WO2003035899A1 - Procede de separation des globules sanguins d'un micro-organisme dans le sens et procede de detection de ce micro-organisme dans le sang - Google Patents

Procede de separation des globules sanguins d'un micro-organisme dans le sens et procede de detection de ce micro-organisme dans le sang Download PDF

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Publication number
WO2003035899A1
WO2003035899A1 PCT/JP2002/010982 JP0210982W WO03035899A1 WO 2003035899 A1 WO2003035899 A1 WO 2003035899A1 JP 0210982 W JP0210982 W JP 0210982W WO 03035899 A1 WO03035899 A1 WO 03035899A1
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WO
WIPO (PCT)
Prior art keywords
blood
bacteria
carbonate
hydroxide
detecting
Prior art date
Application number
PCT/JP2002/010982
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English (en)
Japanese (ja)
Inventor
Yasuo Nakatomi
Hatsue Hatano
Original Assignee
Denka Seiken Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Denka Seiken Co., Ltd. filed Critical Denka Seiken Co., Ltd.
Priority to JP2003538399A priority Critical patent/JP4227019B2/ja
Publication of WO2003035899A1 publication Critical patent/WO2003035899A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components

Definitions

  • 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.
  • the culture solution is collected and subjected to Gram staining and morphological observation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is as follows.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a method for detecting any of the bacteria according to (1) is 0.01 M to 1.0 M.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the culture solution is collected and subjected to Gram staining and morphological observation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bacterial gene in the blood can be detected by contact with a mixed aqueous solution thereof.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • BBL Septicheck TSB registered trademark
  • MRSA strain ANJ-10 [niecA (+) XPCR, MIPPC (R) / KB disc]
  • ATCC43300 [iecA (+) / PCR, MIPPC (R) / KB disc]
  • MSSA strain ATCC25923 [mecA (-) XPCR, MIPPC (S) / KB disc]
  • the sediment obtained here was white, and no blood cells were observed under a microscope.
  • Table 1 shows the results.
  • 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.
  • BBL SeptiCheck BHI registered trademark
  • the sediment obtained here is white and viewed under a microscope. On inspection, no blood cells were observed.
  • Table 2 shows the results. Table 2 Stock judgment
  • T-4 shares Group B streptococcus: Ia strain
  • the sediment obtained here was white, and no blood cells were observed under a microscope.
  • 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
  • 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.
  • 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.

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  • Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de séparation des globules sanguins d'un micro-organisme contenu dans le sang ou un milieu de culture liquide de sang en cas de détection du micro-organisme dans le sang utilisé comme spécimen. L'invention concerne un procédé de détection d'un micro-organisme et un kit prévu à cet effet. Enfin, l'invention concerne un procédé de séparation des globules sanguins d'un micro-organisme contenu dans le sang ou dans un milieu de culture liquide de sang en cas de détection du micro-organisme dans le sang qui consiste à éliminer les globules sanguins en mettant en contact une solution aqueuse d'un hydroxyde de métal alcalin ou de carbonate, un hydroxyde de métal de terre alcaline ou un carbonate ou une amine ou une solution aqueuse d'un mélange de ces derniers. L'invention traite d'un procédé de détection d'un micro-organisme à l'aide de ce procédé de séparation et d'un kit de détection.
PCT/JP2002/010982 2001-10-23 2002-10-23 Procede de separation des globules sanguins d'un micro-organisme dans le sens et procede de detection de ce micro-organisme dans le sang WO2003035899A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2003538399A JP4227019B2 (ja) 2001-10-23 2002-10-23 血液中の血球と菌の分離方法および血液中の菌の検出方法

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JP2001325125 2001-10-23
JP2001-325125 2001-10-23

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011530303A (ja) * 2008-08-13 2011-12-22 ビオメリュー 黄色ブドウ球菌をコアグラーゼ陰性ブドウ球菌と区別することができる培養培地
JP2013512685A (ja) * 2009-12-08 2013-04-18 バイオカルティス、ソシエテ、アノニム 細胞の選択的溶解

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0113103A1 (fr) * 1982-12-27 1984-07-11 EASTMAN KODAK COMPANY (a New Jersey corporation) Inhibition des activités réductrices de leucocytes
WO1993025711A1 (fr) * 1992-06-12 1993-12-23 Gen-Probe Incorporated Preparation d'acide nucleique a partir de sang
WO1994018828A1 (fr) * 1993-02-25 1994-09-01 Abbott Laboratories Systeme de reactifs polyvalent de lyse rapide d'echantillon de sang total

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0635972B2 (ja) * 1986-11-27 1994-05-11 東亜医用電子株式会社 フロ−サイトメトリ−による白血球の分類方法
JP3301646B2 (ja) * 1993-03-19 2002-07-15 シスメックス株式会社 幼若細胞測定用試薬
JP3324050B2 (ja) * 1994-10-31 2002-09-17 日本光電工業株式会社 白血球分類用試薬および白血球分類方法
EP1224258A2 (fr) * 1999-10-29 2002-07-24 Pall Corporation Traitement de fluide biologique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0113103A1 (fr) * 1982-12-27 1984-07-11 EASTMAN KODAK COMPANY (a New Jersey corporation) Inhibition des activités réductrices de leucocytes
WO1993025711A1 (fr) * 1992-06-12 1993-12-23 Gen-Probe Incorporated Preparation d'acide nucleique a partir de sang
WO1994018828A1 (fr) * 1993-02-25 1994-09-01 Abbott Laboratories Systeme de reactifs polyvalent de lyse rapide d'echantillon de sang total

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011530303A (ja) * 2008-08-13 2011-12-22 ビオメリュー 黄色ブドウ球菌をコアグラーゼ陰性ブドウ球菌と区別することができる培養培地
JP2013512685A (ja) * 2009-12-08 2013-04-18 バイオカルティス、ソシエテ、アノニム 細胞の選択的溶解
JP2016127844A (ja) * 2009-12-08 2016-07-14 バイオカルティス、ナームローゼ、フェンノートシャップＢｉｏｃａｒｔｉｓ Ｎｖ 細胞の選択的溶解
US10308976B2 (en) 2009-12-08 2019-06-04 Koninklijke Philips N.V. Selective lysis of cells
US11072813B2 (en) 2009-12-08 2021-07-27 Koninklijke Philips N.V. Selective lysis of cells

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JP4227019B2 (ja) 2009-02-18

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