacteria and enhances its aggregates and / or precipitates as visible as a signal, making it easier to see. And preferably comprises a substrate that interacts with the surface of the mycobacteria and a support that can immobilize the substrate.

  "Substrate" includes polysaccharides such as hyaluronic acid, heparin, heparin sulfate, chondroitin sulfate; antibodies (monoclonal / polyclonal) that use mycolic acid that forms the cell wall of acid-fast bacteria as an antigen; simple substances such as glucose, fructose, ribose, etc. Examples include sugars. Of these, the above antibodies are preferred because of their high affinity with the acid-fast bacterium surface.

  The “support” may be a material used for a conventionally known support or matrix, and includes an organic compound, an inorganic compound, a metal, a metal oxide, or a composite material combining these.

  Examples of the “organic compound” include synthetic organic polymers such as polystyrene, polypropylene, polyacrylate, polymethyl methacrylate, polyethylene, polyamide, and latex; collagen, collagen-glycosaminoglycan coprecipitate, cellulose, chitin, chitosan, Examples include natural organic polymers such as pectin, pectic acid, agar, agarose, xanthan gum, gellan, alginic acid, alginate, polymannan, polyglycan, starch, and natural rubber.

  Examples of the “inorganic compound” include glass, silica, silicon dioxide, silicon nitride and the like.

  Examples of the “metal” include stainless steel and zirconia.

  Examples of the “metal oxide” include zirconium oxide, aluminum oxide, sodium oxide, calcium oxide, magnesium oxide, and zinc oxide.

  Of these, polystyrene, latex, agarose, glass and silica are preferable, and polystyrene is particularly preferable.

  Examples of the shape of the support include a particle shape, a rod (fiber, capillary) shape, a plate shape, and a sheet (film) shape. Among these, the particle shape is preferable because of excellent reaction efficiency.

  The solid support is preferably a particle on which an antibody against the acid-fast bacterium surface is immobilized, and the support may be a magnetic particle having magnetism.

  Examples of the “particulate” form include a bead (sphere) shape, an ellipsoidal shape, a cone shape, a cubic shape, a rectangular parallelepiped shape, and the like. This is also preferable because it is easy to rotate and stir the solid support during use.

  The average particle diameter of the bead-shaped particles is preferably 0.5 to 10 μm, more preferably 2 to 6 μm. When the average particle diameter exceeds 10 μm, the solid support easily settles in an aqueous medium, and thus an operation of stirring the medium may be necessary when capturing the substance or microorganism. In addition, since the surface area relative to the support volume is relatively small, it may be difficult to sufficiently capture the substance or the microorganism.

  As a method of immobilizing the substrate on the support, a conventionally known method can be applied. Of these, the method of covalently binding an anti-mycolic acid antibody to bead-shaped particles is preferred. Specifically, a method of immobilizing an anti-mycolic acid antibody by covalent bonding to a tosyl group exposed on the surface of the bead-shaped particle through a tag made of histidine; cobalt or nickel exposed on the surface of the bead-shaped particle And an anti-mycolic acid antibody having a tag made of histidine by coordinating and immobilizing; a tosyl group, an epoxy group or a carboxyl group (anchored anhydrous) exposed on the surface of the bead-shaped particle A method in which an amino group of an anti-mycolic acid antibody is covalently bonded to and immobilized on maleic acid; an active maleimide group anchored to polystyrene constituting at least the surface of the bead-shaped particle, and an anti-mycolic acid antibody A method of immobilizing sulfhydryl groups by thioether bonding; latex beads Polystyrene constituting at least the surface of, or a method for physically adsorbing the anti-mycolic acids antibodies.

<Method (2): Cell isolation method>
The method (2) of the present invention uses the above-mentioned method (1), that is, a protein or polypeptide that interacts with a mycolic acid-containing glycolipid to aggregate and / or precipitate substances or microorganisms having a mycolic acid-containing glycolipid. A cell isolation method characterized in that a target cell is isolated from a biological specimen, a culture solution or a cell-containing solution using a separation method characterized in that

  The “cell of interest” may not have a mycolic acid-containing glycolipid, but is preferably an acid-fast bacterium, more preferably a pathogenic acid-fast bacterium, and particularly preferably a tuberculosis bacterium.

