WO2013021894A1 - Method and system for detecting bacteria in surgical cleaning solution - Google Patents

Method and system for detecting bacteria in surgical cleaning solution Download PDF

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WO2013021894A1
WO2013021894A1 PCT/JP2012/069584 JP2012069584W WO2013021894A1 WO 2013021894 A1 WO2013021894 A1 WO 2013021894A1 JP 2012069584 W JP2012069584 W JP 2012069584W WO 2013021894 A1 WO2013021894 A1 WO 2013021894A1
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sample
activating substance
platelet activating
luminescent
surgical field
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PCT/JP2012/069584
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French (fr)
Japanese (ja)
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恭大 木村
美雪 小山
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テルモ株式会社
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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
    • C12Q1/22—Testing for sterility conditions
    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase

Definitions

  • the present invention relates to a detection method and a detection system for bacteria in surgical field washing liquid.
  • the present invention relates to a method and a system for easily detecting the presence of bacteria in a surgical field washing liquid mixed with a biological sample.
  • Surgical site infection occurring at the site where surgery is performed is a problem.
  • bacteria presumed to cause SSI include Staphylococcus aureus, coagulase-negative staphylococci, pneumococci, and gram-negative bacteria.
  • the incidence of SSI varies depending on the type of surgery and the site where surgery is performed. 0.01% and 17.1% for rectal surgery, accounting for 14-16% of hospital-acquired hospital infections, second only to urinary tract infections and pneumonia It is.
  • SSI occurs, healing is delayed, which increases the number of days of hospitalization and medical costs, and the burden on the patient is considerably increased. For this reason, efforts to minimize SSI are urgent.
  • SSI countermeasures differ before, during and after surgery.
  • measures such as SSI countermeasures during surgery, such as using synthetic absorbent thread as a suture instead of silk thread, and using wound draping.
  • measures are taken to remove as much of the bacteria that cause infection as possible by washing the surgical field with physiological saline or the like.
  • the detection time takes at least several hours, and the result of detection of the bacteria can be obtained only after the operation. Therefore, the suture is performed without confirming the number of bacteria. For this reason, there is a need for a method that can detect the presence of bacteria in a short time (intraoperative).
  • a method for detecting bacteria there is a method for measuring the concentration of “endotoxin”, which is one of the components constituting the outer membrane of Gram-negative bacteria (for example, Patent Document 1).
  • endotoxin which is one of the components constituting the outer membrane of Gram-negative bacteria
  • Patent Document 1 a sample, a reagent containing factor C activated by binding to endotoxin, and a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide are reacted, and the luminescent synthetic substrate is converted into a luminescent substrate.
  • a sample based on the measurement value obtained by the luminescence substrate release step, the luminescence quantity measurement step in which the luminescent enzyme is allowed to act on the luminescence substrate released in the luminescence substrate release step, and the luminescence amount measurement step is measured. And a concentration determination step for quantifying the endotoxin concentration therein. According to this method, endotoxin in a sample can be measured easily and with high sensitivity.
  • an object of the present invention is to provide a method capable of easily detecting the presence of bacteria in a surgical field washing liquid containing a biological sample.
  • Another object of the present invention is to provide a method capable of detecting the presence of bacteria in a surgical field washing solution containing a biological sample in a short time.
  • the present inventors pre-treat the surgical field washing liquid with a platelet activating substance before measuring the endotoxin concentration in the surgical field washing liquid containing a biological sample. As a result, it was found that the above-mentioned purpose can be achieved by eliminating the false positive problem. Based on the knowledge, the present invention has been completed.
  • the above-mentioned purposes are as follows: (i) mixing and contacting the surgical field washing solution with the platelet activating substance [step (i)]; (ii) removing the platelet activating substance from the surgical field washing liquid and measuring Obtaining a sample [step (ii)]; (iii) reacting the sample to be measured with a C-factor containing reagent activated by binding to endotoxin in the sample to be measured and a luminescent synthesis substrate, thereby producing the luminescent synthesis The luminescent substrate is released from the substrate [Step (iii)]; (iv) A luminescent enzyme is allowed to act on the luminescent substrate released in the step (iii) to measure the amount of luminescence [Step (iv)]; v) The amount of luminescence obtained in the step (iv) is compared with a reference value [step (v)], and this can be achieved by a method for detecting bacteria in the surgical field washing solution.
  • the present invention comprises (i) mixing and contacting an operative field washing solution with a platelet activating substance [step (i)]; (ii) removing the platelet activating substance from the operative field washing solution to obtain a sample to be measured. [Step (ii)]; (iii) reacting the sample to be measured with a C-factor-containing reagent activated by binding to endotoxin in the sample to be measured and a luminescent synthetic substrate, and A luminescent substrate is released [step (iii)]; (iv) a luminescent enzyme is allowed to act on the luminescent substrate released in step (iii) to measure the amount of luminescence [step (iv)]; Comparing the amount of luminescence obtained in step (iv) with a reference value [step (v)], the present invention relates to a method for detecting bacteria in surgical field washing liquid.
  • the method of the present invention is characterized in that the surgical field washing solution is pretreated with a platelet activating substance before measuring the endotoxin concentration in the surgical field washing solution containing a biological sample.
  • the present inventors have discovered that the presence of platelet clotting factors in a biological sample causes light emission even in a sample in which no endotoxin (bacteria) is present. Further, by treating the platelet coagulation factor in the surgical field washing solution with a platelet activating substance, the causative substance of the false positive result as described above can be removed.
  • the method of the present invention enables detection in a short time of 20 minutes or less. For this reason, the burden on the patient can be reduced, and since bacteria can be detected before the epidermis is sutured, SSI can be effectively prevented.
  • the bacteria detected by the method of the present invention are not particularly limited, and may be bacteria that are generally estimated to be the cause of surgical site infection (SSI).
  • SSI surgical site infection
  • Examples include negative bacteria. Of these, it is desirable to adapt to the genus Enterobacter, Escherichia, Pseudomonas, and Bacteroides, which are the main causes of surgical site infections. Although the cause of surgical site infection includes not only Gram-negative bacteria but also Gram-positive bacteria, detection of only Gram-negative bacteria is sufficient for confirming the degree of washing.
  • Step (i) In this step, the surgical field washing solution is mixed and contacted with the platelet activating substance.
  • the surgical field washing liquid refers to the surgical field (for example, intraperitoneal in colorectal surgery) before suturing the affected part in surgical operations such as hip replacement, knee replacement, cardiac surgery, vascular surgery, and colorectal surgery. Is washed with a washing solution, which is a washing solution at that time. For this reason, the surgical field washing liquid to which the method of the present invention is applied usually contains a biological component such as a blood component. After washing, the surgical field washing solution is aspirated / waste, and this washing and aspiration / waste operation are repeated an appropriate number of times, for example, 2 to 25 times (preferably 4 to 20 times) in total.
  • any of the surgical field cleaning solutions obtained in each cleaning step may be used in this step, but generally, the surgical field cleaning solution obtained in the final cleaning step is used.
  • the frequency of cleaning the surgical field varies depending on the surgical method, the severity of the disease, the weight of the patient, etc., and is ultimately left to the judgment of the operator.
  • the surgical field cleaning liquid is not particularly limited, and those usually used for cleaning in a surgical operation can be used in the same manner. Specifically, physiological saline, sterilized water, Ringer's solution, osmotic pressure-retaining agent that is harmless to cells such as 4.5% by weight glucose and sugar, and glycerin are added to substantially the same osmotic pressure as living tissue cells.
  • the amount of the surgical field cleaning solution used per time is not particularly limited, and may be the same as the amount used in normal surgery. Specifically, the amount of the surgical field cleaning solution used at one time is preferably 100 to 1,000 mL, and the total amount is more preferably 100 to 10,000 mL.
  • the surgical field washing solution may be used as it is, but considering the burden on the patient, it is heated to a temperature almost equal to the body temperature (for example, 35 to 40 ° C., preferably around 37 ° C.). It is preferable.
  • the platelet activating substance that can be used in this step is not particularly limited as long as it is a substance that can induce platelet aggregation.
  • a cation exchange resin whose cation is Ca 2+ , Cu 2+ , Zn 2+ , Mg 2+ , K + , NH 4 + , Na + , or H + ; an anion is SO 4 2 ⁇ , I ⁇ , NO 3 -, CrO 4 2- , Br -, Cl -, OH - or F, - a is an anion exchange resin; collagen, fibrin, ADP, arachidonic acid, thrombin, serotonin, protamine, calcium salts, RGD peptide, Or coagulation factors (eg fibrinogen, thrombin, prothrombin, von Willebrand factor, thromboxane, thromboplastin, fifth factor, seventh factor, eighth factor, ninth factor, tenth factor, eleventh factor, twelve Factor, prekallikrein, high
  • cation / anion exchange resin a commercially available product may be used. Specifically, as the cation exchange resin (weakly acidic cation exchange resin, strong acid cation exchange resin), Amberlite (trademark) CG-4000, CG-5000, CG-6000, CG-8000, IR- 116, IR-118, IR-118H, IR-120, IR-120B, IR-122, IR-124, 252, 200CT, 201CT, 200C, IRC-50, IRC-84, XT-1007, XT-1009, Amberlite (trademark) cation exchange resin such as XT-1002 (all are trade names manufactured by Organo Corporation); Diaion (trademark) SK-1A, SK-1B, SK-104, SK-110, SK-112, FMK-10, WK-10, WK-11, WK-20, PA-406, PA-408, PA-412, PA- 16, PA-418, PK-208, PK-212, PK-
  • Diaion (trademark) cation exchange resin As anion exchange resins (weakly basic anion exchange resin, medium basic anion exchange resin, strong basic anion exchange resin), Amberlite (trademark) IRA-400, IRA-400J, IRA-400T , IRA-401, IRA-402BL, IRA-404J, IRA-430, IRA-458, IRA-458, IRA-900, IRA-900J, IRA-904, IRA-910, IRA-910CT, IRA-938, IRA -958, IRA-958RF, IRA-410, IRA-410J, IRA-411, IRA-910, IRA-68, IRA-35, IRA-93 and other Amberlite TM anion exchange resins; Trademarks) WA-10, WA-11, WA-20, WA-21, WA-30, PA-4 6, PA-408, PA-412, PA-416, PA-418, PA-306, PA-306S, PA-308, PA-312, PA-316, PA-318, PA-318L
  • strongly acidic cation exchange resins for example, amberlite (for example) having a styrene-divinylbenzene copolymer, a phenol formalin resin, etc. as a base and having a sulfonic acid group as an ion exchange group, etc.
  • the platelet activating substances other than the cation / anion exchange resin, collagen and polystyrene beads are preferably used.
  • the said platelet activation substance may be used independently or may be used with the form of 2 or more types of mixtures.
  • the mixing ratio of the surgical field washing solution and the platelet activating substance is such that the platelet activating substance is mixed in an amount sufficient to remove substances that cause luminescence in the absence of endotoxin (eg, platelet clotting factor) in the operative field washing solution.
  • endotoxin eg, platelet clotting factor
  • the platelet-activating substance can sufficiently remove the substance that causes luminescence in the absence of endotoxin in the surgical field washing solution.
  • removing a substance that causes luminescence in the absence of endotoxin means preventing or preventing false positives due to the presence of the substance.
  • “removal of a substance causing luminescence in the absence of endotoxin” includes inactivation of the substance in addition to physical removal of the substance.
  • the surgical field washing liquid after removal of the substance contains the substance in a form that does not substantially cause luminescence in the absence of endotoxin and that does not cause luminescence in the absence of endotoxin. It includes any form of form.
