WO2003035899A1

WO2003035899A1 - Method of separating blood cells from microorganism in blood and mehtod of detecting microorganism in blood

Info

Publication number: WO2003035899A1
Application number: PCT/JP2002/010982
Authority: WO
Inventors: Yasuo Nakatomi; Hatsue Hatano
Original assignee: Denka Seiken Co., Ltd.
Priority date: 2001-10-23
Filing date: 2002-10-23
Publication date: 2003-05-01
Also published as: JPWO2003035899A1; JP4227019B2

Abstract

It is intended to provide a method of separating blood cells from a microorganism contained in blood or a liquid culture medium of blood in case of detecting the microorganism in blood employed as a specimen; a method of detecting a microorganism; and a kit therefor. Namely, a method of separating blood cells from a microorganism contained in blood or a liquid culture medium of blood in case of detecting the microorganism in blood which comprises eliminating the blood cells by bringing into contact with an aqueous solution of an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate or an amine or an aqueous solution of a mixture thereof; a method of detecting a microorganism with the use of this separation method; and a detection kit.

Description

Description Method for separating blood cells from bacteria in blood and method for detecting bacteria in blood

TECHNICAL FIELD The present invention relates to a method for separating blood cells and bacteria contained in a liquid culture medium for detecting bacteria contained in the specimen using blood as a specimen. Background art

Culture using blood as a sample to detect bacteria contained in the sample is mainly performed when the presence of bacteria in the bloodstream such as sepsis or bacteremia is suspected. In such cases, patients are often serious and require immediate chemotherapy. In the prior art, a sepsis blood test is performed as follows.

(1) Inoculate 2 to 5 mL of blood collected in about 30 to 100 mL of liquid culture medium for bacterial culture, and immediately incubate at 30 to 37 ° C.

(2) Observe daily for 1 to 14 days until bacterial growth is observed. The growth of the bacteria is determined by the turbidity of the medium, the generation of gas in the medium by the bacteria, and the pH change.

(3) After the growth of the bacteria is confirmed, the culture solution is collected and subjected to Gram staining and morphological observation.The general classification of the bacterial species based on the staining findings and morphological characteristics, and whether only one bacterium is observed, Check if there is more than one.

(4) In order to separate bacteria from blood cells and the like in the culture, a part of the culture is inoculated into a solid medium in which general bacteria grow and cultured. Bacteria grown on the solid medium are subjected to subsequent identification tests, etc.At this time, if the bacteria are a single bacterial species based on gram staining findings, and if the colonies on the solid medium are uniform, they will be present in the blood. Assuming that the type of bacteria used was single (pure culture), the bacteria on the solid medium may be immediately subjected to identification tests, susceptibility tests, etc. On the other hand, if multiple bacterial species are observed on the Gram staining findings, or if colonies with distinctly different shapes proliferate on the solid medium, each colony is picked up and a single fungal colony (community) is collected. Repeat the culture until only the cells develop.

(5) The colonies cultured in pure culture are picked, and the bacterial species is identified by biochemical characterization, or the therapeutic drug is searched by antimicrobial susceptibility testing. In the conventional method for detecting bacteria as described above, it took a number of days from collection of the sample to identification of the bacteria. If sepsis or bacteremia is suspected, the patient is often serious and prompt chemotherapy is required. However, even if the conventional method is simplified, even after the bacteria are detected in the blood culture test, the subsequent test requires at least 3 to 4 days. It was a burden.

In order to solve such problems, separation of bacteria from blood cells and the like in a culture solution has been studied. However, to date, methods for separating blood cells from blood cells in blood cultures have been developed by a simple method of separating bacteria and blood cells using a spitting solution containing a serum-separating agent based on the difference in specific gravity between bacteria and blood cells. However, the separation effect was insufficient and blood cells often remained in the bacterial fraction, and it was difficult to directly test bacteria using the fraction. After all, conventionally, in order to separate bacteria from blood cells and the like in a blood culture solution, it was necessary to further culture, and rapid detection could not be performed. Disclosure of the invention

The present invention relates to a method of inoculating a part of a culture solution into a solid medium in which general bacteria grow in order to separate bacteria from blood cells and the like in the culture solution when detecting bacteria contained in the sample using blood as a sample. An object of the present invention is to provide a method for separating blood cells and bacteria contained in a liquid culture medium without culturing the cells.