(Biological specimen / culture solution / cell-containing solution)
The “biological sample” is not particularly limited as long as a part or all of the living organism is directly collected. Specific examples include blood, sputum, saliva, urine, and alveoli.

  The “culture medium” is not particularly limited as long as it is a part of cells or tissues constituting the biological specimen, or a microorganism grown in an artificial environment. A specific example is a BCG solution.

  The “cell-containing solution” is not particularly limited as long as it is obtained by performing artificial treatment on the biological specimen. Specific examples include plasma, serum, and alveolar tissue disruption lysate.

  These are preferably in the form of fluids and are usually solutions or suspensions.

(Operating procedure)
Although the specific operation procedure is illustrated below as a structure of the cell isolation method of this invention, it is not limited to this aspect.

  MDP1 mixed with PBS as a binding buffer is added to an Eppendorf tube containing a BCG solution as a culture solution, and the tube is mixed by inversion. Thereafter, it may be allowed to stand for several minutes, or may be subjected to flash centrifugation or spin down (centrifugation at 800 × g for about 1 second). Mycobacteria aggregate through MDP1 and settle to the bottom of the Eppendorf tube. The cells can be isolated by removing the supernatant.

  Moreover, in the above-described embodiment, instead of the BCG solution, for example, when a cell-containing solution containing a group of microorganisms having a mycolic acid-containing glycolipid and a cell not having a mycolic acid-containing glycolipid is used, MDP1 is added. However, since the target cells do not precipitate, the target cells can be isolated by removing the precipitate.

<Method (3): Nucleic acid extraction method>
The method (3) of the present invention comprises the above-mentioned method (2), that is, the step of isolating a target cell by using the cell isolation method, and the step of extracting a nucleic acid from the isolated cell. It is the nucleic acid extraction method characterized. In the present invention, “nucleic acid” is a term including DNA, RNA, polynucleotide, and oligonucleotide.

  The “cell of interest” may not have a mycolic acid-containing glycolipid, but is preferably an acid-fast bacterium, more preferably a pathogenic acid-fast bacterium, and particularly preferably a tuberculosis bacterium.

(Step of isolating cells)
“The step of isolating cells” is characterized in that a substance or microorganism having a mycolic acid-containing glycolipid is aggregated and / or precipitated using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid. By using a cell isolation method (method (2)) for isolating target cells from a biological specimen, culture solution or cell-containing solution using a separation method (method (1)) This is a step of isolating cells to be treated.

  The above “biological specimen”, “culture medium”, and “cell-containing solution” have the same meanings as the “biological specimen”, “culture medium”, and “cell-containing solution” described in the invention (2).

(Nucleic acid extraction step)
The “nucleic acid extracting step” in the method (3) of the present invention is a step of extracting nucleic acid from the cells isolated through the above “cell isolating step”. That is, “the step of extracting nucleic acid” is preferably a heat treatment, an ultrasonic irradiation treatment or a chemical reagent treatment, or a combination of these, with respect to cells isolated through the step of isolating cells, The cell membrane is destroyed, the nucleic acid is released outside the cell, and this is separated and recovered as a nucleic acid solution.

  Such a “nucleic acid extracting step” does not include operations such as centrifugation, filtration, and reduced pressure treatment, and therefore nucleic acid can be extracted easily and rapidly.

  Various known physical treatments or chemical treatments can be applied as a treatment for breaking the cell membrane and releasing the nucleic acid out of the cell.

  Examples of the physical treatment include heat treatment, ultrasonic irradiation treatment, freezing / melting treatment, and the like.

  Examples of the chemical treatment include chemical reagent treatment, for example, denaturation treatment using a digestive enzyme, a chaotrope reagent, a surfactant, a lysing agent, and the like. If a chemical reagent that adversely affects the step after the extraction of nucleic acid is not used, it is not necessary to remove the chemical reagent, and the nucleic acid can be extracted from the cells in high yield. Tables 1 and 2 in the specification of Japanese Patent No. 2625340 summarize various protocols for lysing mycobacteria and the like.