  • the mixing / contacting condition of the surgical field washing solution and the platelet activating substance is also a condition that the platelet activating substance can sufficiently remove the substance causing luminescence in the absence of endotoxin in the operative field washing liquid,
  • the mixing / contacting temperature the platelet activating substance is mixed / contacted with the surgical field washing solution, preferably at 15 to 45 ° C., more preferably at 20 to 40 ° C.
  • the mixing / contacting time the platelet activating substance is mixed / contacted with the surgical field washing solution, preferably for 0.5 to 15 minutes, more preferably for 3 to 10 minutes.
  • the platelet-activating substance can sufficiently remove substances that cause luminescence in the absence of endotoxin in the surgical field washing solution.
  • the mixing / contact between the surgical field washing solution and the platelet activating substance may be performed under stirring conditions or standing conditions.
  • Step (ii) This step is a step of preparing the sample to be measured by mixing and contacting the surgical field washing solution with the platelet activating substance in the step (i) and then removing the platelet activating substance from the surgical field washing solution.
  • the method for removing the platelet activating substance is not particularly limited, and a known method is used.
  • a known method is used.
  • the supernatant may be obtained.
  • the surgical field washing solution and the platelet activating substance are separated from the mixed solution in the above step (i) using a known separation method such as centrifugation, filtration, suction filtration, porous membrane filtration, and nonwoven fabric filtration.
  • the supernatant or filtrate may be obtained.
  • the supernatant / filtrate thus obtained may be used as it is as a sample to be measured.
  • the separation method may be applied singly or in appropriate combination of two or more, and is appropriately selected depending on the degree of removal of the platelet activating substance.
  • the sample to be measured may be used in the following step (ii) after being subjected to treatment such as cooling and dilution.
  • the cooling (sample to be measured) temperature for cooling is not particularly limited, but is usually preferably ⁇ 5 to 30 ° C., more preferably 0 to 20 ° C.
  • the cooling time is not particularly limited as long as it can reach the predetermined temperature, but it is usually preferably 3 minutes or more, preferably 5 minutes or more, and preferably 5 to 10 minutes.
  • the diluent used in the case of dilution is not specifically limited, What is normally used for dilution in medical treatment can be used similarly. Specifically, physiological saline, sterilized water, Ringer's solution, osmotic pressure-retaining agent that is harmless to cells such as 4.5% by weight glucose and sugar, and glycerin are added to substantially the same osmotic pressure as living tissue cells. And an isotonic solution, irrigated perfusate for cerebrospinal surgery, and the like.
  • the dilution factor is not particularly limited as long as the amount of luminescence can be measured in the following steps (iii) to (iv), but is usually preferably 1,000 to 1,000,000 times, and preferably 5,000 to 100 times. 1,000 times is more preferable.
  • Step (iii) the sample to be measured obtained in the above step (ii) is activated by binding to the endotoxin in the sample to be measured (hereinafter also simply referred to as “factor C-containing reagent”). And a reaction with a luminescent synthetic substrate to release the luminescent substrate from the luminescent synthetic substrate.
  • the sample to be measured does not need to use the entire amount of the sample to be measured obtained in step (ii), and a part of the sample is usually used, and the amount is the amount of luminescence in step (iv). As long as it is an amount that can be measured, it can be appropriately selected.
  • endotoxin is one of the components constituting the outer membrane of bacteria, particularly gram-negative bacteria, and lipopolysaccharide (LPS) contributes to the activation of factor C.
  • Endotoxins are present as part of the outer membrane on the surface of bacteria, particularly gram-negative bacteria. Endotoxin is usually present in the bloodstream after the death of the bacterium. For this reason, it is possible to detect the presence of bacteria in the sample to be measured by measuring the endotoxin concentration in the sample to be measured.
  • a reaction system for example, a Limulus reaction system
  • endotoxin reaction system when the C-factor-containing reagent is a horseshoe crab blood cell extract (LAL: Limulus ⁇ ⁇ Amebocyte Lysate) is as follows.
  • endotoxin binds to factor C (Factor C) and activates factor C, and activated factor C (active factor C) further activates factor B (Factor B). Subsequently, the activated factor B (active factor B) activates a precoagulase (Preclotting Enzyme) to generate a coagulation enzyme (Clotting Enzyme).
  • This coagulation enzyme is partially hydrolyzed using coagulogen as a substrate to produce coagulin, a coagulation protein, which gels [T. Miyata, M.
  • the C-factor-containing reagent that can be used in this step is not particularly limited as long as a clotting enzyme is generated by reaction with endotoxin.
  • a component of a horseshoe crab blood cell extract (amebocyte lysate) conventionally used in the Limulus test can be suitably used.
  • the horseshoe crab blood cell extract is not particularly limited, and for example, those obtained from blood cells of horseshoe crab belonging to the genus Limulus, Tachypleus, and Carcinoscorpius can be used.
  • the factor C-containing reagent a commercially available product may be used as the factor C-containing reagent.
  • LAL Limulus Amebocyte Lysate
  • LAL reagent included in a kit for measuring endotoxin
  • kits for measuring endotoxin include Kinetic-QCL, QCL-1000, Pyrogent 5000, Pyrogent 06 plus, Pyrogent 03 plus (all are trade names of Lonza Japan Co., Ltd.); Limulus J Test Wako, Limulus J Single Test Wako, Limulus J Single Test Wako, Limulus HS-J Single Test Wako, Limulus ES-J Test Wako, Limulus F Single Test Wako, Limulus HS-F Test Wako, Limulus HS-F Single Test Wako , Limulus ES-II Test Wako, Limulus ES-II Single Test Wako, Limulus HS-T Single Test Wako, Limulus Color KY Test Wako, Limulus Color KY Single Test Wako, Limulus PS Guru Test Wako, endotoxin - (none, Wako Pure Chemical Industries, K.K.) Single Test Wako and the like.
  • horseshoe crab blood cell extract J freeze-dried product horseshoe crab blood cell extract HS-J freeze-dried product, horseshoe crab blood cell extract F freeze-dried product, horseshoe crab blood cell extract HS-F freeze-dried product, horseshoe crab blood cell extract ES-II frozen Dry products (both trade names manufactured by Wako Pure Chemical Industries, Ltd.) and the like may be used as endotoxin measurement dedicated reagents.
  • a horseshoe crab blood cell extract component for example, a commercially available Limulus reagent
  • a sample containing endotoxin for example, a commercially available Limulus reagent
  • Active factor C active factor C
  • active factor B active factor B
  • coagulase proteins having protease activity. Therefore, as the luminescent synthetic substrate, those having an active factor C recognition sequence, those having an active factor B recognition sequence, and those having a coagulation enzyme recognition sequence can be used.
  • one of active factor C, active factor B and clotting enzyme may be used alone as an indicator of protease activity, or two or more may be used as an indicator of protease activity. In consideration of ease of operation, it is preferable to use one type. For this reason, according to each case, the coloring synthetic substrate corresponding to the enzyme used as an index may be appropriately selected and used.
  • a recombinant C factor derived from a recombinant gene synthesized based on part or all of the C factor gene of horseshoe crab is not particularly limited, and for example, a recombinant C factor attached to a commercially available Pilogin rFc (trade name, manufactured by Lonza Japan Co., Ltd.) can be suitably used.
  • recombinant factor C can be recombined by transforming the obtained expression vector into an appropriate host cell by inserting and screening a horseshoe crab factor C gene into an expression vector using known genetic manipulations. It may be produced by expressing and purifying the protein.
  • recombinant factor C when used as a factor C-containing reagent, since factor B and precoagulase are not present in the reagent, the active form is produced by the reaction with a sample containing endotoxin. Recombinant factor C only. Therefore, in this case, a luminescent synthetic substrate having an active factor C recognition sequence may be used.
  • the amount of factor C-containing reagent used is not particularly limited as long as it is sufficient to bind to endotoxin in the sample to be measured. Specifically, the amount of factor C-containing reagent used (protein concentration conversion) is preferably about 1.5 to 3.5 mg, more preferably about 2.0 to 3.3 mg, with respect to 1 mL of the sample to be measured. is there. In addition, when using a commercial item, it can select suitably according to a manufacturer's instruction
  • the luminescent synthetic substrate that can be used in this step may be any substrate formed by binding a luminescent substrate to a peptide.
  • the “luminescent substrate” means a substance that emits light as a reaction substrate by bioluminescence.
  • the luminescent substrate is not particularly limited, and a known luminescent substrate can be used. Examples include firefly luciferin, aminoluciferin represented by the following formula, Renilla luciferin, Cypridina luciferin, vargulin, dinoflagellate luciferin, bacterial luciferin and the like. Among these, when aminoluciferin is used, the amino group in aminoluciferin forms an amide bond with the carboxyl group of the adjacent amino acid.
  • a commercially available luminescent substrate may be used. Specifically, D-Luciferin (D-Luciferin), D-Luciferin sodium (D-Luciferin ⁇ Sodium Salt), D-LuciferinhydrateSodium Salt Monohydrate, D-Luciferin potassium (D-Luciferin) Luciferin Potassium Salt), Luciferase-Luciferin, Lyophilized, Luciferase, recombinant (all trade names made by Wako Pure Chemical Industries, Ltd.), D (-)-Luciferin (Photinus pyralis Luciferin, Roche Diagnostics Inc.) ) And the like.
  • the peptide that binds to aminoluciferin consists of an amino acid sequence in which the amide bond with aminoluciferin at the C-terminal of the peptide is cleaved by protease activity of any one of active factor C, active factor B, and clotting enzyme. Anything is acceptable.
  • the number of amino acid residues and the amino acid sequence are not particularly limited. In view of specificity, synthesis cost, ease of handling, etc., the number of amino acid residues is preferably 2 to 10.
  • the peptide having a recognition sequence for a clotting enzyme is not limited to the following, but includes Gly-Val-Ile-Gly-Arg-, Val-Leu-Gly-Arg-, Leu-Arg-Arg-, Ile. -Glu-Gly-Arg-, Leu-Gly-Arg-, Val-Ser-Gly-Arg-, Val-Gly-Arg- and the like.
  • peptides having a recognition sequence for active factor C are not limited to the following, but include Ile-Glu-Ala-Arg-, Leu-Gly-Asn-Lys-Val-Ser-Arg-, and Ile-Thr-Thr. -Val-Gly-Arg- and the like.
  • Peptides having an active factor B recognition sequence include, but are not limited to, Thr-Thr-Thr-Thr-Arg-, Ser-Arg-Gln-Arg-Arg-, and the like.
  • the peptide may be protected at the N-terminus with a protecting group. Any protecting group that can be used in this field can be used without limitation. Specific examples include N-succinyl group, tert-butoxycarbonyl group, benzoyl group, p-toluenesulfonyl group and the like.
  • the luminescent synthetic substrate can be synthesized by referring to the methods described in Example 6 and Example 7 of JP-T-2005-530485, for example. Further, a luminescent synthetic substrate (benzoyl-Leu-Arg-Arg-aminoluciferin) attached to “Proteasome-Glo TM Assay Systems” commercially available from Promega can be used. When free aminoluciferin is contained in the luminescent synthetic substrate, it is preferable to remove it beforehand. Background luminescence can be suppressed by removing free aminoluciferin from the luminescent synthetic substrate.
  • 0.8 mM coenzyme A 1.5 mM ATP, 250 ⁇ g / ml firefly in 20 mM Tricine, 8 mM Mg 2+ , 0.13 mM EDTA buffer (pH 7.8) can be used.
  • Examples include a method of mixing with a solution containing luciferase and 90 mM DTT and incubating at room temperature (25 ° C.) for 1 to 6 hours.
  • the luminescent synthetic substrate Alternatively, a commercially available product may be used as the luminescent synthetic substrate. Specifically, there is a peptide luciferin for endotoxin (manufactured by Bio-Enex Co., Ltd.).
