JP4813916B2 - Methods for concentration and elution of Legionella - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for concentrating and eluting legionella bacteria enabling the bacteria in a liquid for treating the bacteria to be easily and efficiently retrieved, and to provide a kit for the method. <P>SOLUTION: The method for concentrating and eluting legionella bacteria from a liquid 3 for treating the bacteria containing the same comprises the first step of contacting the liquid 3 with a carrier 1 where an area at least near the surface is composed mainly of a calcium phosphate-based compound to deposit the legionella bacteria on the carrier 1 and the second step of contacting a buffer solution 4 containing a buffering agent in a concentration of 25-500 mM with the resultant carrier 1 to isolate the legionella bacteria into the buffer solution 4 to concentrate and elute the bacteria(retrieve the bacteria into the buffer solution 4). <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a method for concentrating and eluting Legionella.

  For example, Legionella bacteria are respiratory pathogenic bacteria and originally distributed widely in the soil, but are also detected from 24-hour bath water, which is an artificial environment.

  Then, Legionella pneumonia, Pontiac fever, etc., which are caused by scattering from these into the air and causing respiratory infections, are a problem.

  Immunochromatography is known as a method for detecting such microorganisms as Legionella. Immunochromatography is a measurement method that uses an antigen-antibody reaction. An antibody that binds to the antigen to be detected (in this case, a microorganism) is labeled with a chromogenic substance, and the test paper has this antibody immobilized on a local part. To do. Specifically, a liquid specimen is hung on a part of the test paper that is away from the part where the antibody is fixed. Then, the specimen slowly moves on the test paper, and when the antigen is contained in the specimen, the antigen and the antibody are combined to develop a color. Thereby, the antigen in a sample can be detected.

  By the way, in order to detect microorganisms by a microorganism detection method such as immunochromatography, the number of microorganisms per unit volume needs to be equal to or greater than the detection limit (see, for example, Patent Document 1).

Therefore, even when microorganisms live in the liquid to be treated such as bath water, if the number is extremely small, the microorganisms may not be detected and may be overlooked.
In such a case, it can be considered that microorganisms are concentrated and detected from the liquid to be treated.

  However, in reality, no effective method has been found for a method for efficiently concentrating microorganisms.

JP-A-5-322897

  An object of the present invention is to provide a method for concentrating and eluting Legionella bacteria and a method for concentrating and eluting Legionella bacteria, which can concentrate and elute Legionella bacteria in a liquid to be treated easily and with high yield.

Such an object is achieved by the present invention described below.
(1) A method for concentrating and eluting Legionella from a liquid to be treated containing Legionella,
A first step of attaching the Legionella to the carrier by bringing the liquid to be treated into contact with a carrier mainly composed of a calcium phosphate compound at least near the surface;
A second step of releasing and recovering the Legionella bacteria in the buffer solution by contacting the carrier with the Legionella bacteria attached thereto with a buffer solution containing a buffer at a concentration of 25 to 500 mM. A method for concentrating and eluting Legionella, which comprises

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (2) The concentration and elution method of Legionella according to (1) above, wherein the temperature of the liquid to be treated when the liquid to be treated is brought into contact with the carrier is 70 ° C. or less.

  Thereby, Legionella bacteria in a to-be-processed liquid can be made to adhere to a support | carrier with a sufficient yield.

  (3) The method of concentrating and eluting Legionella according to (1) or (2) above, wherein the amount of the buffer solution is less than the amount of the liquid to be treated.

  Thereby, the number of Legionella per unit volume of a buffer solution can be made larger than that of a to-be-processed liquid.

(4) The method for concentrating and eluting Legionella according to any one of (1) to (3), wherein the buffer is a phosphate buffer.
Thereby, concentration and elution of Legionella bacteria can be reliably performed in a shorter time.

  (5) The method of concentrating and eluting Legionella according to any one of (1) to (4) above, wherein the pH of the buffer solution is 6.5 to 8.5.

  Thereby, when detecting Legionella bacteria concentrated and eluted in the buffer solution by immunochromatography, an accurate detection result can be obtained.

  (6) The method of concentrating and eluting Legionella according to any one of (1) to (5) above, wherein the temperature of the buffer solution when the buffer solution is brought into contact with the carrier is 70 ° C. or less.

  Thereby, Legionella bacteria can be concentrated and eluted (recovered in a buffer solution) more efficiently.

  (7) The method of concentrating and eluting Legionella according to any one of (1) to (6), wherein the carrier is granular.

  Thereby, the surface area of a support | carrier can be increased and Legionella bacteria can be made to adhere more efficiently.

  (8) The method of concentrating and eluting Legionella according to (7) above, wherein the granular carrier has an average particle size of 20 to 5000 μm.

  Thereby, since the surface area of a support | carrier can fully be ensured, the adhesion rate of Legionella bacteria to a support | carrier can be improved more.

  (9) The method of concentrating and eluting Legionella according to any one of (1) to (8) above, wherein the carrier is porous at least near the surface.

  Thereby, the surface area of a support | carrier can be increased more and the adhesion rate of Legionella bacteria to a support | carrier can further be improved.

  (10) The above (1), wherein the carrier has a base material mainly composed of a resin material and a coating layer which is provided so as to cover the surface of the base material and is mainly composed of a calcium phosphate compound. The method of concentrating and eluting Legionella according to any one of (9) to (9).

  This facilitates adjustment of the shape, size (average particle size, etc.), physical properties (density, etc.) of the carrier.

  (11) The method of concentrating and eluting Legionella as described in (10) above, wherein the resin material contains at least one of polyamide and epoxy resin as a main component.

  Thus, for example, when a calcium phosphate compound layer is formed by penetrating a part of particles mainly composed of a calcium phosphate compound in the vicinity of the surface of the substrate, the hardness (hardness) of the substrate is reduced. Since it can be made moderate, the coating with the particles can be performed easily and reliably.

(12) The method of concentrating and eluting Legionella according to any one of (1) to (11), wherein the calcium phosphate compound is mainly composed of hydroxyapatite.
Thereby, Legionella bacteria come to adhere to a support | carrier very efficiently.

(13) The method of concentrating and eluting Legionella according to any one of (1) to (12), wherein the carrier has a specific surface area of 5 to 100 m ² / g.

  Thereby, the adhesion efficiency of Legionella bacteria to the carrier can be further improved while preventing the mechanical strength of the carrier from decreasing.

  (14) The method for concentrating and eluting Legionella according to any one of (1) to (13), wherein the liquid to be treated is bath water.

  The method for concentration and elution of Legionella bacteria of the present invention is suitable for application to the concentration and elution of Legionella bacteria contained in bath water.

  (15) The first step is performed by passing the liquid to be treated into the lumen while the carrier is filled in the lumen of the tube. The method for concentrating and eluting Legionella according to any one of 14).

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (16) The concentration of Legionella according to (15) above, wherein the second step is performed by passing the buffer solution through the lumen of the tubular body through which the liquid to be treated is passed. And elution method.

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (17) A flow rate when the buffer solution is passed through the lumen portion is A [mL / min], and a flow rate when the treatment solution is passed through the lumen portion is B [mL / min]. The method for concentrating and eluting Legionella according to (16) above, wherein A / B satisfies the relationship of 0.5 to 3.

  Thereby, Legionella bacteria in the liquid to be treated can be reliably attached to the carrier, and can be reliably released from the carrier and eluted (collected in a buffer solution). That is, the concentration ratio of Legionella bacteria (recovery rate of Legionella bacteria from the to-be-processed liquid) can be raised reliably.

(18) The method of concentrating and eluting Legionella according to any one of (15) to (17), wherein a flow rate when the buffer solution is passed through the lumen is 0.75 to 15 mL / min. .
Thereby, Legionella bacteria can be eluted more reliably.