The present inventors have found that when bacterial growth is observed in a liquid culture test using blood as a specimen, blood cells, which are blood components, hinder the identification of bacterial species when identifying bacteria in the culture solution. In view of this, as a result of diligent studies, it was found that the liquid culture medium containing blood cells, etc. and bacteria was treated with an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an aqueous solution of amine. Alternatively, the present inventors have found that blood cells and the like can be removed by bringing them into contact with a mixed aqueous solution thereof and bacteria can be separated from the blood cells and the like, thereby completing the present invention.

That is, the present invention is as follows.

(1) A method for separating bacteria and blood cells contained in a liquid culture medium of bacteria in blood when detecting bacteria in blood, comprising the steps of: Blood cells are removed by contact with metal hydroxide or carbonate, alkaline earth metal hydroxide or carbonate, or amine aqueous solution, or a mixed aqueous solution of these. A method of separating blood cells and bacteria.

(2) The separation method according to (1), wherein the pH of the aqueous solution is 11 or more.

(3) The concentration of the hydroxide or carbonate of the alkali metal, the hydroxide or carbonate of the alkaline earth metal, or the amine, or a mixture thereof, of 0.01 M to 1.0 M, How to separate blood cells and bacteria.

(4) The alkali metal hydroxide or carbonate, the alkaline earth metal hydroxide or carbonate, or the amine is selected from the group consisting of sodium hydroxide, hydroxylating ability]] triam and triethanolamine. The method for separating blood cells and bacteria according to any one of (1) to (3).

(5) A method for detecting bacteria in blood, wherein the test blood is an aqueous solution of an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine, or a mixed aqueous solution thereof. A method for removing blood cells from said blood by contacting said blood cells and detecting bacteria in blood from which blood cells have been removed. ,

(6) A method for detecting bacteria in blood, comprising culturing a liquid medium inoculated with a test blood to grow the bacteria, and culturing the liquid medium with hydroxide or carbonate of a / potassium metal, alkali metal. A method for detecting bacteria in the liquid medium from which the blood cells have been removed by removing the blood cells in the liquid medium by contacting with an aqueous solution of an earth metal hydroxide or carbonate, or an amine, or a mixed aqueous solution thereof.

(7) A method for detecting the bacterium according to (5) or (6), wherein the pH of the aqueous solution is 11 or more.

(8) The concentration of the alkali hydroxide or carbonate, the alkaline earth metal hydroxide or carbonate, or the amine, or a mixture thereof is 0.01 M to 1.0 M. (7) A method for detecting any of the bacteria according to (1).

(9) the alkali metal hydroxide or carbonate, alkaline earth metal hydroxide or carbonate, or amine is selected from the group consisting of sodium hydroxide, potassium hydroxide and triethanolamine; 5) A method for detecting any of the bacteria of (8).

(10) A method for detecting a bacterium according to any one of (5) to (9), wherein the bacterium to be detected is selected from the group consisting of methicillin-resistant Staphylococcus aureus, Escherichia coli 0157 strain, and group A streptococcus. (11) For separating and detecting bacteria in blood, including hydroxides or carbonates of alkali metals, hydroxides or carbonates of alkaline earth metals, or amines, and reagents for detecting bacteria. kit.

(12) The kit for separating and detecting bacteria in blood according to (11), further comprising a liquid medium for culturing blood containing bacteria.

(13) The hydroxide or carbonate of the alkali metal, the hydroxide or carbonate of the alkaline earth metal, or the amine is selected from the group consisting of sodium hydroxide, potassium hydroxide and triethanolamine. A kit for separating and detecting bacteria in the blood of (11) or (12).