  • the amount of the luminescent synthetic substrate used is not particularly limited as long as it can release a sufficient amount of the luminescent substrate.
  • the use amount of the luminescent synthetic substrate is preferably 50 to 100 ⁇ M, more preferably 70 to 80 ⁇ M.
  • it can select suitably according to a manufacturer's instruction
  • the reaction conditions of the sample to be measured, the C-factor-containing reagent, and the luminescent synthetic substrate are not particularly limited as long as they react to release the luminescent substrate from the luminescent synthetic substrate.
  • the reaction temperature is preferably 15 to 45 ° C, more preferably 20 to 40 ° C.
  • the reaction time is preferably 0.5 to 20 minutes, more preferably 1 to 15 minutes, and particularly preferably 3 to 10 minutes.
  • the reaction sequence of the sample to be measured, the C-factor-containing reagent, and the luminescent synthesis substrate may be the same as the reaction of the above three components or after the sample to be measured and the C-factor-containing reagent are reacted.
  • any reaction sequence may be used, such as adding a substrate, but the latter is preferred.
  • the reaction conditions at that time are not particularly limited as long as they react to release the luminescent substrate from the luminescent synthetic substrate.
  • the sample to be measured and the C-factor-containing reagent are first mixed and the temperature is 15 to 45 ° C., more preferably 20 to 40 ° C., 0.5 to 20 minutes, more preferably 1 to 15 minutes, particularly
  • a luminescent synthetic substrate is added and mixed, and the temperature is 15 to 45 ° C., more preferably 20 to 40 ° C. and 0.5 to 20 minutes, more preferably
  • the reaction (incubation) is performed for 1 to 10 minutes, particularly preferably about 3 to 7 minutes.
  • Step (iv) the amount of luminescence is measured by allowing a luminescent enzyme to act on the luminescent substrate released in the above step (iii).
  • a luminescent enzyme eg, luciferase
  • a luminescent substrate eg, aminoluciferin, luciferin
  • the luminescent enzyme that can be used in this step is not particularly limited, and any luminescent enzyme can be used as long as it catalyzes the luminescence of the luminescent substrate released from the luminescent synthetic substrate to generate light.
  • any luminescent enzyme can be used as long as it catalyzes the luminescence of the luminescent substrate released from the luminescent synthetic substrate to generate light.
  • natural luciferase purified from luminescent organs of organisms such as luminescent bacteria (e.g., Vibrio fischeri), firefly squid (Watasenia scintillans), sea fireflies, insects, etc.
  • recombinant luciferase prepared by genetic engineering techniques
  • amino acids of natural luciferase Mutant luciferase in which mutation such as addition, deletion or substitution is introduced into one or a plurality of amino acids in the sequence can be used.
  • insect-derived luciferases fireflies of North America (Photinus pyralis; accession number M15077), Genji firefly (Luciola cruciata; accession number M26194), Heike firefly (Luciola lateralis; accession number 498Z49891, X66919), acupuncture firefly (Arachnocampa lumina) (Hotaria parvula; accession number L39929), Yaeyama Himebotaru (Yaeyama), madbotaru (Pyrocoelia miyako; accession number L39928, Pyrocoelia pygidialis; accession number EU826678, Pyrocoelia pectoralis; accession number EF155570, Pinaco ), Light beetle (Pyrearinus termitilluminar; accession number AF116843), railway insect (Phrixothrix vivianii; accession number AF139644, Phrixothrix hirtus; accession
  • amino acid sequences of these beetle-derived luciferases and the base sequences of the genes encoding them are registered in a known database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)). As an example, the accession number is shown above.
  • the luciferase is not limited to those having the amino acid sequence of the natural (wild type) luciferase as described above, and has an amino acid sequence different from these amino acid sequences as long as it has a function of catalyzing the bioluminescence of the luminescent substrate. It may be a mutant luciferase.
  • the amino acid sequence different from the wild type amino acid sequence include an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the wild type amino acid sequence.
  • “one or several amino acids have been deleted, inserted, substituted or added” means deletion, insertion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. It means that as many amino acids as possible (preferably 10 or less, more preferably 7 or less, most preferably 5 or less (lower limit is 1)) are deleted, inserted, substituted or added.
  • mutant luciferase When a mutant luciferase is used, it is preferable to use a mutant luciferase modified so as to increase the luminescence intensity. When such a mutant luciferase is used, even a trace amount of endotoxin can be measured with high sensitivity.
  • Mutant luciferases modified to increase the luminescence intensity are known, and are described in, for example, Japanese Patent Application Laid-Open Nos. 2009-77660 and 2007-97577. More specifically, the following mutant luciferases (A) to (E) are mentioned.
  • a mutant firefly luciferase consisting of a substituted amino acid sequence (approximately 18 times the luminescence intensity of wild-type North American firefly luciferase);
  • a mutation comprising an amino acid sequence of wild-type North American firefly luciferase in which isoleucine (Ile) at position 423 is replaced with leucine (Leu) and leucine (Leu) at position 530 is replaced with arginine (Arg)
  • Type firefly luciferase (approximately 18 times the luminescence intensity of wild type North American firefly luciferase);
  • the amino acid sequence of wild-type North American firefly luciferase consists of an amino acid sequence in which aspartic acid (Asp) at position 436 is replaced with glycine (Gly) and leucine (Leu) at position 530 is replaced with arginine (Arg).
  • Asparagine (Asn) in Serine (Ser) Compared to the luminescence intensity of a mutant firefly luciferase consisting of an amino acid sequence in which the methionine (Met) at the position is replaced with threonine (Thr) and the threonine (Thr) at the position 252 is replaced with serine (Ser) About 21 times).
  • the above-mentioned mutant firefly luciferase is a recombinant protein obtained by inserting a mutant firefly luciferase gene obtained by modifying a wild-type firefly luciferase gene into an expression vector by a known method and introducing it into an appropriate host cell. It can be obtained by expression and purification.
  • the gene can be modified by methods well known to those skilled in the art, such as site-directed mutagenesis, random mutagenesis, and organic synthesis.
  • the base sequence of the North American firefly luciferase gene (cDNA) is registered in a database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)) as Accession No. M15077.
  • a database for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)) as Accession No. M15077.
  • the mutant firefly luciferase described in (a) to (e) above can be produced by referring to the examples in JP-A-2007-97577.
  • the person skilled in the art can identify the mutant luciferase with increased luminescence intensity by substituting the amino acid at the equivalent position in the luciferase derived from other beetles. Can be easily obtained.
  • luciferase for endotoxin manufactured by Bio-Enex Co., Ltd.
  • luciferase North American firefly luciferase, manufactured by Roche Diagnostics Inc.
  • luciferase reporter gene assay kit high sensitivity (Roche Diagnostics Inc.) Etc.).
  • the mode of action of the luminescent enzyme on the luminescent substrate released in the above step (iii) is not particularly limited, and a known method can be used. Moreover, when using a commercial item, what is necessary is just to perform reaction (action) and a measurement according to a manufacturer's instruction
  • a luminescent enzyme luciferase
  • luciferase in a buffer containing ATP and magnesium ions and add this luciferase solution.
  • the reaction is performed at 20 to 40 ° C., preferably about 37 ° C., and the luminescence amount is measured for 2 to 10 seconds after the luciferase solution is added.
  • the measuring method of the light emission amount is not particularly limited, and a known method can be used.
  • a commercially available luminometer (luminescence measuring device) or fluorometer can be used for measuring the amount of luminescence.
  • the manufacturer and performance are not particularly limited, but an apparatus capable of measuring a relative light quantity measurement value in a wide range (for example, 0 to 10,000,000) is preferably used. Specifically, specifications such as Lumitester C-110 manufactured by Kikkoman Foods Co., Ltd., Lumitester C1000 manufactured by Kikkoman Foods Co., Ltd., and ARVO Light manufactured by PerkinElmer are suitable. The measurement may be performed according to the instruction manual of the device to be used.
  • an endotoxin concentration measurement kit may be used.
  • the kit for measuring the concentration of endotoxin contains a C-factor-containing reagent, a luminescent synthetic substrate, and a luminescent enzyme as constituent components. If necessary, in addition to the above, necessary reagents and instruments are appropriately selected. It is good also as a structure of a kit. By using the kit, the endotoxin concentration (the amount of luminescence) can be measured easily and rapidly.
  • luciferase FM plus (ATP detection kit) (manufactured by Bio-Enex Co., Ltd.), reagent for bacterial testing CWB-GFP (manufactured by Bio-Enex Co., Ltd.), peptide luciferin for endotoxin (manufactured by Bio-Enex Co., Ltd.), luciferase reporter gene Assay kits, high sensitivity (Roche Diagnostics Inc.) can be used.
  • the above steps (i) to (iv) may be performed as separate steps or continuously.
  • a system (device) capable of performing each process continuously in consideration of ease of operation, handling, and the like.
  • the system (device) that can be used is not particularly limited, but the following system (device) is preferably used. That is, the present invention also provides a platelet activating substance (6), a platelet activating substance placement section (5) that is partitioned so that the platelet activating substance (6) is disposed and can be mixed with the surgical field washing solution.
  • a first hollow tubular body having a platelet activating substance removing section (4) for separating the surgical field washing liquid and the platelet activating substance (6) flowing out from the platelet activating substance placing section (5) 2); a second hollow tubular body (2 ′) having a sample collection unit (3) for collecting the sample to be measured that has passed through the platelet activating substance removing unit; detecting bacteria in the sample to be measured
  • a container (11) for carrying out the reaction a factor C-containing reagent activated by binding to endotoxin in the sample to be measured; a luminescent synthetic substrate that liberates a luminescent substrate by reaction with the factor C-containing reagent; and the luminescent substrate
  • the detection system system
  • the structure of the detection system (device) of the present invention is not particularly limited as long as it has the above-mentioned members.
  • the above steps (i) to (iv) are continuously performed. Can be done.
  • this invention is not limited to the following form.
  • the system (device) shown in Fig. 4 has an upper first hollow tubular body 2 and a lower second hollow tubular body 2 '(Figs. 4A and 4B).
  • a surgical field cleaning liquid (not shown) is injected from the upper part of the first hollow tubular body 2.
  • a platelet activating substance removing unit 4 is installed at the lower part of the first hollow tubular body 2, and a platelet activating substance placing unit 5 is installed above the platelet activating substance removing unit 4.
  • FIG. 4A a section for mixing the platelet activating substance 6 and the surgical field washing solution is provided by the platelet activating substance placing part 5, that is, the platelet activating substance placing part 5 is the step (i).
  • the platelet activating substance placement unit 5 may be a film-like material that does not allow the platelet activating substance 6 and the surgical field washing liquid to pass therethrough.
  • a hole is made in at least a part of the platelet activating substance mounting portion 5, thereby allowing the platelet activating substance 6 and the operative field to be formed.
  • the mixture with the washing solution is moved to the platelet activating substance removing unit 4 (the mixture of the platelet activating substance 6 and the surgical field washing solution is allowed to flow out from the platelet activating substance placing unit 5), and the platelet activating substance removing unit 4
  • the platelet activating substance 6 is removed from the surgical field washing solution (step (ii)).
  • the platelet activating substance mounting part 5 can be a film-like object having a strength that can be easily opened.
  • the platelet activating substance 6 is disposed on the platelet activating substance placing portion 5.
  • the platelet activating substance removing unit 4 separates the surgical field washing liquid and the platelet activating substance that have flowed out from the platelet activating substance mounting unit 5 in step (ii), that is, It is installed to remove the platelet activating substance 6 from the surgical field washing solution (not shown).
  • the platelet activating substance removing unit 4 may be a reticulated material that does not allow the platelet activating substance 6 or the tissue pieces contained in the collected surgical field washing liquid to pass through.