  (19) The concentration of Legionella according to any one of (1) to (14), wherein the first step is performed by immersing a mesh bag containing the carrier in the liquid to be treated. And elution method.

  Thereby, while being able to perform the contact of the to-be-processed liquid to a support | carrier by simpler operation, the Legionella microbe in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (20) The method for concentrating and eluting Legionella according to (19) above, wherein in the first step, the time for immersing the bag containing the carrier in the liquid to be treated is 2.5 hours or more.

  Thereby, even when the number of Legionella bacteria present in the liquid to be treated is relatively small, Legionella bacteria can sufficiently adhere to the carrier, and detection (concentration) of Legionella bacteria in the liquid to be treated is reliably performed. be able to.

  (21) Concentration and elution of Legionella according to (19) or (20) above, wherein the second step is performed by supplying the buffer solution as droplets to a mesh bag containing the carrier. Method.

  Thereby, Legionella bacteria can be efficiently released from the carrier with a relatively small amount of buffer used.

(22) A Legionella concentration and elution kit for use in the method of concentration and elution of Legionella according to any one of (1) to (14) above,
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A container having a storage space for storing the carrier, and an injection port and a discharge port communicating with the storage space;
A concentration and elution kit for Legionella, comprising a filter provided in the storage space so as to close the discharge port.

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (23) The Legionella concentration and elution kit according to (22), wherein at least a part of the container is foldable.

  Thereby, space can be reduced during the concentration of Legionella bacteria and the storage of the elution kit.

(24) A Legionella concentration and elution kit for use in the method of concentration and elution of Legionella according to any one of (15) to (18) above,
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A tubular body comprising a lumen filling the carrier, and an inlet and an outlet communicating with the lumen;
A concentration and elution kit for Legionella, comprising a filter provided in the lumen so as to close the discharge port.

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

(25) A Legionella concentration and elution kit for use in the method of concentration and elution of Legionella according to any of (19) to (21),
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A kit for concentration and elution of Legionella, comprising a reticulated bag for storing the carrier.

  Thereby, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  (26) The concentration and elution kit of Legionella according to (25), wherein the bag has an opening of 20 to 85 μm.

  As a result, it is possible to reliably prevent the carrier from being dissipated from the bag, and when Legionella bacteria are present in the liquid to be treated, the entry and exit of the Legionella bacteria into the bag can be reliably prevented.

  ADVANTAGE OF THE INVENTION According to this invention, Legionella bacteria in a to-be-processed liquid can be concentrated and eluted easily and with sufficient yield.

  Moreover, Legionella can be reliably concentrated by making the quantity of the buffer solution used as a collection | recovery liquid smaller than the quantity of a to-be-processed liquid. For this reason, even if there are only a few Legionella bacteria in the liquid to be treated and the presence thereof cannot be confirmed (cannot be detected), the presence in the buffer can be confirmed. Thereby, the early countermeasure (for example, sterilization etc.) with respect to a to-be-processed liquid can be performed.

  In addition, when the concentration of the buffer solution is regulated within an appropriate range, when the Legionella bacteria eluted (collected in the buffer solution) is detected by, for example, immunochromatography, the buffer agent present in a high concentration in the buffer solution Prevents the antigen-antibody reaction from being inhibited. Thereby, an accurate detection result can be obtained.

  Hereinafter, the concentration and elution method of Legionella bacteria and the concentration and elution kit of Legionella bacteria of this invention are demonstrated in detail based on preferred embodiment shown to an accompanying drawing.

  The method of concentration and elution of Legionella bacteria according to the present invention is a method of recovering Legionella bacteria from a treatment liquid containing Legionella bacteria, and the Legionella bacteria adhere to the carrier by bringing the treatment liquid into contact with the carrier. First, the carrier is attached with Legionella bacteria, and a buffer solution (recovery solution) containing a buffering agent at a concentration of 25 to 500 mM is brought into contact with the carrier to release Legionella bacteria in the buffer solution and elute (buffer) And a second step of collecting in the liquid.

First, the carrier used in the present invention will be described.
FIG. 1 is a cross-sectional view showing an example of a carrier used in the present invention.

  The carrier 1 shown in FIG. 1 is granular (substantially spherical). The carrier 1 can be used in various shapes such as a block shape (lump shape), a pellet shape, and a sheet shape, but by using a granular material, its surface area can be increased and more efficiently. Legionella can be attached.

  The carrier 1 is mainly composed of a calcium phosphate compound at least near the surface. Since the calcium phosphate compound has high affinity (cell affinity) with various cells, the carrier 1 is preferably used as the carrier 1 for attaching Legionella to the surface thereof.

  A carrier 1 shown in FIG. 1 has a base 11 mainly composed of a resin material, and a calcium phosphate-based compound layer 12 provided so as to cover the surface of the base 11 and mainly composed of a calcium phosphate-based compound. Yes. This facilitates adjustment of the shape, size (average particle diameter, etc.), physical properties (density, etc.), etc. of the carrier 1.

  The carrier 1 may be entirely composed of a calcium phosphate compound as a main material. The carrier 1 composed entirely of a calcium phosphate compound can efficiently capture Legionella.

The specific surface area of the carrier 1 is preferably from 5 to 100 m ² / g, and more preferably 15~80m ² / g. When the value is below the lower limit value, Legionella bacteria may not be attached efficiently depending on the amount of the liquid to be treated. Moreover, when the upper limit is exceeded, the carrier 1 is crushed due to a decrease in the mechanical strength of the carrier 1, and the filter may be clogged during the concentration and elution of Legionella described later.

  As the resin material constituting the substrate 11, various thermosetting resins and various thermoplastic resins can be used. Specifically, as the thermoplastic resin, for example, polyamide (for example, nylon 6, nylon 6,. 6, nylon 6,10, nylon 12), polyethylene, polypropylene, polystyrene, polyimide, acrylic resin, thermoplastic polyurethane, etc., as thermosetting resin, for example, epoxy resin, phenol resin, melamine resin, urea resin, unsaturated Examples thereof include polyester, alkyd resin, thermosetting polyurethane, and ebonide, and one or more of these can be used in combination.

  Among these, as a resin material which comprises the base material 11, it is suitable that it is what has at least one of a polyamide and an epoxy resin as a main component. For example, as will be described later, when the calcium phosphate compound layer 12 is formed by penetrating a part of particles mainly composed of the calcium phosphate compound near the surface of the substrate 11, the substrate 11 is described above. By using the material, the hardness (hardness) can be made moderate, so that the coating with the particles can be performed easily and reliably.

As the calcium phosphate-based compound constituting the calcium phosphate-based compound layer 12 is not particularly limited, Ca / P ratio can be used various compounds of 1.0 to 2.0, for _{example, Ca 10 (PO 4) 6} ( _{OH) 2, Ca 10 (PO} 4) 6 F 2, Ca 10 (PO 4) 6 Cl 2, Ca 3 (PO 4) 2, Ca 2 P 2 O 7, Ca (PO 3) 2, CaHPO 4 , etc. One or two or more of them can be used in combination.

Among them, the calcium phosphate-based compound, hydroxyapatite _{(Ca 10 (PO 4) 6} (OH) 2) as a main component is preferable. Hydroxyapatite is used as a biomaterial, and Legionella bacteria adhere to the carrier 1 very efficiently. In addition, since hydroxyapatite has a particularly low possibility of causing damage to Legionella, it can be eluted (recovered in a buffer) as described later in a state where Legionella is not disrupted (not destroyed). Thereby, Legionella bacteria can be detected more reliably.

  These calcium phosphate compounds can be synthesized by a known wet synthesis method, dry synthesis method, or the like. In this case, the calcium phosphate compound may contain a substance (raw material, etc.) remaining during the synthesis or a secondary reaction product generated during the synthesis.