  The heat treatment refers to application at a temperature range of 70 to 120 ° C., preferably 80 to 120 ° C., more preferably 80 to 100 ° C., for 20 seconds to 10 minutes, preferably 20 seconds to 300 seconds. The heating temperature and time vary depending on the type of cells or bacteria (size, composition and thickness of the cell membrane, etc.), and thus are appropriately selected from the above ranges. The heating means is performed by any appropriate heating means, and examples thereof include, but are not limited to, a dry heat block, a hot water bath, a microwave oven, and various heaters.

  When performing ultrasonic irradiation treatment, it is necessary to appropriately set ultrasonic irradiation conditions effective for lysis. The irradiation amount can be calculated from the rated output of the ultrasonic irradiation apparatus to be used, the amount of cells to be irradiated, and the irradiation time. For example, the frequency of the ultrasonic wave is selected depending on the amount of the cell-containing solution, stirring conditions, etc., but is preferably 10 to 100 kHz, more preferably 15 to 45 kHz. At this frequency, at least 5 minutes, preferably 5 to 5 kHz. It is desirable to irradiate for about 30 minutes.

  A more preferable treatment for destroying the cell membrane and releasing the nucleic acid out of the cell is an embodiment using a combination of heat treatment and ultrasonic irradiation treatment. In such an embodiment, lysis can be achieved under milder conditions than the above treatment conditions. However, the nucleic acid is not denatured at least by heat treatment or ultrasonic irradiation treatment.

<Method (4): Cell detection method>
The method (4) of the present invention uses the above-mentioned method (1), that is, a protein or polypeptide that interacts with a mycolic acid-containing glycolipid to aggregate and / or precipitate substances or microorganisms having a mycolic acid-containing glycolipid. A solid that interacts with the surface of an acid-fast bacterium, preferably by aggregation and / or precipitation, in the presence or absence of cells of interest from a biological specimen, culture solution or cell-containing solution using a separation method characterized in that It is a cell detection method characterized by detecting aggregation by enhancing the aggregation and / or precipitation to a level that can be visually recognized as a signal.

  The above “target cells” are preferably acid-fast bacteria, more preferably pathogenic acid-fast bacteria, and particularly preferably tuberculosis bacteria.

  The above “biological specimen”, “culture medium”, and “cell-containing solution” have the same meanings as the “biological specimen”, “culture medium”, and “cell-containing solution” described in the invention (2).

(Detection of cells by aggregation / precipitation)
The presence or absence of the target cell due to aggregation and / or precipitation can be detected, for example, by measuring turbidity. That is, the effectiveness of the aggregated cells can be measured in terms of the turbidity reduction rate (% RT). “% RT” refers to a decrease in the absorbance (600 nm) of the suspension medium after aggregation compared to the absorbance before aggregation, and is obtained from the following formula.

% RT = ((A ₁ −A ₂ ) × 100) / A ₁
(Where A ₁ represents the absorbance at 600 nm of the suspension medium prior to aggregation, and A ₂ is the result of adding a protein or polypeptide that interacts with a mycolic acid-containing glycolipid and allowing it to settle for 15 minutes. Represents the absorbance of the suspending medium at 600 nm.)
Such a turbidity reduction rate (% RT) is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably substantially 100%.

  Alternatively, as a preferred embodiment, a solid support that interacts with the acid-fast bacterium surface can be added, and cell aggregation and / or precipitation can be enhanced as a visible signal.

<Kit (5)>
The kit (5) of the present invention is used in any one of the above methods (1) to (4).