  • a sample collection unit 3 for collecting a sample to be measured that has passed through the platelet activating substance removing unit 4 is disposed at the bottom of the second hollow tubular body 2 '(FIG. 4B).
  • the sample collection unit 3 has a hollow tubular structure having the volume of the sample to be measured in the step (iii) (50 ⁇ L in the form of FIG. 1), the upper part is opened, and the lower part is sealed with a rubber valve or the like.
  • the first hollow tubular body 2 has an integral structure, but may be divided into a plurality of members.
  • the first hollow tubular body 2 includes a hollow tubular body 12 having a platelet activating substance mounting portion 5 on which the platelet activating substance 6 is disposed, and a platelet activating substance. It may be composed of a hollow tubular body 13 having the removing portion 4.
  • the detection system (device) of the present invention may further include a platelet activating substance removing member 8 (FIG. 4C).
  • the platelet activating substance removing member 8 can be used to make a hole in the platelet activating substance mounting portion 5 as described above. In this way, when the platelet activating substance mounting member 5 is pierced by the platelet activating substance removing member 8, the surgical field washing solution and the platelet activating substance 6 move to the platelet activating substance removing unit 4. At this time, the platelet activating substance 6 and the tissue pieces contained in the collected surgical field washing liquid remain on the platelet activating substance removing unit 4, and only the remaining surgical field washing liquid is the second hollow tubular body 2 ′ in the lower part. Move to. The liquid obtained in the second hollow tubular body 2 'becomes the sample to be measured.
  • the detection system (device) of the present invention further includes a container 11 for detecting bacteria in the sample to be measured (FIG. 4F).
  • a container 11 for detecting bacteria in the sample to be measured FIG. 4F.
  • the sample to be measured obtained above is placed in the container, and steps (iii) to (iv) are performed on the sample to be measured to measure the amount of luminescence.
  • the detection system (device) of the present invention is a factor C-containing reagent activated by binding to endotoxin in a sample to be measured, the factor C-containing reagent, A luminescent synthetic substrate that liberates the luminescent substrate by the above reaction, and a luminescent enzyme for measuring the amount of luminescence of the luminescent substrate.
  • the detection system (device) of the present invention only needs to have a Minatooki configuration, but may further include a sealing member 9 (FIG. 4D).
  • the sealing member 9 is used to seal the opening (upper part of FIG. 4B) of the sample collection unit 3. In this way, by sealing the opening of the sample collection unit 3 with the sealing member 9, the sample collection is performed even if the surgical field cleaning liquid is present in the second hollow tubular body 2 ′ other than the sample collection unit 3. Only the surgical field washing liquid in part 3 can be collected. For this reason, a predetermined amount of the sample to be measured can be collected more accurately and with better reproducibility.
  • the detection system (device) of the present invention may further include a syringe 10 in place of or in addition to the sealing member 9 (FIG. 4E).
  • the syringe 10 can be used to puncture a rubber valve (not shown) installed at the lower part of the sample collection unit 3 to collect the surgical field washing liquid 7 ′ inside the sample collection unit 3.
  • the detection system (device) 1 of the present invention is prepared (FIG. 6A).
  • step (i) a predetermined amount of surgical field washing solution 7 is added to the first hollow tubular body 2 (FIG. 6B).
  • the surgical field washing solution 7 is mixed and contacted with the platelet activating substance 6 on the platelet activating substance placing portion 5.
  • a hole is made in the platelet activating substance placing part 5 with the platelet activating substance removing member 8. (FIG. 6C).
  • the platelet activating substance removing member 8 only needs to have a structure in which a hole is formed in at least a part of the platelet activating substance placing portion 5, and a rod-like (rod-like object as shown in FIG.
  • the platelet-activating substance placement unit 5 may be perforated with scissors or the like.
  • the surgical field washing solution 7 passes through the platelet activating substance removing unit 4 and is stored in the second hollow tubular body 2 '.
  • the platelet activating substance 6 is held in the platelet activating substance removing unit 4. That is, the platelet activating substance is removed from the surgical field washing liquid, and the surgical field washing liquid 7 'is obtained.
  • the first hollow tubular body 2 and the second hollow tubular body 2 ′ are separated (FIG. 6D), and the opening of the second hollow tubular body 2 ′ is sampled by the sealing member 9.
  • the opening of the part 3 is sealed, and a syringe 10 is punctured into a rubber valve (not shown) installed in the second part of the sample collecting part 3 to collect the surgical field washing liquid 7 ′ inside the sample collecting part 3 (FIG. 6E).
  • the sample collected in this way becomes the sample 7 ′′ to be measured in step (ii).
  • this sample 7 ′′ is put in the container 11 (FIG. 6F), and the container 11 contains the C-factor-containing reagent and A luminescent synthetic substrate (indicated collectively as “12” in FIG.
  • FIG. 6G (FIG. 6G) is sequentially added, and the sample 7 ′′ to be measured is reacted with a C-factor-containing reagent and the luminescent synthetic substrate to obtain a luminescent synthetic substrate.
  • the C-factor-containing reagent and the luminescent synthetic substrate may be placed in advance in the container 11 and the sample 7 ′′ to be measured may be added to the container 11.
  • the luminescent enzyme 13 is allowed to act on the released luminescent substrate (FIG. 6H), and the amount of luminescence is measured with an appropriate measuring device (for example, luminometer) 14 (FIG. 6I).
  • Step (v) the light emission amount obtained in the step (iv) is compared with a reference value.
  • the “reference value” can be the amount of luminescence corresponding to the number of bacteria (endotoxin concentration) that can be expected to kill the bacteria by the self-help recovery ability.
  • the number of bacteria that can be expected to be killed by self-help resilience varies depending on the procedure, procedure, severity of illness, patient weight, etc., and is ultimately left to the judgment of the operator.
  • Method (1) This method describes a method for measuring a reference value when a sample with a known number of bacteria is used.
  • a standard substance containing a known number of bacteria in the case of this embodiment, 20 CFU / mL is prepared, and in the above steps (iii) and (iv), the substance is used in place of the sample to be measured.
  • the same operations as in the above steps (iii) and (iv) are performed, and the obtained light emission amount is set as a reference value. That is, the reference value is obtained by performing the steps (iii) and (iv) using a standard substance containing a known number of bacteria instead of the sample to be measured.
  • a standard substance containing a known number of bacteria for example, Bio Ball (registered trademark) series (manufactured by Sysmex Corporation) can be used.
  • Bio Ball registered trademark
  • SingleShot 30 Escherichia coli NCTC12923 manufactured by Sysmex Corporation
  • Sysmex Corporation is a ball-shaped product (microbe quantitative test) prepared to contain about 30 CFU / piece (28-33 CFU / piece) of Escherichia coli NCTC12923 Standard strain). Therefore, a standard substance containing 20 CFU / mL can be prepared by adding the product to 75 ⁇ L of physiological saline or distilled water and collecting 50 ⁇ L of the uniformly mixed solution.
  • Method (2) This method explains a method for measuring a reference value when a sample with an unknown number of bacteria is used.
  • the bacteria to be detected (bacteria presumed to cause surgical site infection (SSI)) are cultured by a general culture method.
  • the cells are separated from the obtained culture solution using a known method such as centrifugation.
  • 1 mL of distilled water is added to the bacterial sample whose cell number (bacterial concentration) is unknown, and turbidity (OD600) is measured.
  • the turbidity (OD600) of 20 CFU / mL bacterial solution is measured using a sample (reference sample) belonging to the same species and having a known number of bacteria. Based on this result, the bacterial sample is diluted with distilled water so that the turbidity of the known 20 CFU / mL bacterial solution is the same.
  • Bio Ball (registered trademark) series manufactured by Sysmex Corporation
  • the 20 CFU / mL bacterial solution thus obtained is used in place of the sample to be measured in the above steps (iii) and (iv), and the same operations as in the above steps (iii) and (iv) are performed.
  • the obtained light emission amount can be used as a reference value.
  • the light emission amount obtained in the step (iv) is compared with the reference value obtained above, as a result, the light emission amount obtained in the step (iv) is less than the reference value.
  • the operative field washing solution collected in step (i) there are only the number of bacteria that can be expected to be killed by self-help recovery. Therefore, in such a case, as shown by “Yes” in FIG. 2, the surgical field cleaning is finished, the surgical field cleaning liquid is removed from the surgical field by suction or the like, and the epidermis is sutured. End the surgery.
  • the operative field washing solution collected in step (i) contains bacteria that cannot be killed by self-help recovery ability. There is a possibility. Therefore, in such a case, as indicated by “No” in FIG. 2, the surgical field cleaning is performed again, and the steps (i) to (iv) are repeated again for the obtained surgical field cleaning liquid. The operative field cleaning is repeated until the amount of luminescence obtained in step (iv) is below the reference value. Even in such a case, steps (i) to (iv) can be performed in a short time, so that the burden on the patient is reduced. Also, by using such a method, it is possible to confirm the presence of bacteria presumed to be the cause of SSI during the operation, and thus it is possible to suppress / prevent surgical site infections.
  • the amount of luminescence may be detected in a small amount. .
  • the amount of luminescence in the background is measured in advance, and whether or not the surgical field cleaning is completed according to the value (calculation result) obtained by the calculation formula as shown in FIG. May be judged. That is, the light emission amount obtained in the step (iv), the reference value obtained in the step (v) and the light emission amount of the background are applied to the following calculation formula, and the result (calculation result) is 1 or less.
  • the number of bacteria that can be expected to be killed by self-help recovery force is present in the surgical field washing liquid collected in step (i). Therefore, in such a case, as shown by “Yes” in FIG. 3, the surgical field cleaning is finished, the surgical field cleaning liquid is removed from the surgical field by suction or the like, drained, sutured, and operated. Exit.
  • the light emission amount obtained in the step (iv), the reference value obtained in the step (v) and the light emission amount in the background are applied to the following calculation formula, and the obtained result (calculation result) is 1. In the case of exceeding, there may be bacteria in the surgical field washing liquid collected in step (i) so that the bacteria cannot be killed by self-help recovery ability.
  • the amount of light emitted from the background can be obtained, for example, as follows. That is, a blood sample of a patient is collected, and a sample obtained by diluting the blood appropriately with physiological saline (eg, 10,000 to 1,000,000 times) is used as a background sample. ) To (iv), and the obtained light emission amount is defined as “background light emission amount”.
  • the method of the present invention it is possible to confirm whether or not the presence of bacteria in the surgical field washing liquid can be killed by self-help recovery force even in the surgical field washing liquid containing a biological sample such as a blood component.
  • the method of the present invention allows detection in a short time of 60 minutes or less. For this reason, the burden on the patient can be significantly reduced, and since bacteria can be detected during the operation, bacteria can be detected before the epidermis is sutured, and SSI can be effectively prevented.
  • the detection time according to the method of the present invention can be shortened to preferably within 35 minutes, more preferably within 30 minutes, even more preferably within 20 minutes, even more preferably within 15 minutes, and particularly preferably within 12 minutes.
  • Reference example 1 Heparinized human whole blood was diluted 10,000 times with physiological saline to prepare a diluted sample. Using this diluted sample, Escherichia coli liquid cultures that had been cultured in total were prepared so that the bacterial concentrations would be 13 CFU / mL, 130 CFU / mL, and 1173 CFU / mL, respectively.
  • the respective samples are referred to as sample A-13 (bacterial concentration is 13 CFU / mL), sample A-130 (bacterial concentration is 130 CFU / mL), and sample A-1173 (bacterial concentration is 1173 CFU / mL).
  • sample C-1 a diluted sample itself to which no bacteria are added is used as a control, and this is referred to as sample C-1.