  In addition, as shown in FIG. 1, in the calcium phosphate compound layer 12, a part of particles 13 (hereinafter simply referred to as “particles 13”) mainly composed of a calcium phosphate compound are formed near the surface of the substrate 11. It is preferably formed by penetrating. Thereby, the adhesiveness of the calcium-phosphate type compound layer 12 and the base material 11 can be made excellent. For this reason, peeling from the surface of the base material 11 of the calcium-phosphate-type compound layer 12 can be prevented suitably, ie, the intensity | strength of the support | carrier 1 can be made excellent.

  In this case, the calcium phosphate compound layer 12 is formed by, for example, colliding porous particles mainly composed of a calcium phosphate compound (hereinafter simply referred to as “porous particles”) on the surface of the substrate 11. be able to. According to this method, the calcium phosphate compound layer 12 can be formed easily and reliably.

  The collision between the substrate 11 and the porous particles can be performed by a dry method using, for example, a commercially available hybridization device or mechanofusion device. The conditions at this time are, for example, a mixing ratio of the base material 11 and the porous particles in a weight ratio of about 400: 1 to 20: 1, and the temperature in the apparatus is a resin material used as the main material of the base material 11 Or less (usually 80 ° C. or less).

Such a carrier 1 preferably has a density (specific gravity) close to that of water. Specifically, the density of the carrier 1 is preferably from 0.8~2g / cm ³ or so, and more preferably 0.9~1.35g / cm ³ order. Thereby, the support | carrier 1 can be more uniformly suspended in the to-be-processed liquid and buffer solution mentioned later. As a result, these liquids and the carrier 1 can be brought into contact with each other more reliably and uniformly, and the efficiency of attaching Legionella to the carrier 1 and the yield of Legionella in the buffer solution can be further improved. .

  The average particle size of the carrier 1 is preferably about 20 to 5000 μm, and more preferably about 40 to 200 μm. Thereby, since the surface area of the support | carrier 1 can fully be ensured, the adhesion rate of Legionella bacteria to the support | carrier 1 can be improved more. If the particle size is too small, the liquid may not easily flow between the carriers 1.

  Further, it is preferable that the carrier 1 is porous at least near the surface (in this embodiment, the calcium phosphate compound layer 12). Thereby, the surface area of the support | carrier 1 can be increased more and the adhesion rate of Legionella bacteria to the support | carrier 1 can further be improved.

Next, the concentration and elution method of Legionella bacteria of this invention is demonstrated.
<First Embodiment>
First, a first embodiment of the method for concentration and elution of Legionella bacteria according to the present invention will be described.

  Here, the Legionella concentration and elution kit (hereinafter simply referred to as “kit”) used in the Legionella concentration and elution method of the first embodiment will be described.

  2 is a schematic diagram (internal perspective view) showing a kit used in the method of concentration and elution of Legionella bacteria according to the first embodiment, and FIG. 3 is a schematic diagram showing a state in which the kit shown in FIG. 2 is folded. is there. In the following description, the upper side in FIGS. 2 and 3 is referred to as “upper” and the lower side is referred to as “lower”.

  The kit 2 shown in FIG. 2 has the carrier 1 as described above, a storage space 20 for storing the carrier 1, and a container 21 having an inlet 22 and an outlet 23 communicating with the storage space 20. Yes.

  The amount of the carrier 1 stored in the container 21 (storage space 20) is not particularly limited, but is preferably about 0.3 to 5 g per 500 mL volume of liquid to be processed (liquid to be processed), 0.5 to More preferably, it is about 3 g. If the amount of the carrier 1 is too small, the number of Legionella bacteria that can adhere to the carrier 1 is limited, and there is a risk that the concentration ratio of Legionella bacteria (recovery rate in the buffer 4) may be low. If the amount is too large, the carrier 1 becomes difficult to move in the liquid to be treated and the buffer solution (eluent), so that contact between these liquids and the carrier 1 becomes insufficient, and the concentration ratio of Legionella bacteria may be reduced. There is.

  In addition, such a kit 2 may have a form in which the carrier 1 is stored in the container 21 in advance, and the carrier 1 is attached to the container 21 separately, and the carrier 1 is put in the container 21 at the time of use. It may be in the form of being put in and used.

  The container 21 has a side wall portion 211 having a substantially cylindrical shape, and an upper wall portion 212 and a bottom wall portion 213 having a substantially disk shape.

  The side wall portion 211 has a bellows shape (bellows shape) and can be folded as shown in FIG. Thereby, space can be reduced when the kit 2 is stored.

  The upper wall 212 and the bottom wall 213 are provided with an inlet 22 and an outlet 23 that communicate with the storage space 20, respectively.

  These inlet 22 and outlet 23 can be closed (sealed) by plugs 24 and 25, respectively.

  The shape of the side wall portion 211 is not limited to a cylindrical shape, and may be any shape such as a cube, a rectangular parallelepiped, or a bag, but the carrier 1, the liquid to be treated, the buffer solution, and the carrier 1 A cylindrical shape is preferred because contact can be made uniformly and strength is excellent.

  The constituent material of the container 21 (the side wall part 211, the upper wall part 212, and the bottom wall part 213) is not particularly limited. For example, ABS resin, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinylidene chloride resin, polyphenylene oxide , Thermoplastic polyurethane, polymethyl methacrylate, polyoxyethylene, fluororesin, polycarbonate, polyamide, acetal resin, acrylic resin, polyethylene terephthalate, and other thermoplastic resins, phenol resin, epoxy resin, silicone resin, unsaturated polyester, etc. Examples thereof include curable resins. Further, as the constituent material of the container 21, for example, various ceramic materials, various metal materials, and the like can be used.

  Further, a filter 26 is provided in the storage space 20 (the bottom wall portion 213 of the container 21) so as to close the discharge port 23.

  The filter 26 has an opening diameter that prevents the carrier 1 from passing through and allows the liquid to be processed and the buffer solution to pass through. The opening diameter is preferably about 0.1 to 90% of the average particle diameter of the carrier 1, and more preferably about 1 to 50% of the average particle diameter of the carrier 1. If the opening diameter is too small, depending on the composition and viscosity of the liquid to be treated and the buffer, the passage resistance of these liquids may be increased, and the time required for the processing may be increased. The carrier 1 may easily pass through the filter 26.

  Next, a method of concentration and elution of Legionella bacteria (first embodiment of the method of concentration and elution of Legionella bacteria of the present invention) performed using the kit 2 shown in FIG. 2 will be described.

  FIG. 4 is a schematic diagram for explaining a first embodiment of a method for concentrating and eluting Legionella. In the following description, the upper side in FIG. 4 is referred to as “upper” and the lower side is referred to as “lower”.

[1] A step of bringing the liquid to be treated into contact with the carrier (first step)
First, the to-be-processed liquid 3 containing Legionella bacteria (Legionella genus microbe) is prepared.

  Although it does not specifically limit as the to-be-processed liquid 3, Bath water (hot spring water), Cooling tower cooling water, humidification water, hot water storage tank water with a temperature of 50 ° C. or less, river water, lake water, well water, pool water, etc. However, bath water (especially circulating bath water) is preferred. In recent years, generation of Legionella in bath water has become a problem. In order to solve such a problem, it is important to detect Legionella before the growth. If the present invention is used, even if the number of Legionella bacteria present in the bath water is small, Legionella bacteria can be efficiently concentrated and eluted, and can be reliably detected. For this reason, the early coping with respect to bathtub water is attained.

  Next, Legionella is adhered to the carrier 1 by bringing the liquid 3 to be treated into contact with the carrier 1.

  Specifically, first, the kit 2 as described above is prepared, the outlet 23 of the container 21 is closed with a stopper 25, and the inlet 22 is opened. It is assumed that the carrier 1 is stored in the container 21 (storage space 20).