(14) The reagent for detecting bacteria is a reagent for detecting bacteria selected from the group consisting of methicillin-resistant Staphylococcus aureus, Escherichia coli 0157 strain, and group A streptococcus,

A kit for separating and detecting bacteria in blood according to any one of (11) to (13).

Preferred embodiments of the present invention will be described in detail with reference to the effects thereof. In the present invention, the animal species to be used as a subject is not limited, and an animal species having a disease such as sepsis or bacteremia, such as human, pest, poma, bush, dog, cat, etc. is used. Examples of the bacterium to be used in the present invention include methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, Enterococcus, Candida, Streptococcus such as streptococcus, etc., but are not particularly limited. All bacteria and fungi correspond to this.

First, inoculate 2 to 10 mL of blood collected from a subject into approximately 30 to 100 mL of a liquid medium for bacterial culture, and immediately culture at 30 to 37 ° C. In this case, the liquid medium includes, but is not limited to, trypticase soy broth, brain heart infusion broth, ordinary bouillon, Mueller-Hinton broth, heart infusion broth, and the like. Next, observe daily for 1 to 14 days until bacterial growth is observed. Bacterial growth is determined by the turbidity of the medium, the generation of gas in the medium by the bacteria, changes in pH, and changes in the concentration of carbon dioxide in the medium. After the growth of the bacteria is confirmed, the culture solution is collected and subjected to Gram staining and morphological observation.The general classification of the bacterial species based on the staining findings and morphological characteristics, and whether or not the observed bacteria is single or multiple, etc. Check.

Thereafter, the liquid culture medium containing blood cells and bacteria is mixed with an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine aqueous solution, or a mixed aqueous solution thereof. Mix and contact. Examples of alkali metal or alkaline earth metal hydroxides include lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, and hydroxide. Barium can be mentioned, and among these, lithium hydroxide, sodium hydroxide and potassium hydroxide are preferable. Examples of alkali metal or alkaline earth metal carbonates include lithium carbonate, sodium carbonate, potassium carbonate, rubidium carbonate, cesium carbonate, magnesium carbonate, calcium carbonate, strontium carbonate, and barium carbonate. Of these, lithium carbonate, sodium carbonate and potassium carbonate are preferred. Examples of the amine that can be used in the method of the present invention include triethanolamine, tris (hydroxymethyl) aminomethane, and hydroxylamine.

The liquid culture broth may be directly contacted with an alkali metal hydroxide or the like, or, once, an appropriate amount of the liquid culture broth is centrifuged, and the supernatant is discarded to obtain a precipitate in which blood cells and bacteria are mixed. Then, the precipitate may be brought into contact with an alkali metal hydroxide or the like. The centrifugal separation at this time is desirably performed at 800 to 5000G. The sediment contains blood cells and bacteria. When bacteria are detected by an immunological method, that is, by a detection method using an antibody against the bacteria, a mixture (solution or precipitate) of the blood cells and the bacteria is converted to an alkali metal hydroxide or carbonate, alkaline earth metal, or the like. It is preferable to grow the bacteria as described above before contacting with an aqueous solution of a metal hydroxide or carbonate, or an amine, or a mixed aqueous solution thereof. On the other hand, genetic tests such as PCR that amplify bacterial genes and detect bacteria, such as PCR, use a mixture of blood cells and bacteria with hydroxides or carbonates of alkali metals and hydroxides of alkaline earth metals without growing the bacteria. By contacting it with an aqueous solution of a substance, carbonate, or amine, or a mixed solution of these, it is possible to remove only blood cells and amplify bacterial genes without the influence of blood cells, and detect the presence of bacteria more quickly can do. At this time, the blood is used as it is or after being diluted with physiological saline or a suitable buffer, etc., and then an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an aqueous solution of amine. Alternatively, the bacterial gene in the blood can be detected by contact with a mixed aqueous solution thereof. In addition, blood or blood diluted with physiological saline or an appropriate buffer is centrifuged to obtain a precipitate containing blood cells and bacteria, and the precipitate is subjected to alkali metal hydroxide or carbonate, alkaline force. Bacterial genes in the blood can also be detected by contact with an aqueous solution of a hydroxide or carbonate of an alkaline earth metal, or an amine, or a mixed aqueous solution thereof. The temperature and time for contacting are not particularly limited, but any conditions may be used as long as blood cells in the blood break down and components constituting the blood cells are dissolved, preferably 2 to 37: several seconds to several minutes, and stirring is performed. It is desirable to make the contact more uniform, in order to further enhance the effect. As a result, the blood cells are destroyed, and the components constituting the blood cells are dissolved in the aqueous solution, but the bacteria are not dissolved.Thus, by centrifuging the cells, it is possible to efficiently separate contaminants such as blood cells. High-purity bacteria can be obtained from the sediment of centrifugation.