  • cation exchange resin Amberlite IR-120 as a platelet activating substance was added to human whole blood treated with heparin so as to have a concentration of 0.2 g / mL, and the mixture was incubated at 37 ° C. for 10 minutes. After mixing and contacting for 5 minutes, the supernatant was recovered and ice-cooled for 5 minutes or more. Further, the human whole blood sample after ice cooling is diluted 10,000 times with physiological saline, and the obtained sample to be measured is referred to as C-2. In the above operation, ice cooling can be omitted.
  • Samples A-13, A-130, A-1173, C-1 and C-2 were sampled in 50 ⁇ L each, and each sample was taken as an endotoxin kit (trade name: endotoxin-single test wako (turbidimetric time). Analysis method), a Limulus reagent attached to Wako Pure Chemical Industries, Ltd.) was added to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) containing 50 ⁇ L, and heated in an incubator at 37 ° C. for 10 minutes. .
  • endotoxin luciferase (Luciferase FM, manufactured by Bio-Enex Inc.) (luminescent enzyme) dissolved in 50 mM Tris-Cl (pH 7.5) containing 1 mM MgSO 4 and 10% trehalose was added to the reaction solution. Thereafter, 50 ⁇ L of 10 ⁇ 5 M ATP solution dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added, and the tube was tapped and stirred several times.
  • a luminometer (trade name: Luminescence tester C-110 (manufactured by Kikkoman Foods Co., Ltd.) was used to measure the luminescence (RLU).
  • sample C-1 not treated with the platelet activating substance shows a light emission level substantially equivalent to that of sample A-13 having a bacterial concentration of 13 CFU / mL. From this, samples containing biological samples (blood components) emit light even when bacteria (endotoxin) are not present when they are not treated with platelet-activating substances, whereas platelet-activating substances By processing, the amount of luminescence of sample C-2 in which no bacteria (endotoxin) is present is significantly reduced, and can be significantly distinguished from the amount of luminescence of sample A-13 having a bacterium concentration of 13 CFU / mL.
  • Example 1 Human whole blood treated with heparin was prepared as a human whole blood sample.
  • a cation exchange resin Amberlite IR-120 as a platelet activating substance was added to the human whole blood sample so as to have a concentration of 0.2 g / mL. Mix and contact for 3, 5, 10 minutes. After mixing and contacting for a predetermined time, the supernatant was collected and cooled on ice for 5 minutes or more. Furthermore, by diluting the human whole blood sample after ice cooling 10,000 times with physiological saline, sample B-1 to be measured (mixing / contact for 1 minute), B-3 (mixing / contact for 3 minutes), B-5 (mixing / contact for 5 minutes) and B-10 (mixing / contact for 10 minutes) were prepared. A diluted sample that has not been brought into contact with the platelet activating substance is used as a control, and this is referred to as sample C-3. In the above operation, ice cooling can be omitted.
  • Samples B-1, B-3, B-5, B-10, and C-3 were each collected at 50 ⁇ L, and each sample was taken as an endotoxin kit (trade name: endotoxin-single test Wako (turbidimetric time). Analysis method), a Limulus reagent attached to Wako Pure Chemical Industries, Ltd.) was added to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) containing 50 ⁇ L, and heated in an incubator at 37 ° C. for 10 minutes. .
  • endotoxin kit trade name: endotoxin-single test Wako (turbidimetric time). Analysis method
  • a Limulus reagent attached to Wako Pure Chemical Industries, Ltd. was added to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) containing 50 ⁇ L, and heated in an incubator at 37 ° C. for 10 minutes. .
  • endotoxin luciferase (Luciferase FM, manufactured by Bio-Enex Inc.) (luminescent enzyme) dissolved in 50 mM Tris-Cl (pH 7.5) containing 1 mM MgSO 4 and 10% trehalose was added to the reaction solution. Thereafter, 50 ⁇ L of 10 ⁇ 5 M ATP solution dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added, and the tube was tapped and stirred several times.
  • a luminometer (trade name: Luminescence tester C-110 (manufactured by Kikkoman Foods Co., Ltd.) was used to measure the luminescence (RLU).
  • samples B-1, B-3, B-5, and B-10 treated with the platelet activating substance are luminescence of sample C-3 not treated with the platelet activating substance.
  • the amount of light emission can be reduced compared to the amount. From this result, in sample C-3 which is not treated with the platelet activating substance, the components contained in the blood react with the reagent and the luminescence reaction proceeds, but by treating the blood with the platelet activating substance, It is considered that the amount of light contained in the blood can be reduced, and as a result, the amount of light emitted from the background when bacteria are not present can be reduced.

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Abstract

Provided are a method and a system, each of which can detect the presence of bacteria in a surgical cleaning solution containing a biological sample in a simple manner. The present invention relates to a method for detecting bacteria in a surgical cleaning solution, comprising: (i) mixing the surgical cleaning solution with a platelet-activating substance to allow the surgical cleaning solution and the platelet-activating substance to contact with each other; (ii) removing the platelet-activating substance from the surgical cleaning solution to produce a sample of interest; (iii) reacting the sample of interest with a C factor-containing reagent that can be activated through the binding to an endotoxin contained in the sample of interest and a luminescent synthetic substrate to cause the release of the luminescent substrate from the luminescent synthetic substrate; (iv) allowing a luminescent enzyme to act on the luminescent substrate that has been released in the above-mentioned step (iii) and measuring the quantity of emitted light; and (v) comparing the quantity of emitted light obtained in the above-mentioned step (iv) with a reference value.

Description

術野洗浄液中の菌の検出方法およびシステムMethod and system for detecting bacteria in surgical field washing liquid
　本発明は、術野洗浄液中の菌の検出方法および検出システムに関する。生体試料が混入した術野洗浄液中の菌の存在を簡易に検出する方法およびシステムに関する。 The present invention relates to a detection method and a detection system for bacteria in surgical field washing liquid. The present invention relates to a method and a system for easily detecting the presence of bacteria in a surgical field washing liquid mixed with a biological sample.
　股関節置換術、膝関節置換術、心臓手術、血管手術、結腸直腸手術等の外科手術時には、微生物に対してバリアとして機能している皮膚が切開され、本来無菌である内部組織が開放されるため、手術を行った部位に発生する手術部位感染（surgical site infection：ＳＳＩ）が問題となっている。ＳＳＩの原因と推定される細菌としては、黄色ブドウ球菌、コアグラーゼ陰性ブドウ球菌、肺炎球菌、グラム陰性菌などが挙げられる。また、ＳＳＩの発生率は手術の種類や手術を行う部位によって異なり、ＪＡＮＩＳ（厚生労働省院内感染対策サーベイランス事業）２００７年季報（１月～６月）のデータによると、例えば、胃の手術では４．０％、直腸の手術では１７．１％となっており、入院患者の院内感染の中で、ＳＳＩが占める割合は１４～１６％を占め、尿路感染や肺炎に次いで２番目の多さである。さらに、ＳＳＩが起こると、治癒が遅くなることで、入院日数や医療コストが増え、患者への負担がかなり大きくなってしまう。このため、ＳＳＩを最小限にする取り組みが急務である。 During surgical operations such as hip replacement, knee replacement, heart surgery, vascular surgery, and colorectal surgery, skin that functions as a barrier to microorganisms is incised, and inherently sterile internal tissue is released. Surgical site infection (SSI) occurring at the site where surgery is performed is a problem. Examples of bacteria presumed to cause SSI include Staphylococcus aureus, coagulase-negative staphylococci, pneumococci, and gram-negative bacteria. In addition, the incidence of SSI varies depending on the type of surgery and the site where surgery is performed. 0.01% and 17.1% for rectal surgery, accounting for 14-16% of hospital-acquired hospital infections, second only to urinary tract infections and pneumonia It is. Furthermore, when SSI occurs, healing is delayed, which increases the number of days of hospitalization and medical costs, and the burden on the patient is considerably increased. For this reason, efforts to minimize SSI are urgent.
　一般的にＳＳＩ対策としては、手術前・中・後で異なる。このうち、手術中のＳＳＩ対策としては、絹糸ではなく合成吸収糸を縫合糸として使用する、創縁ドレープを用いる、など様々な対策があるが、上記対策に加えて、疾患部を縫合する前に、術野を生理食塩水などで洗浄し、感染の原因となる菌を可能な限り除去する対策がとられている。また、術野での菌数がある程度以下であれば自助回復力によってその菌を死滅させることが期待できる。しかしながら、従来の方法では、検出時間に少なくとも数時間を要し、術後にしか細菌の検出結果を得られないため、細菌数の確認が行われないまま縫合されていた。このため、短時間で（術中に）細菌の存在を検出できる方法が求められている。 Generally, SSI countermeasures differ before, during and after surgery. Among these measures, there are various measures such as SSI countermeasures during surgery, such as using synthetic absorbent thread as a suture instead of silk thread, and using wound draping. In addition to the above countermeasures, before suturing the diseased part In addition, measures are taken to remove as much of the bacteria that cause infection as possible by washing the surgical field with physiological saline or the like. In addition, if the number of bacteria in the surgical field is below a certain level, it can be expected that the bacteria will be killed by self-help recovery ability. However, in the conventional method, the detection time takes at least several hours, and the result of detection of the bacteria can be obtained only after the operation. Therefore, the suture is performed without confirming the number of bacteria. For this reason, there is a need for a method that can detect the presence of bacteria in a short time (intraoperative).
　一方、細菌の検出方法としては、グラム陰性菌の外膜を構成する成分の一つである「エンドトキシン(endotoxin)」の濃度を測定する方法がある（例えば、特許文献１）。特許文献１の方法は、試料と、エンドトキシンとの結合により活性化されるＣ因子を含有する試薬と、ペプチドに発光基質が結合してなる発光合成基質とを反応させ、発光合成基質から発光基質を遊離させる発光基質遊離工程と、発光基質遊離工程により遊離した発光基質に発光酵素を作用させ、発光量を測定する発光量測定工程と、発光量測定工程により得られた測定値に基づいて試料中のエンドトキシン濃度を定量する濃度定量工程とを包含することを特徴としている。当該方法によると、試料中のエンドトキシンを、簡便かつ高感度に測定できる。 On the other hand, as a method for detecting bacteria, there is a method for measuring the concentration of “endotoxin”, which is one of the components constituting the outer membrane of Gram-negative bacteria (for example, Patent Document 1). In the method of Patent Document 1, a sample, a reagent containing factor C activated by binding to endotoxin, and a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide are reacted, and the luminescent synthetic substrate is converted into a luminescent substrate. A sample based on the measurement value obtained by the luminescence substrate release step, the luminescence quantity measurement step in which the luminescent enzyme is allowed to act on the luminescence substrate released in the luminescence substrate release step, and the luminescence amount measurement step is measured. And a concentration determination step for quantifying the endotoxin concentration therein. According to this method, endotoxin in a sample can be measured easily and with high sensitivity.
米国特許出願公開第２０１１／０１１４４１号明細書US Patent Application Publication No. 2011/011441
　しかしながら、特許文献１に記載の方法は、血液等の生体試料を含む場合には、エンドトキシンが存在しない試料でも発光してしまい、少量のエンドトキシン（細菌）が存在する試料の場合には、当該試料とエンドトキシン（細菌）が存在しない試料とを識別することができないという問題がある。 However, in the method described in Patent Document 1, when a biological sample such as blood is included, even a sample without endotoxin emits light, and in the case of a sample with a small amount of endotoxin (bacteria), the sample There is a problem that it cannot be distinguished from a sample in which endotoxin (bacteria) is not present.
　したがって、本発明は、上記事情を鑑みてなされたものであり、生体試料を含む術野洗浄液中の菌の存在を簡便に検出できる方法を提供することを目的とする。 Therefore, the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method capable of easily detecting the presence of bacteria in a surgical field washing liquid containing a biological sample.