  Next, the liquid 3 to be treated is injected (supplied) into the storage space 20 through the injection port 22 (see (a) in FIG. 4).

  Next, the inlet 22 is closed with a stopper 24, and the carrier 21 is suspended in the liquid 3 to be treated by shaking (swinging), stirring, applying ultrasonic waves, etc. ((b) in FIG. 4). )reference). Thereby, the to-be-processed liquid 3 is made to contact the support | carrier 1, and Legionella bacteria are made to adhere to the surface of the support | carrier 1. FIG.

  Here, since the carrier 1 is composed mainly of a calcium phosphate compound having a high affinity for Legionella at least near the surface, Legionella adheres efficiently.

  In this embodiment, the time for which the liquid 3 to be treated is brought into contact with the carrier 1 is preferably about 1 to 240 minutes per 500 mL, and more preferably about 10 to 180 minutes. If this time is too short, the number of Legionella bacteria adhering to the carrier 1 is reduced, and there is a possibility that Legionella bacteria cannot be sufficiently recovered. In addition to not expecting an increase in the effect, Legionella bacteria collapse, and when Legionella bacteria are detected in the steps described later, it may be difficult to accurately detect them.

  Further, the temperature of the liquid 3 to be treated at this time (during treatment) is preferably 70 ° C. or less, more preferably 50 ° C. or less, and further preferably about 5 to 50 ° C. If the temperature of the liquid 3 to be treated at the time of treatment is too low, depending on the type of Legionella bacteria, there is a possibility that Legionella bacteria cannot be efficiently attached to the carrier 1, while the temperature of the liquid 3 to be treated at the time of treatment. Even if the value exceeds the above upper limit, not only an increase in the effect cannot be expected, but Legionella bacteria are destroyed (destroyed), and Legionella bacteria are detected in the process described later. Therefore, it may be difficult to accurately perform the detection.

  Next, the stopper 25 is removed, and the discharge port 23 of the container 21 is opened. Thereby, the liquid 3 to be processed in the container 21 is discharged through the discharge port 23.

Since the carrier 1 cannot pass through the filter 26, the carrier 1 to which Legionella bacteria are adhered remains in the storage space 20 (see (c) in FIG. 4).
Next, the discharge port 23 is closed with a plug 25.

[2] A step of bringing the buffer solution into contact with the carrier (second step)
Next, the carrier 1 to which Legionella bacteria are attached is brought into contact with a buffer solution 4 containing a buffer at a concentration of 25 to 500 mM (particularly 50 to 300 mM), and Legionella bacteria attached to the carrier 1 are released into the buffer solution 4. Elute.

Specifically, first, the stopper 24 is removed, and the inlet 22 of the container 21 is opened.
Next, the buffer solution 4 is injected (supplied) into the storage space 20 through the injection port 22 (see FIG. 4D).

  Next, the inlet 22 is closed with a stopper 24, and the carrier 1 is suspended in the buffer solution 4 by shaking (swinging), stirring, applying ultrasonic waves, etc. ((e) in FIG. 4). reference). Thereby, the buffer solution 4 is brought into contact with the carrier 1 to release (remove) Legionella bacteria from the surface of the carrier 1.

  When the buffer solution 4 is brought into contact with the carrier 1, for example, the charge state on the surface of the carrier 1 is changed, and Legionella bacteria attached to the carrier 1 are efficiently released from the carrier 1.

  Here, in the concentration and elution method of Legionella bacteria of this invention, it is important that the density | concentration of the buffering agent of the buffer solution 4 shall be 25-500 mM. Legionella bacteria adhering to the carrier 1 are more efficiently released from the carrier 1 as the buffer 4 has a higher concentration. If the concentration of the buffering agent in the buffer solution 4 is too low, the number of Legionella bacteria released from the carrier 1 is small, and Legionella bacteria cannot be sufficiently eluted (collected in the buffer solution 4). On the other hand, if the concentration of the buffering agent in the buffer 4 is too high, the buffering agent affects the detection system (measurement system) when detecting the eluted Legionella, and an accurate detection result cannot be obtained. The reproducibility of the detection result is lowered.

  The pH of the buffer 4 to be used is preferably 6.5 to 8.5, and more preferably 7 to 8. When the pH of the buffer solution 4 is out of the above range, that is, when the liquid property of the buffer solution 4 is alkaline or acidic, when detecting the eluted Legionella, There is a risk that a correct detection result cannot be obtained. In particular, when an immunochromatography method is used as a detection method, the antigen-antibody reaction is inhibited, and there is a tendency that an accurate measurement result cannot be obtained.

  The buffer solution 4 is preferably an isotonic solution (a liquid having an osmotic pressure substantially equal to the osmotic pressure of the intracellular solution of Legionella bacteria). Thereby, collapse of Legionella bacteria by the difference in osmotic pressure can also be prevented.

  Examples of such buffer 4 include triethanolamine hydrochloric acid-sodium hydroxide buffer, veronal (sodium 5,5-diethylbarbiturate) -hydrochloric acid buffer, tris-hydrochloric acid buffer, glycylglycine. -Sodium hydroxide buffer, 2-amino-2-methyl-1,3-propanediol-hydrochloric acid buffer, diethanolamine-hydrochloric acid buffer, boric acid buffer, sodium borate-hydrochloric acid buffer, glycine-sodium hydroxide Buffer, sodium carbonate-sodium bicarbonate buffer, sodium borate-sodium hydroxide buffer, sodium bicarbonate-sodium hydroxide buffer, potassium phosphate-disodium phosphate buffer, disodium phosphate-hydroxyl Sodium buffer, potassium chloride-sodium hydroxide buffer, briton-robinson buffer, Various buffers TA buffer solution (liquid containing a buffering agent) and the like. Thereby, while Legionella bacteria can be more reliably released from the support | carrier 1, collapse of Legionella bacteria can be prevented more reliably. As a result, Legionella bacteria can be detected more reliably.

  Of these buffer solutions 4, it is particularly preferable to use a phosphate buffer solution. Thereby, concentration and elution of Legionella bacteria can be reliably performed in a shorter time.

  Moreover, when eluting Legionella bacteria liberated in the buffer solution 4 in a living state (collected in the buffer solution 4), various additives necessary for the growth of Legionella bacteria are added to the buffer solution 4. You may do it. Thereby, it can prevent effectively that the survival number of Legionella bacteria reduces.

  Examples of such additives include amino acids such as glucose, various vitamins and glutamine, electrolytes such as NaCl and KCl, and the like, and one or more of these are used in combination. be able to.

  In this embodiment, the time for which the buffer solution 4 is brought into contact with the carrier 1 is preferably about 0.1 second to 5 minutes per 2.5 mL, and more preferably about 1 second to 1 minute. If this time is too short, Legionella bacteria may not be sufficiently eluted in the buffer solution 4. On the other hand, even if this time is increased beyond the upper limit, an increase in the effect cannot be expected. When Legionella bacteria are destroyed (destroyed) and Legionella bacteria are detected in the process described later, it may be difficult to accurately detect the Legionella bacteria.

  Further, the temperature of the buffer solution 4 at this time (during treatment) is preferably 70 ° C. or lower, more preferably 50 ° C. or lower, and further preferably about 5 to 50 ° C. If the temperature of the buffer solution 4 at the time of treatment is too low, there is a risk that Legionella bacteria cannot be efficiently released from the carrier 1 depending on the type of Legionella bacteria, while the temperature of the buffer solution 4 at the time of treatment is Even if the value exceeds the upper limit, not only an increase in the effect cannot be expected, but Legionella bacteria are destroyed (destroyed), and when Legionella bacteria are detected in the process described later, There is a risk that it is difficult to accurately detect the detection.

  Next, the stopper 25 is removed, and the discharge port 23 of the container 21 is opened. Thereby, the buffer solution 4 in the container 21 is discharged and collected through the discharge port 23 (see (f) in FIG. 4).