The pH of the alkali metal hydroxide or carbonate, the alkaline earth metal hydroxide or carbonate, or the aqueous solution of amine, or the pH of the mixed aqueous solution thereof is such that blood cells in blood break down and blood cells The pH may be any pH at which the constituents of the bacteria are dissolved but the constituents of the bacteria are not dissolved.If the mixture of blood cells and bacteria to be brought into contact with the aqueous solution is sedimented, the pH is preferably 9 or more, and more preferably 10 or more. And particularly preferably 11 or more. When a liquid culture medium containing blood cells and bacteria is brought into contact with an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine aqueous solution, or a mixed aqueous solution thereof, or As is, or after dilution with physiological saline or a suitable buffer, etc., and then an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine aqueous solution, or a mixture thereof. When bringing into contact with an aqueous solution, the final pH of the solution after the contact is preferably 9 or more, more preferably 10 or more, particularly preferably 11 or more, so that the final pH of the solution is at least 11. An aqueous solution of hydroxide or carbonate of alkaline earth metal or amine, or a mixed aqueous solution thereof may be prepared. This adjustment depends on the volume ratio of the solution containing blood cells and bacteria used for the contact and the hydroxide or carbonate of an alkali metal, the hydroxide or carbonate of an alkaline earth metal, or the aqueous solution of amine, or a mixed aqueous solution thereof. It can be changed as appropriate. At this time, the concentration of the hydroxide or carbonate of the alkali metal, the hydroxide or carbonate of the alkaline earth metal, or the aqueous solution of the amine is preferably such that the pH falls within the above range. When the mixture of blood cells and bacteria to be brought into contact with the aqueous solution is sedimentation, the concentration is preferably 0.01 M to 1.0 M. Liquid medium containing blood cells and bacteria When the culture solution is brought into contact with an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an aqueous solution of amine, or a mixed aqueous solution thereof, or the blood as it is or physiological saline When contacting with an aqueous solution of an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine, or a mixed aqueous solution thereof after dilution with an appropriate buffer, etc. The concentration of the alkali metal hydroxide or carbonate, the alkaline earth metal hydroxide or carbonate, or the amine, or the mixed aqueous solution thereof is adjusted so that the final concentration of the solution after the contact is the above concentration. It should be adjusted. This adjustment is carried out using a solution containing blood cells and bacteria used for the contact, and a hydroxide or carbonate of alkaline metal, a hydroxide or carbonate of alkaline earth metal, or an aqueous solution of amine or a mixture thereof. It can be appropriately changed depending on the volume ratio of the aqueous solution. For example, to mix lOmL of test solution containing blood cells and bacteria with lOmL of sodium hydroxide solution to make the final concentration of sodium hydroxide 0.1M, add lOmL of sodium hydroxide solution adjusted to 0.2M to blood cells. What is necessary is just to contact and mix with 10 mL of test liquid containing bacteria.