　また、本発明の他の目的は、生体試料を含む術野洗浄液中の菌の存在を短時間で検出できる方法を提供することである。 Another object of the present invention is to provide a method capable of detecting the presence of bacteria in a surgical field washing solution containing a biological sample in a short time.
　本発明者らは、上記の問題を解決すべく、鋭意研究を行った結果、生体試料を含む術野洗浄液中のエンドトキシン濃度を測定する前に、術野洗浄液を血小板活性化物質で予め処理することによって、偽陽性の問題を解消し、上記目的が達成できることを知得した。当該知見に基づいて、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors pre-treat the surgical field washing liquid with a platelet activating substance before measuring the endotoxin concentration in the surgical field washing liquid containing a biological sample. As a result, it was found that the above-mentioned purpose can be achieved by eliminating the false positive problem. Based on the knowledge, the present invention has been completed.
　すなわち、上記諸目的は、（ｉ）術野洗浄液を血小板活性化物質と混合・接触させ［工程（ｉ）］；（ｉｉ）前記血小板活性化物質を前記術野洗浄液から除去して、被測定試料を得［工程（ｉｉ）］；（ｉｉｉ）前記被測定試料を、前記被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬および発光合成基質と反応させて、前記発光合成基質から前記発光基質を遊離させ［工程（ｉｉｉ）］；（ｉｖ）前記工程（ｉｉｉ）で遊離した発光基質に発光酵素を作用させて、発光量を測定し［工程（ｉｖ）］；さらに（ｖ）前記工程（ｉｖ）で得られた発光量を、基準値と比較する［工程（ｖ）］、ことを有する術野洗浄液中の菌の検出方法によって達成できる。 That is, the above-mentioned purposes are as follows: (i) mixing and contacting the surgical field washing solution with the platelet activating substance [step (i)]; (ii) removing the platelet activating substance from the surgical field washing liquid and measuring Obtaining a sample [step (ii)]; (iii) reacting the sample to be measured with a C-factor containing reagent activated by binding to endotoxin in the sample to be measured and a luminescent synthesis substrate, thereby producing the luminescent synthesis The luminescent substrate is released from the substrate [Step (iii)]; (iv) A luminescent enzyme is allowed to act on the luminescent substrate released in the step (iii) to measure the amount of luminescence [Step (iv)]; v) The amount of luminescence obtained in the step (iv) is compared with a reference value [step (v)], and this can be achieved by a method for detecting bacteria in the surgical field washing solution.
　本発明の方法によれば、生体試料を含む術野洗浄液であっても、術野洗浄液中の細菌を容易にかつ短時間で検出できる。 According to the method of the present invention, even in an operative field washing solution containing a biological sample, bacteria in the operative field washing solution can be detected easily and in a short time.
　本発明のさらに他の目的、特徴および特質は、以後の説明および添付図面に例示される好ましい実施の形態を参酌することによって、明らかになるであろう。 Further objects, features, and characteristics of the present invention will become apparent by referring to the following description and preferred embodiments exemplified in the accompanying drawings.
本発明の方法の第一の実施形態を示すフローチャートである。It is a flowchart which shows 1st embodiment of the method of this invention. 本発明の方法の第二の実施形態を示すフローチャートである。It is a flowchart which shows 2nd embodiment of the method of this invention. 本発明の方法の第三の実施形態を示すフローチャートである。It is a flowchart which shows 3rd embodiment of the method of this invention. 本発明の方法に好適に使用できる検出システム（デバイス）の一実施形態を示す図である。It is a figure which shows one Embodiment of the detection system (device) which can be used suitably for the method of this invention. 本発明の方法に好適に使用できる検出システム（デバイス）の第１の中空の管状体の他の実施形態を示す図である。It is a figure which shows other embodiment of the 1st hollow tubular body of the detection system (device) which can be used suitably for the method of this invention. 図４の検出システム（デバイス）を用いて本発明の方法を実施する工程を説明するための図である。It is a figure for demonstrating the process of implementing the method of this invention using the detection system (device) of FIG. 参考例１において、菌数と発光量との関係を示す図である。In Reference Example 1, it is a figure which shows the relationship between the number of bacteria and the amount of luminescence. 実施例１において、血小板活性化物質との混合・接触時間と発光量との関係を示す図である。In Example 1, it is a figure which shows the relationship between mixing and contact time with a platelet activation substance, and light-emission quantity.
　本発明は、（ｉ）術野洗浄液を血小板活性化物質と混合・接触させ［工程（ｉ）］；（ｉｉ）前記血小板活性化物質を前記術野洗浄液から除去して、被測定試料を得［工程（ｉｉ）］；（ｉｉｉ）前記被測定試料を、前記被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬および発光合成基質と反応させて、前記発光合成基質から前記発光基質を遊離させ［工程（ｉｉｉ）］；（ｉｖ）前記工程（ｉｉｉ）で遊離した発光基質に発光酵素を作用させて、発光量を測定し［工程（ｉｖ）］；さらに（ｖ）前記工程（ｉｖ）で得られた発光量を、基準値と比較する［工程（ｖ）］、ことを有する術野洗浄液中の菌の検出方法に関する。本発明の方法は、生体試料を含む術野洗浄液中のエンドトキシン濃度を測定する前に、術野洗浄液を血小板活性化物質で予め処理することを特徴とする。本発明者らは、生体試料中の血小板凝固因子が存在すると、エンドトキシン（細菌）が存在しない試料であっても発光してしまうことを発見した。さらに、術野洗浄液中の血小板凝固因子を血小板活性化物質で処理することによって、上記したような偽陽性結果の原因物質を除去できる。このため、当該処理後の術野洗浄液中のエンドトキシン濃度を測定すると、エンドトキシン（細菌）が存在しない試料の発光量を有意に抑制でき、エンドトキシン（細菌）の存在を正確に検出することができる。また、２０ＣＦＵ／ｍＬ以下という少数の細菌をも検出することができる。上記利点に加えて、本発明の方法によると、２０分以下という短時間での検出が可能である。このため、患者への負担を軽減でき、また、表皮を縫合する前に細菌が検出できるため、ＳＳＩを有効に予防することができる。 The present invention comprises (i) mixing and contacting an operative field washing solution with a platelet activating substance [step (i)]; (ii) removing the platelet activating substance from the operative field washing solution to obtain a sample to be measured. [Step (ii)]; (iii) reacting the sample to be measured with a C-factor-containing reagent activated by binding to endotoxin in the sample to be measured and a luminescent synthetic substrate, and A luminescent substrate is released [step (iii)]; (iv) a luminescent enzyme is allowed to act on the luminescent substrate released in step (iii) to measure the amount of luminescence [step (iv)]; Comparing the amount of luminescence obtained in step (iv) with a reference value [step (v)], the present invention relates to a method for detecting bacteria in surgical field washing liquid. The method of the present invention is characterized in that the surgical field washing solution is pretreated with a platelet activating substance before measuring the endotoxin concentration in the surgical field washing solution containing a biological sample. The present inventors have discovered that the presence of platelet clotting factors in a biological sample causes light emission even in a sample in which no endotoxin (bacteria) is present. Further, by treating the platelet coagulation factor in the surgical field washing solution with a platelet activating substance, the causative substance of the false positive result as described above can be removed. For this reason, if the endotoxin concentration in the surgical field washing liquid after the said process is measured, the light-emission quantity of the sample which does not have endotoxin (bacteria) can be suppressed significantly, and presence of endotoxin (bacteria) can be detected correctly. In addition, a small number of bacteria of 20 CFU / mL or less can be detected. In addition to the above advantages, the method of the present invention enables detection in a short time of 20 minutes or less. For this reason, the burden on the patient can be reduced, and since bacteria can be detected before the epidermis is sutured, SSI can be effectively prevented.
　なお、本発明の方法により検出される菌としては、特に制限されず、一般的に手術部位感染（ＳＳＩ）の原因と推定される細菌でありうる。具体的には、Budvicia属、Buttauxella属、Cedecea属、Citrobacter属、Enterobacter属、Escherichia属、Edwardsiella属、Erwinia属、Ewingella属、Hafnia属、Klebsiella属、Kluyvera属、Leclercia属、Leminorella属、Morganella属、Obesumbacterium属、Pragia属、Proteus属、Providencia属、Rahnella属、Salmonella属、Serratia属、Shigella属、Tatumella属、Trabulsiella属、Xenorhabdus属、Yersinia属、Yokenella属、Acinetobacter属、Actinobacillus属、Aeromonas属、Agrobacterium属、Alcaligenes属、Arcobacter属、Bordetella属、Brucella属、Branhamella属、Burkholderia属、Calymmatobacterium属、Campylobacter属、Capnocytophage属、Cardiobacterium属、Chromobacterium属、Chryseomonas属、Comamonas属、Eikenella属、Flavimonas属、Flavobacterium属、Flexispira属、Francisella属、Haemophilus属、Helicobacter属、Kingella属、Legionella属、Methylobacterium属、Moraxella属、Neisseria属、Ochrobactrum属、Oligella属、Pasteurella属、Pectobacterium属、Plesiomonas属、Pseudomonas属、Shewanella属、Sphingobacterium属、Sphingomonas属、Stenotrophomonas属、Streptobacillus属、Viblio属、Weeksalla属、Xanthomonas属、Acidaminococcus属、Anaerobiospirillum属、Anaerorhabdus属、Bacteroides属、Biophila属、Centipeda属、Desulfomonas属、Desulfovibrio属、Dichelobacter属、Fusobacterium属、Leptotrichia属、Megasphaera属、Mitsuokella属、Mobiluncus属、Porphyromonas属、Prevotella属、Selenomonas属、Tissierella属、Veillonella属、及びWolinella属等に属するグラム陰性菌などが挙げられる。これらのうち、手術部位感染の主要原因となるEnterobacter属、Escherichia属、Pseudomonas属、Bacteroides属に適応するのが望ましい。なお、手術部位感染の原因としてはグラム陰性菌だけではなく、グラム陽性菌も含まれるが、洗浄の度合いを確認するためにはグラム陰性菌のみの検出で十分である。 Note that the bacteria detected by the method of the present invention are not particularly limited, and may be bacteria that are generally estimated to be the cause of surgical site infection (SSI). Specifically, the genus Budvicia, Buttauxella, Cedecea, Citrobacter, Enterobacter, Escherichia, Edwardsiella, Erwinia, Ewingella, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Morganella, Obesumbacterium, Pragia, Proteus, Providencia, Rahnella, Salmonella, Serratia, Shigella, Tatumella, Trabulsiella, Xenorhabdus, Yersinia, Yokenella, Acinetobacter, Actinobacillus, Gromona, Agromonas , Alcaligenes, Arcobacter, Bordetella, Brucella, Branhamella, Burkholderia, Calymmatobacterium, Campylobacter, Capnocytophage, Cardiobacterium, Chromobacterium, Chryseomonas, Comamonas, Eikenella, Flavomonas, Flavomonas Genus, Francisella, Haemophilus, Helicobacter, Kingella, Legionella, Methylobacterium, Moraxella, Neisseria, Ochrobactrum, Oligella, Pasteure lla, Pectobacterium, Plesiomonas, Pseudomonas, Shewanella, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptobacillus, Viblio, Weeksalla, Xanthomonas, Acidaminococcus, Anaerobiospirillum, Anaerobiospirillum , Centipeda genus, Desulfomonas genus, Desulfovibrio genus, Dichelobacter genus, Fusobacterium genus, Leptotrichia genus, Megasphaera genus, Mitsuokella genus, Mobiluncus genus, Porphyromonas genus, Prevotella genus, Selenomonas genus, Tissierella genus, Veillonella genus, and Wolinella genus, etc. Examples include negative bacteria. Of these, it is desirable to adapt to the genus Enterobacter, Escherichia, Pseudomonas, and Bacteroides, which are the main causes of surgical site infections. Although the cause of surgical site infection includes not only Gram-negative bacteria but also Gram-positive bacteria, detection of only Gram-negative bacteria is sufficient for confirming the degree of washing.