  Here, the number of the Legionella per unit volume of the buffer solution 4 is set by making the amount of the buffer solution 4 used in this step [2] smaller than the amount of the liquid 3 to be treated in the step [1]. Can be made larger than that of the liquid 3 to be treated. That is, it is possible to reliably concentrate Legionella. For this reason, even if the number of Legionella bacteria in the liquid to be treated 3 is small and its presence cannot be confirmed (detected), the presence of Legionella bacteria in the buffer 4 by appropriately setting the volume of the buffer 4 Will be able to confirm.

  Such treatment with the buffer solution 4 may be performed in a plurality of times. Thereby, the yield of Legionella bacteria collect | recovered in the buffer solution 4 can be made higher.

Second Embodiment
Next, a second embodiment of the method for concentration and elution of Legionella bacteria according to the present invention will be described.

  Hereinafter, the method of concentration and elution of Legionella bacteria according to the second embodiment will be described with a focus on the differences from the method of concentration and elution of Legionella bacteria according to the first embodiment, and the description of the same matters will be omitted. .

  Here, the Legionella concentration and elution kit used in the Legionella concentration and elution method of the second embodiment will be described.

  FIG. 5 is a schematic view (internal perspective view) showing a Legionella concentration and elution kit used in the Legionella concentration and elution method of the second embodiment. In the following description, the upper side in FIG. 5 is referred to as “upper” and the lower side is referred to as “lower”.

  A column (kit) 2 ′ shown in FIG. 5 is provided with two filters 26a ′ and 26b ′ in the inner cavity of a column main body (tubular body) 21 ′ having a substantially cylindrical shape. A filling space 20 ′ for filling (accommodating) the carrier 1 is defined by 26a ′ and 26b ′.

  In the column 2 ′, the upper end of the column main body 21 ′ in FIG. 5 constitutes the injection port 22 ′, and the lower end constitutes the discharge port 23 ′.

  Further, as the filters 26a 'and 26b', the same filters as those described in the first embodiment can be used.

  Next, a method for concentrating and eluting Legionella using the column 2 'shown in FIG. 5 (second embodiment of the method for concentrating and eluting Legionella of the present invention) will be described.

  FIG. 6 is a schematic diagram for explaining a second embodiment of a method for concentrating and eluting Legionella. In the following description, the upper side in FIG. 6 is referred to as “upper” and the lower side is referred to as “lower”.

[1] A step of bringing the liquid to be treated into contact with the carrier (first step)
First, the column 2 ′ as described above is prepared. In addition, it is set as the state which accommodated the support | carrier 1 in column main body 21 '(packing space 20').

Next, the packing space 20 ′ of the column 2 ′ is filled with a buffer solution.
Specifically, the buffer solution is injected (supplied) into the filling space 20 ′ through the injection port 22 ′.

The buffer solution injected from the inlet 22 'passes through the filter 26b' by its own weight, is supplied into the filling space 20 ', and passes downward through the filling space 20'. Then, the buffer solution that has reached the lower end of the filling space 20 ′ passes through the filter 26a ′ and is gradually discharged from the discharge port 23 ′.
Then, when the filling space 20 ′ is filled with the buffer solution, the injection of the buffer solution is stopped.

  As this buffer solution, the same buffer solution 4 used in the next step [2] may be used, or a different one may be used.

  Next, the to-be-processed liquid 3 containing the Legionella bacteria similar to the said 1st Embodiment is made to contact the support | carrier 1, and Legionella bacteria are made to adhere to the support | carrier 1. FIG.

  Specifically, first, a recovery container 27a 'for recovering the liquid discharged from the discharge port 23' is disposed below the discharge port 23 '.

  Next, the liquid 3 to be processed is injected (supplied) into the filling space 20 ′ through the injection port 22 ′ (see FIG. 6A).

  The liquid to be treated 3 injected from the injection port 22 ′ passes through the filter 26 b ′ by its own weight and is supplied into the filling space 20 ′. The liquid to be treated 3 supplied into the filling space 20 ′ contacts the carrier 1 while passing downward in the filling space 20 ′, and Legionella bacteria adhere to the surface of the carrier 1.

  Then, the component of the liquid 3 to be treated that has not adhered to the carrier 1 reaches the lower end of the filling space 20 ′, passes through the filter 26a ′, and is gradually discharged from the discharge port 23 ′. To be recovered.

  Then, when a desired amount of the liquid 3 to be processed is passed through the storage space 20, the injection of the liquid 3 to be processed is stopped.

  Here, since the carrier 1 is composed mainly of a calcium phosphate compound having a high affinity for Legionella at least near the surface, Legionella adheres efficiently.

  In this case (during treatment), the temperature of the liquid 3 to be treated is preferably the same as that in the first embodiment.

[2] A step of bringing the buffer solution into contact with the carrier (second step)
Next, the same buffer solution 4 as in the first embodiment is brought into contact with the carrier 1 to which Legionella bacteria are adhered, and Legionella bacteria adhered to the carrier 1 are released into the buffer solution 4 and collected.
Specifically, first, the recovery container 27a ′ is replaced with an empty recovery container 27b ′.

  Next, the buffer solution 4 is injected (supplied) into the filling space 20 ′ through the injection port 22 ′ (see FIG. 6B).

  The buffer solution 4 injected from the injection port 22 'passes through the filter 26b' by its own weight and is supplied into the filling space 20 '. The buffer solution 4 supplied into the filling space 20 ′ contacts the carrier 1 while passing downward through the filling space 20 ′.

  When the buffer 4 comes into contact with the carrier 1, for example, the charge state of the surface of the carrier 1 is changed, and Legionella bacteria attached to the carrier 1 are efficiently released from the carrier 1.

  Then, after reaching the lower end of the filling space 20 ′, the buffer solution 4 in which Legionella bacteria are suspended passes through the filter 26 a ′, is gradually discharged from the discharge port 23 ′, and is collected in the collection container 27 b ′.

  Then, when the desired amount of the buffer solution 4 has passed through the filling space 20 ′, the injection of the buffer solution 4 is stopped.

  Here, the type of buffer solution 4, the osmotic pressure, the temperature, and the type of additive are preferably the same as those in the first embodiment.

  The effect similar to that of the first embodiment can be obtained by the kit (column 2 ') of the second embodiment.

<Third Embodiment>
Next, a third embodiment of the method of concentration and elution of Legionella bacteria according to the present invention will be described.

  Hereinafter, the method of concentration and elution of Legionella bacteria according to the third embodiment will be described with a focus on differences from the method of concentration and elution of Legionella bacteria according to the first and second embodiments, and the same matters will be described. Is omitted.

  Here, the concentration and elution kit of Legionella used in the concentration and elution method of Legionella of 3rd Embodiment is demonstrated.

  FIG. 7 is a schematic diagram (internal perspective view) showing a Legionella concentration and elution kit used in the concentration and elution method of Legionella according to the third embodiment. In the following description, the upper side in FIG. 7 is referred to as “upper” and the lower side is referred to as “lower”.

  The third embodiment is the same as the second embodiment except that the configuration (shape) of the column body 21 'is different.

  That is, in the column 2 ′ shown in FIG. 7, the inner diameter and outer diameter of the upper end of the column main body 21 ′ in the drawing are reduced, and the portion constitutes the inlet 22 ′.

  The size (inner diameter) of the injection port 22 ′ is set so that the distal end portion (mouth portion) 110 of the normal syringe 100 can be attached.

  By attaching (connecting) the syringe 100 to the column main body 21 ′ and pressing the syringe 100, the liquid stored in the syringe 100 can be injected into the column main body 21 ′. Thereby, the flow velocity (velocity) at the time of letting the liquid pass through the column main body 21 '(packing space 20') can be adjusted.