Bacteria finally obtained by centrifugation from the solution in which the blood cells were disrupted and lysed were MRSA-LA “Seiken” (manufactured by Denrikseiken), pathogenic Escherichia coli immune serum 0157 “Seiken” (manufactured by Denkiseiken), It can be detected using a commercially available agglutination method such as a group A streptococcus detection kit, A-Strept AD “Seiken” (manufactured by Denrikseiken), an ELISA method, or a Western blot method. In addition, when bacterial DNA is detected by PCR or the like, the DNA of the bacteria can be extracted by known means and detected using a commercially available DNA test reagent. Therefore, by combining the hydroxide or carbonate of an alkali metal, the hydroxide or carbonate of an alkaline earth metal, or a reagent for detecting an amine or a bacterium, which can be used in the present invention, it is possible to rapidly and accurately detect the blood or blood. A bacteria detection kit capable of detecting the above bacteria can be configured.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a more specific description will be given based on examples of the present invention. However, the present invention is not limited to the following examples.

[Example 1] Detection of MRSA (methicillin-resistant Staphylococcus aureus) in blood Blood cultures: anticoagulant was added © Ma blood lOmL the anonymous SA (methicillin-resistant staphylococci), or MSSA 1 loopful of bacterial suspension containing 10 ⁸ ciuZmL the (methicillin-susceptible Staphylococcus aureus) (0.001 .0015 mL), 8 mL of which was inoculated into a bottle (BBL Septicheck TSB (registered trademark)) containing a liquid culture medium for blood culture, and cultured overnight at 35 ° C.

MRSA strain: ANJ-10 [niecA (+) XPCR, MIPPC (R) / KB disc]

ANJ-12 [fflecA (+) / PCR, MIPPC (R) / KB disc]

ANJ-131 [mecA (+) PCR, MIPPC (R) / KB disc]

ATCC43300 [iecA (+) / PCR, MIPPC (R) / KB disc]

MSSA strain: ATCC25923 [mecA (-) XPCR, MIPPC (S) / KB disc]

FDA209P [mecA (—) / PCR, MIPPC (S) / KB disk]

After collecting 10 mL of the culture solution, this was centrifuged at 1500 G for 15 minutes. The sediment obtained here was red due to the presence of erythrocytes. When a part of the sediment was sampled and observed under a microscope after gram staining, gram-positive cocci and blood cells were observed.

After centrifugation, the supernatant was discarded, and 5 mL of a 0.1 M sodium hydroxide solution was added to the remaining precipitate, followed by stirring for 30 seconds using a test tube mixer.

After stirring, the mixture was centrifuged again at 1500 G for 15 minutes.

After centrifugation, the supernatant was discarded to obtain a sediment. The sediment obtained here was white, and no blood cells were observed under a microscope.

Two platinum loops of sediment were collected from the obtained sediment, and this was used as a sample for PBP2 '(Penicillin in binding protein) using MRSA-LA “Seiken” (Denrik Seiken). in) In the detection test, bacteria derived from blood cultures inoculated with MRSA-added blood were determined to be PBP2'-positive, and bacteria from blood cultures inoculated with MSSA-added blood were PBP2'-negative. there were.

Table 1 shows the results. table 1 Strain PBP2 'detection result

RSA ANJ-10 sex methicillin resistant bacteria

ANJ-12 sex methicillin resistant bacteria

ANJ-131 methicillin resistant bacteria

ATCC43300 methicillin-resistant bacteria

MSSA ATCC25923 methicillin-sensitive bacteria

FDA209P 1 Raw methicillin-sensitive bacteria

[Example 2] Detection of E. coli 0157 in blood

The following examples do not represent a clinical test method that can be immediately applied to actual clinical diagnosis, but are examples performed to specifically explain the present invention.

Blood culture: Add 1 loopful (0.001 to 0.0015 mL) of a bacterial solution containing Escherichia coli 0157 or 10 ⁸ cfu / mL of Escherichia coli of another serotype to 10 mL of poma blood supplemented with an anticoagulant. 8 mL of this was inoculated into a bottle (BBL SeptiCheck BHI (registered trademark)) containing a liquid culture medium for blood culture, and cultured overnight at 35 ° C.