　以下、本発明の実施の形態を図１～３を参照しながら説明する。なお、各図中では、液の量、反応条件などが記載されているが、これらは好ましい一実施形態を示すものであり、本発明はこれらに限定されるものではない。 Hereinafter, embodiments of the present invention will be described with reference to FIGS. In each figure, the amount of liquid, reaction conditions, and the like are described, but these show one preferred embodiment, and the present invention is not limited to these.
　１．工程（ｉ）
　本工程では、術野洗浄液を血小板活性化物質と混合・接触させる。 1. Step (i)
In this step, the surgical field washing solution is mixed and contacted with the platelet activating substance.
　ここで、術野洗浄液とは、股関節置換術、膝関節置換術、心臓手術、血管手術、結腸直腸手術等の外科手術で患部の縫合前に、術野（例えば、結腸直腸手術では腹腔内）を洗浄液で洗浄するが、その際の洗浄液をいう。このため、本発明の方法が適用される術野洗浄液は、通常、血液成分等の生体由来の成分を含む。洗浄後は、術野洗浄液を吸引・廃液し、この洗浄および吸引・廃液操作を、適当回数、例えば、合計２～２５回（好ましくは４～２０回）程度、繰り返す。各洗浄工程で得られる術野洗浄液のいずれを本工程で使用してもよいが、一般的には、最終洗浄工程で得られた術野洗浄液が使用される。なお、術野の洗浄回数は術式、病気の重篤度、患者の体重などによって異なり、最終的には術者の判断にゆだねられる。また、術野洗浄液は、特に制限されず、通常、外科手術で洗浄に使用されるものが同様にして使用できる。具体的には、生理食塩水、滅菌水、リンガー溶液、４．５重量％のブドウ糖等の糖やグリセリン等の細胞に無害な浸透圧保持剤を加えて生体組織細胞と実質的に同じ浸透圧に等張した溶液、脳脊髄手術用洗浄灌流液などが挙げられる。また、１回あたりに使用される術野洗浄液の量は、特に制限されず、通常外科手術における使用量と同様でありうる。具体的には、１回あたりに使用される術野洗浄液の量は、１００～１，０００ｍＬが好ましく、総量としては、１００～１０，０００ｍＬがより好ましい。また、上記術野洗浄液は、そのまま使用されてもよいが、患者への負担をかんがみると、体温とほぼ同等の温度（例えば、３５～４０℃、好ましくは３７℃前後）に加温されることが好ましい。 Here, the surgical field washing liquid refers to the surgical field (for example, intraperitoneal in colorectal surgery) before suturing the affected part in surgical operations such as hip replacement, knee replacement, cardiac surgery, vascular surgery, and colorectal surgery. Is washed with a washing solution, which is a washing solution at that time. For this reason, the surgical field washing liquid to which the method of the present invention is applied usually contains a biological component such as a blood component. After washing, the surgical field washing solution is aspirated / waste, and this washing and aspiration / waste operation are repeated an appropriate number of times, for example, 2 to 25 times (preferably 4 to 20 times) in total. Any of the surgical field cleaning solutions obtained in each cleaning step may be used in this step, but generally, the surgical field cleaning solution obtained in the final cleaning step is used. The frequency of cleaning the surgical field varies depending on the surgical method, the severity of the disease, the weight of the patient, etc., and is ultimately left to the judgment of the operator. In addition, the surgical field cleaning liquid is not particularly limited, and those usually used for cleaning in a surgical operation can be used in the same manner. Specifically, physiological saline, sterilized water, Ringer's solution, osmotic pressure-retaining agent that is harmless to cells such as 4.5% by weight glucose and sugar, and glycerin are added to substantially the same osmotic pressure as living tissue cells. And an isotonic solution, irrigated perfusate for cerebrospinal surgery, and the like. Further, the amount of the surgical field cleaning solution used per time is not particularly limited, and may be the same as the amount used in normal surgery. Specifically, the amount of the surgical field cleaning solution used at one time is preferably 100 to 1,000 mL, and the total amount is more preferably 100 to 10,000 mL. The surgical field washing solution may be used as it is, but considering the burden on the patient, it is heated to a temperature almost equal to the body temperature (for example, 35 to 40 ° C., preferably around 37 ° C.). It is preferable.
　本工程において、術野洗浄液は、全量を血小板活性化物質と混合・接触させる必要はなく、通常、その一部が使用される。 In this step, it is not necessary to mix and contact the entire amount of the surgical field washing solution with the platelet activating substance, and a part thereof is usually used.
　本工程で使用できる血小板活性化物質は、血小板の凝集を誘導できる物質であれば特に制限されない。具体的には、陽イオンがＣａ２＋、Ｃｕ２＋、Ｚｎ２＋、Ｍｇ２＋、Ｋ＋、ＮＨ４ ＋、Ｎａ＋、またはＨ＋である陽イオン交換樹脂；陰イオンがＳＯ４ ２－、Ｉ－、ＮＯ３ －、ＣｒＯ４ ２－、Ｂｒ－、Ｃｌ－、ＯＨ－、またはＦ－である陰イオン交換樹脂；コラーゲン、フィブリン、ＡＤＰ、アラキドン酸、トロンビン、セロトニン、プロタミン、カルシウム塩、ＲＧＤペプチド、若しくは凝固因子（例えば、フィブリノゲン、トロンビン、プロトロンビン、フォンビルブランドファクター、トロンボキサン、トロンボプラスチン、第五因子、第七因子、第八因子、第九因子、第十因子、第十一因子、第十二因子、プレカリクレイン、高分子キニノゲン等）などの血小板を活性化して血栓を生じやすくする物質から構成されるまたは前記少なくとも一の物質で被覆されるビーズ；珪砂、例えば、大きさが０．４～２０μｍの珪砂、結晶シリカ、例えば、大きさが５μｍ以下で平均粒径が１．１μｍの結晶シリカ（例えば、ペンシルバニア・グラスサンド社製、商品名：Ｍｉｎ－ＵＳｉｌ）、珪藻土、ガラス微粉末、カオリン、ベントナイト；ならびにポリスチレン、ポリオレフィン、ポリイミド、ポリカーボネート、ポリアリレート、ポリエステル、ポリアクリロニトリル、ポリメタクリル酸メチル、およびポリアクリル酸等の樹脂などが挙げられる。陽／陰イオン交換樹脂は、市販品を使用してもよい。具体的には、陽イオン交換樹脂（弱酸性陽イオン交換樹脂、強酸性陽イオン交換樹脂）としては、アンバーライト（商標）ＣＧ－４０００、ＣＧ－５０００、ＣＧ－６０００、ＣＧ－８０００、ＩＲ－１１６、ＩＲ－１１８、ＩＲ－１１８Ｈ、ＩＲ－１２０、ＩＲ－１２０Ｂ、ＩＲ－１２２、ＩＲ－１２４、２５２、２００ＣＴ、２０１ＣＴ、２００Ｃ、ＩＲＣ－５０、ＩＲＣ－８４、ＸＴ－１００７、ＸＴ－１００９、ＸＴ－１００２（以上、いずれも株式会社オルガノ製の商品名）等のアンバーライト（商標）系陽イオン交換樹脂；ダイヤイオン（商標）ＳＫ－１Ａ、ＳＫ－１Ｂ、ＳＫ－１０４、ＳＫ－１１０、ＳＫ－１１２、ＦＭＫ－１０、ＷＫ－１０、ＷＫ－１１、ＷＫ－２０、ＰＡ－４０６、ＰＡ－４０８、ＰＡ－４１２、ＰＡ－４１６、ＰＡ－４１８、ＰＫ－２０８、ＰＫ－２１２、ＰＫ－２１６、ＰＫ－２１８、ＰＫ－２２０、ＰＫ－２２８、ＵＢＫ０８、ＵＢＫ１０、ＵＢＫ１２（以上、いずれも三菱化学株式会社製の商品名）等のダイヤイオン（商標）系陽イオン交換樹脂などが挙げられる。また、陰イオン交換樹脂（弱塩基性陰イオン交換樹脂、中塩基性陰イオン交換樹脂、強塩基性陰イオン交換樹脂）としては、アンバーライト（商標）ＩＲＡ－４００、ＩＲＡ－４００Ｊ、ＩＲＡ－４００Ｔ、ＩＲＡ－４０１、ＩＲＡ－４０２ＢＬ、ＩＲＡ－４０４Ｊ、ＩＲＡ－４３０、ＩＲＡ－４５８、ＩＲＡ－４５８、ＩＲＡ－９００、ＩＲＡ－９００Ｊ、ＩＲＡ－９０４、ＩＲＡ－９１０、ＩＲＡ－９１０ＣＴ、ＩＲＡ－９３８、ＩＲＡ－９５８、ＩＲＡ－９５８ＲＦ、ＩＲＡ－４１０、ＩＲＡ－４１０Ｊ、ＩＲＡ－４１１、ＩＲＡ－９１０、ＩＲＡ－６８、ＩＲＡ－３５、ＩＲＡ－９３等のアンバーライト（商標）系陰イオン交換樹脂；ダイヤイオン（商標）ＷＡ－１０、ＷＡ－１１、ＷＡ－２０、ＷＡ－２１、ＷＡ－３０、ＰＡ－４０６、ＰＡ－４０８、ＰＡ－４１２、ＰＡ－４１６、ＰＡ－４１８、ＰＡ－３０６、ＰＡ－３０６Ｓ、ＰＡ－３０８、ＰＡ－３１２、ＰＡ－３１６、ＰＡ－３１８、ＰＡ－３１８Ｌ、ＰＡ－３１８Ｌ、ＰＡ－４０８、ＰＡ－４１２、ＰＡ－４１８、ＨＰＡ２５、ＳＡ－１０Ａ、ＳＡ－１１Ａ、ＳＡ－１２Ａ、ＳＡ－２０Ａ、ＳＡ－２１Ａ、ＮＳＡ１００、ＵＢＡ１２０（以上、いずれも三菱化学株式会社製の商品名）等のダイヤイオン（商標）系陰イオン交換樹脂；ＯＰＴＩＰＯＲＥ－ＸＵＳ４０２８５．００、ＯＰＴＩＰＯＲＥ－ＸＵＳ　４０３９０．００（以上、いずれもダウケミカル株式会社製の商品名）等の弱塩基性陰イオン交換樹脂などが挙げられる。上記陽／陰イオン交換樹脂のうち、スチレン－ジビニルベンゼン共重合体、フェノールホルマリン樹脂などを基体とし、イオン交換基としてスルホン酸基を有するもの等の強酸性陽イオン交換樹脂（例えば、アンバーライト（商標）ＩＲ－１２０、ＩＲ－１２０Ｂ、ＩＲ－１２４、２５２、２００ＣＴ、；ダイヤイオン（商標）ＳＫ－１Ａ、ＳＫ－１Ｂ、ＳＫ－１０４、ＳＫ－１１０、ＳＫ－１１２、ＵＢＫ０８、ＵＢＫ１０、ＵＢＫ１２、ＦＭＫ－１０、ＰＫ－２０８、ＰＫ－２１２、ＰＫ－２１６、ＰＫ－２１８、ＰＫ－２２０、ＰＫ－２２８）等の、強酸性陽イオン交換樹脂、およびスチレン－ジビニルベンゼン共重合体などを基体とし、イオン交換基としてトリメチルアンモニウム基、β―ヒドロキシエチルジメチルアンモニウム基を有するもの等の強塩基性陰イオン交換樹脂（例えば、アンバーライト（商標）ＩＲＡ－４００、ＩＲＡ－４００Ｊ、ＩＲＡ－４０２ＢＬ、ＩＲＡ－４０４Ｊ、ＩＲＡ－４１０、ＩＲＡ－４１０Ｊ、ＩＲＡ－４１１、ＩＲＡ－９００Ｊ、ＩＲＡ－９０４、ＩＲＡ－９１０ＣＴ、ＩＲＡ－９５８、ＩＲＡ－９５８ＲＦ、；ダイヤイオン（商標）ＳＡ－１０Ａ、ＳＡ－１１Ａ、ＳＡ－１２Ａ、ＳＡ－１０Ｂ、ＦＭＡ－１０、ＮＳＡ１００、ＵＢＡ１２０、ＰＡ－３０６Ｓ、ＰＡ－３０８、ＰＡ－３１２、ＰＡ－３１６、ＰＡ－３１８Ｌ、ＨＰＡ２５、ＳＡ－２０Ａ、ＳＡ－２１Ａ、ＰＡ－４０８、ＰＡ－４１２、ＰＡ－４１８）等の、強塩基性陰イオン交換樹脂が好ましく使用される。また、上記陽／陰イオン交換樹脂以外の血小板活性化物質のうちでは、コラーゲンやポリスチレンビーズが好ましく使用される。なお、上記血小板活性化物質は、単独で使用されてもあるいは２種以上の混合物の形態で使用されてもよい。 The platelet activating substance that can be used in this step is not particularly limited as long as it is a substance that can induce platelet aggregation. Specifically, a cation exchange resin whose cation is Ca 2+ , Cu 2+ , Zn 2+ , Mg 2+ , K + , NH 4 + , Na + , or H + ; an anion is SO 4 2− , I − , NO 3 -, CrO 4 2- , Br -, Cl -, OH - or F, - a is an anion exchange resin; collagen, fibrin, ADP, arachidonic acid, thrombin, serotonin, protamine, calcium salts, RGD peptide, Or coagulation factors (eg fibrinogen, thrombin, prothrombin, von Willebrand factor, thromboxane, thromboplastin, fifth factor, seventh factor, eighth factor, ninth factor, tenth factor, eleventh factor, twelve Factor, prekallikrein, high molecular weight kininogen, etc.) Beads formed or coated with at least one substance; silica sand, eg, silica sand having a size of 0.4-20 μm, crystalline silica, eg, crystals having a size of 5 μm or less and an average particle size of 1.1 μm Silica (for example, Pennsylvania Glass Sand, trade name: Min-USil), diatomaceous earth, glass fine powder, kaolin, bentonite; and