  In this case, the flow rate when the buffer solution 4 is passed through the filling space 20 ′ (lumen portion) and the flow rate when the solution 3 is passed through the filling space 20 ′ (lumen portion) It is preferable to set so as to satisfy the following relationship.

  That is, the flow rate A [mL / min] when the buffer solution 4 is passed through the filling space 20 ′ is set, and the flow rate when the solution 3 is passed through the filling space 20 ′ is B [mL / min]. In this case, it is preferable to satisfy the relationship in which A / B is about 0.5 to 3, and it is more preferable to satisfy the relationship in which about 0.5 to 1.5. Thereby, Legionella bacteria can be reliably attached to the carrier 1 and can be reliably released from the carrier 1 and eluted (collected in the buffer solution 4). That is, the concentration rate (recovery rate) of Legionella bacteria from the to-be-processed liquid 3 can be raised reliably. Even if A / B is set to be lower than the lower limit value, that is, even if the flow rate when the buffer solution 4 is passed through the filling space 20 ′ is slower than necessary, the concentration of Legionella and Only a long time is required for elution, and no further increase in the concentration ratio of Legionella can be expected.

  Specifically, the flow rate when the liquid 3 to be treated is passed through the filling space 20 ′ is preferably about 1.5 to 5 mL / min, more preferably about 2 to 4 mL / min. . On the other hand, the flow rate when the buffer solution 4 is passed through the filling space 20 ′ is preferably about 0.75 to 15 mL / min, more preferably about 1 to 6 mL / min, and 1 to 5 mL. More preferably, it is about / min. Thereby, Legionella bacteria can be eluted reliably.

  Further, in this case, after elution of the liquid 3 to be processed and the buffer solution 4 that have passed through the column main body 21 ′, the syringe 100 that sucks a gas such as air is attached to the column 2 ′, and the gas in the syringe 100 is removed from the column. You may make it inject | pour into main body 21 '. Due to the pressure generated by this gas injection, the liquid remaining in the column main body 21 ′ is pushed out from the discharge port 23 ′ and recovered in the recovery container 27 b ′. Thereby, the concentration rate (recovery rate) of Legionella bacteria can be raised more.

  The gas to be injected is preferably air or an inert gas such as nitrogen or argon gas. Thereby, it is prevented that a support | carrier and Legionella bacteria receive damage by contact with gas.

  The effect similar to that of the first and second embodiments can be obtained by the kit (column 2 ') of the third embodiment.

<Fourth embodiment>
Next, a fourth embodiment of the method for concentration and elution of Legionella bacteria of the present invention will be described.

  Hereinafter, the method of concentration and elution of Legionella bacteria according to the fourth embodiment will be described with a focus on differences from the method of concentration and elution of Legionella bacteria according to the first embodiment, and the description of the same matters will be omitted. .

  Here, the concentration and elution kit of Legionella used in the concentration and elution method of Legionella of 4th Embodiment is demonstrated.

  FIG. 8 is a schematic diagram showing a Legionella concentration and elution kit used in the Legionella concentration and elution method of the fourth embodiment. In the following description, the upper side in FIG. 8 is referred to as “upper” and the lower side is referred to as “lower”.

  A bag (kit) 2 ″ shown in FIG. 8 has the carrier 1 and a net-like bag 21 ″, and the carrier 1 is housed in the bag 21 ″ for use.

  Further, the bag 21 ″ has a rectangular parallelepiped shape, and a string (a linear body or a belt-like body) that is gripped with fingers or the like when a processing operation is performed using the bag 2 ″ at the center of one long side thereof. ) 22 ″ is fixed. The overall shape of the bag 21 ″ is not limited to a rectangular parallelepiped shape, and may be any shape such as a cubic shape, a triangular pyramid shape (a pyramid shape), and a conical shape.

  The opening of the bag 21 ″ may be set smaller than the average particle diameter of the carrier 1, and is not particularly limited, but is preferably about 20 to 85 μm, and more preferably about 40 to 80 μm. By setting the opening of the bag 21 ″ within the above range, when the carrier 1 is dissipated from the bag 21 ″ or when Legionella bacteria are present in the liquid to be treated, the Legionella bag 21 ″ It is possible to surely prevent entry / exit from being hindered. The length of Legionella is about 2 to 5 μm.

  Further, as the constituent material of the bag 21 ″, the same resin material as that described in the container 21 in the first embodiment can be used.

  Next, a method of concentration and elution of Legionella bacteria (fourth embodiment of the method of concentration and elution of Legionella bacteria of the present invention) performed using the kit 2 ″ shown in FIG. 8 will be described.

  FIG. 9 is a schematic diagram for explaining a fourth embodiment of a method of concentrating and eluting Legionella. In the following description, the upper side in FIG. 9 is referred to as “upper” and the lower side is referred to as “lower”.

[1] A step of bringing the liquid to be treated into contact with the carrier (first step)
First, the bag 2 "as described above is prepared. The carrier 21 is stored in the bag 21".

  Next, the to-be-processed liquid 3 containing the Legionella bacteria similar to the said 1st Embodiment is made to contact the support | carrier 1, and Legionella bacteria are made to adhere to the support | carrier 1. FIG.

  Specifically, first, a container 27a ″ containing the liquid 3 to be processed is prepared, and the bag 2 ″ is immersed in the liquid 3 to be processed (see FIG. 9A).

  As a result, the liquid 3 to be processed passes through the opening of the bag 21 ″ and enters the bag 21 ″, contacts the carrier 1, and Legionella bacteria adhere to the surface of the carrier 1.

  Then, after the bag 2 ″ is immersed in the liquid 3 to be processed for a predetermined time, the bag 2 ″ is taken out from the liquid 3 to be processed.

  Here, since the carrier 1 is composed mainly of a calcium phosphate compound having a high affinity for Legionella at least near the surface, Legionella adheres efficiently.

  The time for immersing the bag 2 ″ in the liquid to be treated 3 is preferably 2.5 hours or more, and more preferably 7.5 hours or more. Even when the number of Legionella bacteria present in the liquid 3 is relatively small, Legionella bacteria can be sufficiently adhered to the carrier 1 and the detection (concentration) of Legionella bacteria in the liquid 3 to be treated can be reliably performed. The upper limit of the immersion time is not particularly limited, but is preferably 25 hours or less, even if the immersion time is increased beyond the upper limit, the number of Legionella bacteria adhering to the carrier 1 The increase cannot be expected.

  In this case (during treatment), the temperature of the liquid 3 to be treated is preferably the same as that in the first embodiment.

  In addition, this immersion is preferably performed while applying vibration or rocking to at least one of the bag 2 ″ and the liquid to be treated 3. Thereby, Legionella can be efficiently attached to the carrier 1.

  In this step [1], instead of immersing the bag 2 ″ in the liquid 3 to be processed, a method of supplying (spraying or showering) the liquid 3 to the bag 2 ″ as droplets may be used. Good. However, by using the former method, there is an advantage that the liquid 3 to be treated can be brought into contact with the carrier 1 by a simpler operation.

[2] A step of bringing the buffer solution into contact with the carrier (second step)
Next, the buffer solution 4 is brought into contact with the carrier 1 to which Legionella bacteria are adhered, and the Legionella bacteria adhered to the carrier 1 are released into the buffer solution 4 and collected.

  Specifically, first, a bag 2 ″ is placed (placed) in an empty collection container (for example, a petri dish or the like) 27b ″, and the buffer solution 4 is supplied (sprayed or sprayed) to the bag 2 ″. Shower) (see FIG. 9B).

  As a result, the supplied buffer solution 4 passes through the opening of the bag 21 ″ and enters the bag 21 ″ to come into contact with the carrier 1.