  That is, the kit (5) of the present invention is a set of equipment required for carrying out any of the methods (1) to (4) of the present invention, specifically, various reagents, solid supports, cells. It includes equipment necessary for isolation and nucleic acid extraction / purification. These reagents include lysis (or dilution) solutions for dissolving (or diluting) samples, washing solutions, various buffers, and the like. Solid support, binding buffer, cell washing buffer (cell washing solution), lysis buffer (resuspension), nucleic acid washing buffer required for carrying out any of the methods (1) to (4) of the present invention (Nucleic acid washing solution) and nucleic acid elution buffer (elution solution) are preferably kits in which the respective containers are enclosed in advance. In the necessary equipment set, a dedicated instrument for attaching or binding cells to a solid support and breaking the cell membrane to extract nucleic acids contained therein may be included as a component of the kit. Any of the above methods (1) to (4) of the present invention can be carried out using these instruments.

  In order to carry out any of the methods (1) to (4) of the present invention, tools or equipment other than those described above may be required, but these are appropriately included as components of the kit of the present invention. Centrifugal force may be used for solid-liquid separation, and in that case, a small centrifuge such as a desktop centrifuge can be used.

  In the cell isolation method (2) and the nucleic acid extraction method (3) of the present invention, at least two kinds of buffer solutions are used. Examples of the binding buffer include phosphate-based, acetate-based, or Tris-based buffer solutions that may contain sodium chloride, potassium acetate, EDTA, a surfactant, and the like as salts. Specifically, PBS, TBS, and the like It is. As the cell washing buffer, a buffer obtained by diluting the binding buffer 4 to 5 times may be used, but different types of buffers may be separately prepared. Examples of the lysis buffer include water, a buffer solution, and a buffer solution containing a salt, and TE buffer is particularly preferable. Kit (5) does not include organic solvents such as chloroform and phenol, chaotrope reagents, lysing agents and the like that have been used for nucleic acid extraction in the prior art.

  When carrying out any of the methods (1) to (4) of the present invention, a consumable and a mixer for stirring, a stirrer, a heater or ultrasonic generator for heating, solid-liquid separation by magnetic treatment Magnets, magnetic sorters, etc. used in the above are also used as necessary. Specifically, the stirring is desirably vibration by a test tube mixer or inversion mixing that rotates a container, and the magnet may be a bar magnet. The type of magnet may be either an electromagnet or a permanent magnet, but a permanent magnet is preferred from the viewpoint of simplicity and operability, and a neodymium magnet is particularly preferred from the strength of magnetic force.

  These are devices normally provided in the examination room, and these devices can be used with the kit (5) to carry out any of the methods (1) to (4) of the present invention. Further, if necessary, some of such devices may be included as part of the kit. The configuration of the kit (5) used in each of the methods (1) to (4) of the present invention may be the same or different.

  The whole or a part of the solid support or kit can be variously modified so long as the structure, configuration, arrangement, shape, size, material, method, method, and the like are consistent with the gist of the present invention.

  Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these.

[Preparation Example 1: Preparation of rMDP1]
RMDP1 with a histidine tag was prepared according to a previously reported method (Aoki et al., J. Biol. Chem., 279: 39798-39806 (2004)). The outline is as follows.

  First, the rMDP1 expression vector (pET22B-MDP1) was introduced into E. coli competent cells (BL21 (DE3) pLysS competent cell / Invitrogen) and transformed. When this transformant was cultured at 22 ° C. in an LB liquid medium containing 50 μg / mL carbenicillin and 34 μg / mL chloramphenicol, the absorbance at 600 nm reached 0.3 to 0.6. IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added so that the final concentration was 0.1 mM. By culturing for 16 hours, rMDP1 was expressed.

  The Escherichia coli recovered by centrifugation is pulverized by sonication using an ultrasonic cell disruption apparatus (trade name “Bioruptor UCD-200T” / manufactured by Tosoh Corporation), and again 8,000 × g, 30 After centrifugation for 1 minute, the supernatant was recovered as eluted protein. The supernatant was passed through a membrane filter (0.22 μm) to remove dust, and then 50 mM sodium dihydrogen phosphate (pH 8.0), 10 mM imidazole, 0.5 M NaCl, It was added to a metal chelate affinity chromatography carrier (trade name “Ni-NTA Agarose” / Qiagen Co., Ltd.) equilibrated with 0.01% Tween. After removing the non-specifically adsorbed protein by washing the column, 50 mM sodium dihydrogen phosphate (pH 8.0), 300 mM imidazole, 0.5 M NaCl, 0.01 RMDP1 was eluted with an elution buffer consisting of% Tween. Thereafter, the elution buffer was replaced with PBS by dialysis. As a result of measuring by staining with Coomassie Brilliant Blue (CBB) after SDS-PAGE, the concentration of rMDP1 obtained was 1 mg / mL.