Escherichia coli 0157 strain: M-02 strain [0157: H7]

M-03 shares [0157: H7]

DK- 04 strain [0157: H-]

M-05 stock [0157: H7]

E. coli 026 strain DK-01 strain [026: H-]

DK-06 share 1 ： H-]

M-EC- 1 share [01: H-]

After collecting 10 mL of the culture solution, this was centrifuged at 1500 G for 15 minutes. The sediment obtained here was red due to the presence of erythrocytes. When a part of the sediment was sampled and observed under a microscope after gram staining, gram-negative short bacilli and blood cells were observed.

After centrifugation, the supernatant was discarded, and 5 niL of a 0.2 M potassium hydroxide solution was added to the remaining precipitate, followed by stirring for 30 seconds using a test tube mixer. After stirring, the mixture was centrifuged again at 1500 G for 15 minutes.

After centrifugation, the supernatant was discarded to obtain a sediment. The sediment obtained here is white and viewed under a microscope. On inspection, no blood cells were observed.

0.1 ml of 0.1 M phosphate buffer pH 7.0 was added to the obtained sediment to prepare a concentrated solution of bacteria, and pathogenic Escherichia coli immune serum 0157 “Seiken” (manufactured by Denriki Seiken) was used as the specimen. In a slide agglutination test, bacteria from blood cultures inoculated with E. coli 0157-added blood were determined to be positive for agglutination, while bacteria from blood cultures inoculated with strains other than 0157 were negative. there were.

Table 2 shows the results. Table 2 Stock judgment

E. coli 0157 strain DK-02 strain

DK-03 stock

DK-0 stock

D 05 stock sex

E. coli 026 DK-01 strain

E. coli Ollll DK-06 strain

E. coli Ol DK-EC 1 strain

(Example 3) Detection of group A streptococcus

Blood cultures: anticoagulant was added Usagi blood lOinU this group A streptococci or other loopful of bacterial suspension containing 10 ⁸ c iuZmL the streptococcus group (. 0. 00 1~0 0015mL) was added, this 8 mL of the mixture was inoculated into a bottle (BBL SeptiCheck TSB (registered trademark)) containing a liquid culture medium for blood culture, and cultured overnight at 35 ° C.

Group A streptococcal strain T-1

T-2 shares

T-3 shares

T-4 shares Group B streptococcus: Ia strain

: I I shares

: I I I shares

After collecting 10 mL of the culture solution, this was centrifuged at 1500 G for 15 minutes. The sediment obtained here was red due to the presence of erythrocytes. When a part of the sediment was collected and observed under a microscope after gram staining, gram-positive linked cocci and hemocytes were observed.

After centrifugation, the supernatant was discarded, and 5 mL of a 0.05 M triethanolamine and 0.1 M NaCl solution was added to the remaining sediment, followed by stirring for 30 seconds using a test tube mixer. After stirring, the mixture was centrifuged again at 1500 G for 15 minutes.

Add 5 mL of 1% acetic acid solution to the obtained sediment, centrifuge, and use the obtained sediment as a sample, slide using kit for detection of group A streptococcus, A Strept AD “Seiken” (Denrik Seiken) In the latex agglutination test, a sample derived from a blood culture inoculated with blood to which group A streptococci had been added was determined to be positive, and a sample derived from a blood culture inoculated with group B streptococcus was negative. Table 3 shows the results. Table 3 Bacteria one strain

Group A streptococcal strain T-1

T-2 shares

T-3 shares

T-4 strain

Group B streptococcus Ia strain

Stock

These results indicate that blood cells were removed from a culture containing blood cells and bacteria by the method of the present invention. This shows that bacteria can be detected quickly and accurately.

Industrial applicability

As shown in the Examples, when the bacteria in the blood are detected by contacting with an aqueous solution of an alkali metal hydroxide or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine, By removing the blood cells contained in the liquid culture medium of the bacteria in the blood or in the blood, the blood cells and the bacteria can be efficiently separated. In particular, by setting the pH of the aqueous solution to 11 or more, blood cells and bacteria can be more efficiently separated.