  When the buffer 4 comes into contact with the carrier 1, for example, the charge state of the surface of the carrier 1 is changed, and Legionella bacteria attached to the carrier 1 are efficiently released from the carrier 1.

  Then, the buffer solution 4 in which Legionella bacteria float is passed through the opening of the bag 21 ", flows out of the bag 21", and is gradually stored in the collection container 27b ".

  Here, the type of buffer solution 4, the osmotic pressure, the temperature, and the type of additive are preferably the same as those in the first embodiment. Further, the amount of the buffer solution 4 used is preferably the same as that of the first embodiment or a relatively small amount of the buffer solution less than that. The specific amount of the buffer solution is preferably about 500 to 1200 μL, and more preferably about 700 to 900 μL.

  In this step [2], instead of supplying the buffer solution 4 as droplets (spraying or showering) to the bag 2 ″, a method of immersing the bag 2 ″ in the buffer solution 4 may be used. However, by using the former method, there is an advantage that Legionella can be efficiently released from the carrier 1 with a relatively small amount of the buffer 4 used.

  The effect similar to that of the first embodiment can be obtained by the kit (bag 2 ″) of the fourth embodiment.

  As mentioned above, although the concentration and elution method of Legionella bacteria and the concentration and elution kit of Legionella bacteria of this invention were demonstrated, this invention is not limited to these, Even if arbitrary processes are added as needed. Good.

  In addition, this invention is applicable when not only Legionella but also microorganisms, such as colon_bacillus | E._coli O157, Staphylococcus aureus, Vibrio cholerae, anthrax, etc. are concentrated and eluted from a to-be-processed liquid.

  The purpose of concentrating and eluting Legionella is not limited to the purpose of detecting Legionella. For example, the kit of the second embodiment can be used as an application for removing Legionella from circulating water in a facility that circulates and uses water such as a public bath or a pool. Moreover, if the kit of the said 4th Embodiment is immersed in the water stored in a bathtub or a pool in the said facility, it can be used similarly for the use which removes Legionella bacteria from stored water. In any kit, it is possible to repeatedly use Legionella by releasing Legionella in the second step.

  Moreover, if this invention is used, it can apply not only for the objective of concentrating and eluting Legionella bacteria from to-be-processed liquid but when collect | recovering Legionella bacteria from to-be-processed liquids simply.

Next, specific examples of the present invention will be described.
1-1. Preparation of kit 1 g of hydroxyapatite porous particles having an average particle size of 80 μm (specific surface area of 40 m ² / g, volume: about 1.6 mL) is packed in an Econopack column (manufactured by Bio-Rad) having a capacity of 20 mL. A column (kit) as shown in FIG. Each of the two filter openings has a diameter of 10 μm.

1-2. Concentration of Legionella bacteria Bath water (treatment liquid) containing Legionella bacteria was prepared.

Example 1
First, 25 ± 1 ° C., pH 7.5, 10 mM phosphate buffer (KH ₂ PO ₄ —Na ₂ HPO ₄ ) was passed through the container of the kit, and the column body was filled with phosphate buffer.

  Next, bath water (stock solution) of 25 ± 1 ° C .: 500 mL was supplied from the inlet into the column body, and the bathtub water flowing out from the outlet was collected. At this time, the passage time of the bath water is about 180 minutes, and the flow rate of liquid flow is about 2.7 mL / min.

Next, 25 ± 1 ° C., pH 7.5, 50 mM phosphate buffer (KH ₂ PO ₄ -Na ₂ HPO ₄ ): 2.5 mL is injected into the column body from the inlet and flows out from the outlet. A phosphate buffer solution (recovery solution) was recovered. At this time, the passage time of the phosphate buffer is about 50 seconds, and the flow rate of the phosphate buffer is about 2 mL / min.

(Examples 2 to 4)
Legionella was concentrated in the same manner as in Example 1 except that the phosphate buffer concentration was changed as shown in Table 1.

(Examples 5 to 8)
Legionella was concentrated in the same manner as in Example 1 except that the phosphate buffer concentration and flow rate were changed as shown in Table 1.

(Comparative Examples 1 and 2)
Legionella was concentrated in the same manner as in Example 1 except that the phosphate buffer concentration was changed as shown in Table 1.

1-3. Evaluation A part of the stock solution and the collected solution were collected, and the number of Legionella bacteria was measured for each sample by a culture method. Moreover, about some recovery liquids, the microbial cell was detected by the immunochromatography method.

  FIG. 10 shows the results of measurement of the number of bacteria by the culture method, and Table 1 shows the results of detection of bacterial cells by the immunochromatography method. Moreover, the relationship between the flow rate and the concentration factor is shown in FIG. Here, the numerical value on the vertical axis of the graph in FIG. 11 represents how many times the number of bacteria in the recovered solution per unit volume is larger than the number of bacteria in the stock solution. The larger the value, the greater the degree of concentration. It shows that.

  In the results of detection of bacterial cells by immunochromatography, “+” indicates positive, “±” indicates weakly positive (false positive), and “−” indicates negative.

  As shown in FIG. 10 and Table 1, Legionella was detected in the collected liquid of each Example by any of the culture method and the immunochromatography method.

  On the other hand, Legionella spp. Were hardly detected in the collected liquid of Comparative Example 1 by any of the culture method and the immunochromatography method.

  In the recovered solution of Comparative Example 2, Legionella was detected by the culture method, but Legionella was not detected by the immunochromatography method. This is presumably because, in the immunochromatography method, the antigen-antibody reaction was inhibited by a buffer agent present in a high concentration in the recovered solution, thereby inhibiting the detection of bacteria.

  Moreover, in each Example, the number of Legionella bacteria per unit volume of the collected liquid was clearly higher than that of Legionella per unit volume of the stock solution.

  From this, by using a buffer containing a buffer at a concentration of 25 to 500 mM (invention), Legionella bacteria can be efficiently released from the carrier and concentrated and eluted in the buffer (recovered solution). It was revealed that the concentrated bacteria can be detected by the immunochroma method.

  Moreover, as shown in FIG. 11, the tendency for the concentration rate of Legionella bacteria to become low is seen, so that the flow rate of a buffer solution becomes high.

  And it became clear that the concentration rate of Legionella increases by setting the flow rate of the buffer solution to 0.5 to 3 times (especially 0.5 to 1.5 times) the flow rate of the liquid to be treated. .

  In addition, nylon particles (base material) and hydroxyapatite particles were prepared, and these were put into NARA hybridization system NHS-1 (manufactured by Nara Machinery Co., Ltd., rated power 5.5 kW, rated current 23 A). The apparatus was run at 6400 rpm for 5 minutes at 45 ° C. As a result, a carrier as shown in FIG. 1 was obtained. The obtained carrier was porous around the surface.

  Then, using this carrier, Legionella was concentrated from bath water in the same manner as in Examples 1 to 8 and Comparative Examples 1 and 2, and the same results as described above were obtained.

2-1. Production of Kit 1 g of hydroxyapatite porous particles having an average particle size of 100 μm (specific surface area of 40 m ² / g) are stored in a nylon (polyamide) bag having an opening of 76 μm, and a bag (kit) as shown in FIG. A was produced. Six kits A were prepared.
Kits B and C were prepared using nylon bags having a mesh size of 42 μm and 20 μm, respectively.

2-2. Concentration of Legionella The Legionella bacteria solution was diluted 1000 times with a pH 6.8, 1 mM phosphate buffer to prepare a solution to be treated.

(Example 11)
First, 500 mL of the liquid to be processed was placed in a beaker, and the kit A was immersed in this liquid to be processed (25 ± 1 ° C.) while shaking.
Next, after 2.5 hours had elapsed, kit A was taken out of the liquid to be treated, and excess water was removed.
Next, the kit A was placed in a petri dish, and 800 μL of a phosphate buffer solution of 25 ± 1 ° C., pH 7.5, 250 mM was supplied in a shower shape, and left for 3 minutes. Then, kit A was taken out from the petri dish.