[Example 1]
Add 0.8 μg, 2.4 μg, and 8.0 μg of rMDP1 mixed in binding buffer (PBS) to 500 μL of BCG solution (5 × 10 ⁶ cfu / mL), and mix by inverting (25 ° C. × 5 minutes). did. The mixed solution was allowed to stand for 10 minutes to visually confirm bacterial aggregation and / or precipitation.

[Example 2]
The mixture immediately after inversion in Example 1 was centrifuged (flushed) at 800 × g for 1 second to visually confirm bacterial aggregation and / or precipitation. The result is shown in FIG.

[Comparative Example 1]
In Example 1, instead of rMDP1, an anti-MDP1 monoclonal antibody (reference: Katsube et al., Control of cell wall assembly by a histone-like protein in Mycobacteria .; J Bacteriol., 189 (22): 8241-8249, 2007) was added 10 μg, or rMDP1 was not added. Aggregation of bacteria was not confirmed after standing for 10 minutes.

[Comparative Example 2]
In Example 2, 10 μg of anti-MDP1 antibody was added instead of rMDP1, or rMDP1 was not added. Even after centrifugation (flash) at 800 × g for 1 second, bacterial aggregation and / or precipitation was not observed. The result is shown in FIG.

  The method of the present invention is suitable as a simple and rapid test method for Mycobacterium tuberculosis infection that satisfies the needs of POCT.

α-MDP1 Ab: anti-MDP1 antibody (-) ... system in which rMDP1 is not added in Example 2

Claims (14)

A separation method comprising aggregating and / or precipitating a substance or microorganism having a mycolic acid-containing glycolipid using a protein or polypeptide that interacts with the mycolic acid-containing glycolipid.
The protein and the polypeptide are MDP1, or a recombinant protein comprising an amino acid sequence in which one or several amino acids of the MDP1 are deleted, substituted or added, and the recombinant protein is an acid-fast bacterium 2. The method of claim 1 having immunogenicity against.
The method according to claim 1 or 2, wherein the aggregation and / or precipitation is promoted by flash centrifugation or spinning down.
The method according to any one of claims 1 to 3, wherein the microorganism having the mycolic acid-containing glycolipid is a mycobacteria.
The method according to any one of claims 1 to 4, wherein the aggregation and / or precipitation is enhanced as a signal by a solid support that interacts with the acid-fast bacterium surface.
The method according to claim 5, wherein the solid support is a particle on which an antibody against an acid-fast bacterium is immobilized.
The method according to any one of claims 4 to 6, wherein the acid-fast bacterium is a pathogenic acid-fast bacterium.
The method according to claim 7, wherein the pathogenic mycobacteria are Mycobacterium tuberculosis.
A cell isolation method comprising isolating a target cell from an organism-derived specimen, a culture solution or a cell-containing solution using the method according to claim 1.
A nucleic acid extraction method comprising the steps of: isolating a target cell by using the cell isolation method according to claim 9; and extracting a nucleic acid from the isolated cell.
The nucleic acid extraction method according to claim 10, wherein the step of extracting the nucleic acid is performed by performing a heat treatment, an ultrasonic irradiation treatment, or a chemical reagent treatment.
A cell characterized by detecting the presence or absence of a target cell from an organism-derived specimen, culture solution or cell-containing solution by aggregation and / or precipitation using the method according to claim 1. Detection method.
The cell detection method according to claim 13, wherein the cell is Mycobacterium tuberculosis.
  A kit for use in the method according to claim 1.