The concentration of the hydroxide or carbonate of the alkali metal, the hydroxide or carbonate of the alkaline earth metal, or the concentration of the amine is 0.01 M to 1.0 M, so that the blood cells can be more efficiently used. And bacteria can be separated.

All publications cited herein are incorporated by reference in their entirety. It will be readily understood by those skilled in the art that various modifications and changes of the present invention are possible without departing from the technical concept and the scope of the invention described in the appended claims. . The present invention is intended to cover such modifications and variations.

Claims

The scope of the claims

1. A method for separating bacteria and blood cells contained in a liquid culture medium of bacteria in blood when detecting bacteria in blood, the method comprising: A method for separating blood cells from bacteria, which comprises removing blood cells by contacting an aqueous solution of a substance or carbonate, an alkaline earth metal hydroxide or carbonate, or an amine, or a mixed aqueous solution thereof.

2. The separation method according to claim 1, wherein ρΗ of the aqueous solution is 11 or more.

3. The concentration of the hydroxide or carbonate of an alkali metal, the hydroxide or carbonate of an alkaline earth metal, or an amine, or a mixture thereof, is 0.011 to 1.0Μ. The method for separating blood cells and bacteria according to the above.

4. The alkali metal hydroxide or carbonate, alkaline earth metal hydroxide or carbonate, or amine is selected from the group consisting of sodium hydroxide, potassium hydroxide and triethanolamine. The method for separating blood cells and bacteria according to any one of claims 1 to 3.

5. A method for detecting bacteria in blood, which comprises subjecting the test blood to a hydroxide or carbonate of an alkali metal, a hydroxide or carbonate of an alkaline earth metal, an aqueous solution of amine, or a mixed aqueous solution thereof. A method of removing blood cells from the blood by contacting the blood cells with bacteria and detecting bacteria in the blood from which the blood cells have been removed.

6. A method for detecting bacteria in blood, comprising culturing a liquid medium inoculated with a test blood to proliferate the bacteria, and forming the liquid medium from a hydroxide or carbonate of an alkali metal or an alkaline earth metal. A method of removing blood cells in the liquid medium by contacting with an aqueous solution of hydroxide, carbonate, or amine, or a mixed aqueous solution thereof, and detecting bacteria in the liquid medium from which the blood cells have been removed.

7. The method for detecting bacteria according to claim 5, wherein ρΗ of the aqueous solution is 11 or more.

8. The concentration of the alkali metal hydroxide or carbonate, alkaline earth metal hydroxide or carbonate, or amine, or a mixture thereof, is 0.01 M to 1.0 M. A method for detecting the bacterium according to any one of claims 1 to 7.

9. The hydroxide or carbonate of the alkali metal, the hydroxide or carbonate of the alkaline earth metal, or the amine is sodium hydroxide, potassium hydroxide or triethanol. The method for detecting bacteria according to any one of claims 5 to 8, which is selected from the group consisting of ethanolamine.

10. The method for detecting a bacterium according to any one of claims 5 to 9, wherein the bacterium to be detected is selected from the group consisting of methicillin-resistant Staphylococcus aureus, Escherichia coli 0157 strain, and group A streptococcus.

Kits for separating and detecting bacteria in blood, including hydroxides or carbonates of alkali metals, hydroxides or carbonates of alkaline earth metals, or amines, and reagents for detecting bacteria. Uru.

12. The kit for separating and detecting bacteria in blood according to claim 11, further comprising a liquid medium for culturing blood containing bacteria.

13. The hydroxide or carbonate of an alkali metal, the hydroxide or carbonate of an alkaline earth metal, or the amine is selected from the group consisting of sodium hydroxide, potassium hydroxide and triethanolamine. The kit for separating and detecting bacteria in blood according to claim 11 or 12.

14. The reagent according to any one of claims 11 to 13, wherein the reagent for detecting the bacterium is a reagent for detecting a bacterium selected from the group consisting of methicillin-resistant Staphylococcus aureus, Escherichia coli 0157 strain, and group A streptococcus. Or a kit for separating and detecting bacteria in blood according to claim 1.