(Examples 12 to 16)
Legionella was concentrated in the same manner as in Example 11 except that the immersion time of kit A in the liquid to be treated was changed as shown in Table 2.

(Example 17)
Using the kit B, Legionella was concentrated in the same manner as in Example 11 except that the immersion time of the kit B in the liquid to be treated was as shown in Table 2.

(Example 18)
Using the kit C, Legionella was concentrated in the same manner as in Example 11 except that the immersion time of the kit C in the liquid to be treated was as shown in Table 2.

2-3. Evaluation 100 μL of a phosphate buffer solution (collected solution) in each petri dish was collected and the cells were detected by immunochromatography.
Table 2 shows the results of detection of bacterial cells by immunochromatography.

  In the results of detection of bacterial cells by immunochromatography, “+” indicates positive, “±” indicates weakly positive (false positive), and “−” indicates negative.

  As shown in Table 2, when the same kit A was used, it was confirmed that Legionella concentration (detection) can be more accurately performed by setting the immersion time to 7.5 hours or longer.

  Moreover, in the range of 20-85 micrometers of opening of a bag, a difference was not confirmed by the concentration (detection) result of Legionella bacteria.

It is sectional drawing which shows an example of the support | carrier used by this invention. It is a schematic diagram (internal perspective view) showing a kit used in the first embodiment of the method for concentrating and eluting Legionella bacteria of the present invention. It is a schematic diagram which shows the state by which the kit shown in FIG. 2 was folded. It is a schematic diagram for demonstrating 1st Embodiment of the concentration and elution method of Legionella bacteria of this invention. It is a schematic diagram (internal perspective view) showing the concentration and elution kit of Legionella used in the second embodiment of the method of concentration and elution of Legionella of the present invention. It is a schematic diagram for demonstrating 2nd Embodiment of the concentration and elution method of Legionella bacteria of this invention. It is a schematic diagram (internal perspective view) showing a kit used in the third embodiment of the method for concentration and elution of Legionella bacteria of the present invention. It is a schematic diagram which shows the concentration and elution kit of Legionella used in 4th Embodiment of the concentration and elution method of Legionella of this invention. It is a schematic diagram for demonstrating 4th Embodiment of the concentration and elution method of Legionella bacteria of this invention. It is a graph which shows the number of Legionella bacteria in each liquid. It is a graph which shows the relationship between the flow rate of a phosphate buffer, and a concentration rate.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Support | carrier 11 Base material 12 Calcium-phosphate type compound layer 13 Particle | grain 2 Kit 20 Storage space 21 Container 211 Side wall part 212 Upper wall part 213 Bottom wall part 22 Inlet 23 Outlet 24, 25 Plug 26 Filter 2 'Column 20' Packing space 21 'Column body 22' inlet 23 'outlet 26a', 26b 'filter 27a', 27b 'collection container 2 "bag 21" bag 22 "string 27a" container 27b "collection container 3 processed liquid 4 buffer solution 100 Syringe 110 tip

Claims (26)

A method of concentrating and eluting the Legionella bacteria from a liquid to be treated containing Legionella bacteria,
A first step of attaching the Legionella to the carrier by bringing the liquid to be treated into contact with a carrier mainly composed of a calcium phosphate compound at least near the surface;
A second step of releasing and recovering the Legionella bacteria in the buffer solution by contacting the carrier with the Legionella bacteria attached thereto with a buffer solution containing a buffer at a concentration of 25 to 500 mM. A method for concentrating and eluting Legionella, which comprises   The method of concentrating and eluting Legionella according to claim 1, wherein the temperature of the liquid to be treated when the liquid to be treated is brought into contact with the carrier is 70 ° C or lower.   The method of concentrating and eluting Legionella according to claim 1 or 2, wherein the amount of the buffer solution is smaller than the amount of the liquid to be treated.   The method for concentrating and eluting Legionella according to any one of claims 1 to 3, wherein the buffer is a phosphate buffer.   The method for concentrating and eluting Legionella according to any one of claims 1 to 4, wherein the pH of the buffer solution is 6.5 to 8.5.   The method of concentrating and eluting Legionella according to any one of claims 1 to 5, wherein the temperature of the buffer solution when the buffer solution is brought into contact with the carrier is 70 ° C or lower.   The method of concentrating and eluting Legionella according to any one of claims 1 to 6, wherein the carrier is granular.   The method for concentrating and eluting Legionella according to claim 7, wherein the average particle size of the granular carrier is 20 to 5000 µm.   The method of concentrating and eluting Legionella according to any one of claims 1 to 8, wherein the carrier is porous at least near the surface.   10. The carrier according to any one of claims 1 to 9, wherein the carrier has a base material mainly composed of a resin material, and a coating layer provided so as to cover the surface of the base material and mainly composed of a calcium phosphate compound. A method for concentrating and eluting Legionella as described above.   The method for concentrating and eluting Legionella bacteria according to claim 10, wherein the resin material contains at least one of polyamide and epoxy resin as a main component.   The method of concentrating and eluting Legionella according to any one of claims 1 to 11, wherein the calcium phosphate compound is mainly composed of hydroxyapatite. The carrier concentration and elution process of Legionella according to any one of the specific surface area of claims 1 a 5~100m 2 / ^g 12.   The method for concentrating and eluting Legionella according to any one of claims 1 to 13, wherein the liquid to be treated is bath water.   The said 1st process is performed by flowing the said to-be-processed liquid in the said cavity part in the state with which the said carrier was filled in the cavity part of the pipe body. The method for concentration and elution of Legionella as described.   The method of concentrating and eluting Legionella according to claim 15, wherein the second step is performed by passing the buffer solution through the lumen of the tubular body through which the liquid to be treated is passed.   When the flow rate when the buffer solution is passed through the lumen part is A [mL / min] and the flow rate when the treatment liquid is passed through the lumen part is B [mL / min] The method for concentrating and eluting Legionella according to claim 16, wherein A / B satisfies the relationship of 0.5 to 3.   The method for concentrating and eluting Legionella according to any one of claims 15 to 17, wherein a flow rate when the buffer solution is passed through the lumen is 0.75 to 15 mL / min.   15. The method of concentrating and eluting Legionella according to any one of claims 1 to 14, wherein the first step is performed by immersing a mesh bag containing the carrier in the liquid to be treated.   20. The method of concentrating and eluting Legionella according to claim 19, wherein, in the first step, the time for immersing the bag containing the carrier in the liquid to be treated is 2.5 hours or more.   21. The method of concentrating and eluting Legionella according to claim 19 or 20, wherein the second step is performed by supplying the buffer solution as droplets to a net-like bag containing the carrier. Legionella concentration and elution kit for use in the method of concentration and elution of Legionella according to any one of claims 1 to 14,
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A container having a storage space for storing the carrier, and an injection port and a discharge port communicating with the storage space;
A concentration and elution kit for Legionella, comprising a filter provided in the storage space so as to close the discharge port.   The concentration and elution kit for Legionella according to claim 22, wherein at least a part of the container is foldable. A Legionella concentration and elution kit for use in the method of concentration and elution of Legionella according to any one of claims 15 to 18,
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A tubular body comprising a lumen filling the carrier, and an inlet and an outlet communicating with the lumen;
A concentration and elution kit for Legionella, comprising a filter provided in the lumen so as to close the discharge port. A concentration and elution kit for Legionella for use in the method for concentration and elution of Legionella according to any one of claims 19 to 21,
A carrier at least near the surface mainly composed of a calcium phosphate compound;
A kit for concentration and elution of Legionella, comprising a reticulated bag for storing the carrier. 26. The concentration and elution kit of Legionella according to claim 25, wherein the bag has an opening of 20 to 85 [mu] m.

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