polystyrene, polyolefin, polyimide, polycarbonate, polyarylate, polyester, polyacrylonitrile, polymethyl methacrylate And resins such as polyacrylic acid. As the cation / anion exchange resin, a commercially available product may be used. Specifically, as the cation exchange resin (weakly acidic cation exchange resin, strong acid cation exchange resin), Amberlite (trademark) CG-4000, CG-5000, CG-6000, CG-8000, IR- 116, IR-118, IR-118H, IR-120, IR-120B, IR-122, IR-124, 252, 200CT, 201CT, 200C, IRC-50, IRC-84, XT-1007, XT-1009, Amberlite (trademark) cation exchange resin such as XT-1002 (all are trade names manufactured by Organo Corporation); Diaion (trademark) SK-1A, SK-1B, SK-104, SK-110, SK-112, FMK-10, WK-10, WK-11, WK-20, PA-406, PA-408, PA-412, PA- 16, PA-418, PK-208, PK-212, PK-216, PK-218, PK-220, PK-228, UBK08, UBK10, UBK12 (all are trade names manufactured by Mitsubishi Chemical Corporation), etc. And Diaion (trademark) cation exchange resin. As anion exchange resins (weakly basic anion exchange resin, medium basic anion exchange resin, strong basic anion exchange resin), Amberlite (trademark) IRA-400, IRA-400J, IRA-400T , IRA-401, IRA-402BL, IRA-404J, IRA-430, IRA-458, IRA-458, IRA-900, IRA-900J, IRA-904, IRA-910, IRA-910CT, IRA-938, IRA -958, IRA-958RF, IRA-410, IRA-410J, IRA-411, IRA-910, IRA-68, IRA-35, IRA-93 and other Amberlite ™ anion exchange resins; Trademarks) WA-10, WA-11, WA-20, WA-21, WA-30, PA-4 6, PA-408, PA-412, PA-416, PA-418, PA-306, PA-306S, PA-308, PA-312, PA-316, PA-318, PA-318L, PA-318L, PA-408, PA-412, PA-418, HPA25, SA-10A, SA-11A, SA-12A, SA-20A, SA-21A, NSA100, UBA120 (all are trade names manufactured by Mitsubishi Chemical Corporation) Diaion (trademark) type anion exchange resins such as OPTIPORE-XUS40285.00, OPTIPORE-XUS40390.00 (both are trade names manufactured by Dow Chemical Co., Ltd.), etc. Is mentioned. Among the above cation / anion exchange resins, strongly acidic cation exchange resins (for example, amberlite (for example) having a styrene-divinylbenzene copolymer, a phenol formalin resin, etc. as a base and having a sulfonic acid group as an ion exchange group, etc. Trademarks) IR-120, IR-120B, IR-124, 252, 200CT; Diaion (trademark) SK-1A, SK-1B, SK-104, SK-110, SK-112, UBK08, UBK10, UBK12, FMK-10, PK-208, PK-212, PK-216, PK-218, PK-220, PK-228) and other strongly acidic cation exchange resins and styrene-divinylbenzene copolymers , Trimethylammonium group and β-hydroxyethyldimethylammonium group as ion exchange groups Strongly basic anion exchange resins such as Amberlite (trademark) IRA-400, IRA-400J, IRA-402BL, IRA-404J, IRA-410, IRA-410J, IRA-411, IRA-900J , IRA-904, IRA-910CT, IRA-958, IRA-958RF; DIAION ™ SA-10A, SA-11A, SA-12A, SA-10B, FMA-10, NSA100, UBA120, PA-306S , PA-308, PA-312, PA-316, PA-318L, HPA25, SA-20A, SA-21A, PA-408, PA-412, PA-418), etc. Preferably used. Of the platelet activating substances other than the cation / anion exchange resin, collagen and polystyrene beads are preferably used. In addition, the said platelet activation substance may be used independently or may be used with the form of 2 or more types of mixtures.
　術野洗浄液と血小板活性化物質との混合比は、血小板活性化物質が、術野洗浄液中のエンドトキシン不存在下で発光の原因となる物質（例えば、血小板凝固因子）を十分除去できる量混合される限り、特に制限されない。具体的には、血小板活性化物質を、術野洗浄液１ｍＬに対して、好ましくは０．０５～１．０ｇ、より好ましくは０．１～０．５ｇの量で、混合・接触させることが好ましい。上記量であれば、血小板活性化物質により、術野洗浄液中のエンドトキシン不存在下で発光の原因となる物質を十分除去できる。なお、本明細書において、「エンドトキシン不存在下で発光の原因となる物質を除去する」とは、当該物質の存在による偽陽性を予防・防止することを意味する。このため、「エンドトキシン不存在下で発光の原因となる物質の除去」には、当該物質の物理的な除去に加えて、当該物質の不活性化をも包含する。このため、当該物質の除去後の術野洗浄液は、エンドトキシン不存在下で発光の原因となる物質を実質的に含まない形態、およびエンドトキシン不存在下では発光の原因とならない形態で当該物質を含む形態のいずれの形態をも包含する。 The mixing ratio of the surgical field washing solution and the platelet activating substance is such that the platelet activating substance is mixed in an amount sufficient to remove substances that cause luminescence in the absence of endotoxin (eg, platelet clotting factor) in the operative field washing solution. As long as it is not limited. Specifically, it is preferable to mix and contact the platelet activating substance in an amount of 0.05 to 1.0 g, more preferably 0.1 to 0.5 g, with respect to 1 mL of the surgical field washing solution. . With the above amount, the platelet-activating substance can sufficiently remove the substance that causes luminescence in the absence of endotoxin in the surgical field washing solution. In the present specification, “removing a substance that causes luminescence in the absence of endotoxin” means preventing or preventing false positives due to the presence of the substance. For this reason, “removal of a substance causing luminescence in the absence of endotoxin” includes inactivation of the substance in addition to physical removal of the substance. For this reason, the surgical field washing liquid after removal of the substance contains the substance in a form that does not substantially cause luminescence in the absence of endotoxin and that does not cause luminescence in the absence of endotoxin. It includes any form of form.
　また、術野洗浄液と血小板活性化物質との混合・接触条件もまた、血小板活性化物質が、術野洗浄液中のエンドトキシン不存在下で発光の原因となる物質を十分除去できる条件であれば、特に制限されない。具体的には、混合・接触温度については、血小板活性化物質を、術野洗浄液と、好ましくは１５～４５℃で、より好ましくは２０～４０℃で、混合・接触する。また、混合・接触時間については、血小板活性化物質を、術野洗浄液と、好ましくは０．５～１５分間、より好ましくは３～１０分間、混合・接触する。このような条件であれば、血小板活性化物質により、術野洗浄液中のエンドトキシン不存在下で発光の原因となる物質を十分除去できる。この際、術野洗浄液と血小板活性化物質との混合・接触は、攪拌条件下で行ってもまたは静置条件下で行ってもよい。 In addition, the mixing / contacting condition of the surgical field washing solution and the platelet activating substance is also a condition that the platelet activating substance can sufficiently remove the substance causing luminescence in the absence of endotoxin in the operative field washing liquid, There is no particular limitation. Specifically, regarding the mixing / contacting temperature, the platelet activating substance is mixed / contacted with the surgical field washing solution, preferably at 15 to 45 ° C., more preferably at 20 to 40 ° C. Regarding the mixing / contacting time, the platelet activating substance is mixed / contacted with the surgical field washing solution, preferably for 0.5 to 15 minutes, more preferably for 3 to 10 minutes. Under such conditions, the platelet-activating substance can sufficiently remove substances that cause luminescence in the absence of endotoxin in the surgical field washing solution. At this time, the mixing / contact between the surgical field washing solution and the platelet activating substance may be performed under stirring conditions or standing conditions.
　２．工程（ｉｉ）
　本工程は、上記工程（ｉ）で術野洗浄液を血小板活性化物質と混合・接触した後、血小板活性化物質を術野洗浄液から除去して、被測定試料を調製する工程である。 2. Step (ii)
This step is a step of preparing the sample to be measured by mixing and contacting the surgical field washing solution with the platelet activating substance in the step (i) and then removing the platelet activating substance from the surgical field washing solution.
　本工程において、血小板活性化物質の除去方法は、特に制限されず、公知の方法が使用される。例えば、上記工程（ｉ）の混合液を所定時間（例えば、３分間以上）静置して血小板活性化物質を沈殿させた後、その上澄み液を得てもよい。または、上記工程（ｉ）の混合液を、遠心分離、濾過、吸引濾過、多孔質膜濾過、不織布濾過等の公知の分離方法を用いて術野洗浄液と血小板活性化物質とを分離して、その上澄み液や濾液を得てもよい。ここで、このようにして得られた上澄み液／濾液をそのまま被測定試料として使用してもよい。また、上記分離方法は、１種を単独で適用してもあるいは２種以上を適宜組み合わせて適用してもいずれでもよく、血小板活性化物質の除去程度によって適宜選択される。なお、被測定試料は、必要であれば、冷却、希釈等の、処理を施した後、下記工程（ｉｉ）で使用してもよい。ここで、冷却する場合の冷却（被測定試料）温度は、特に制限されないが、通常、－５～３０℃が好ましく、０～２０℃がより好ましい。また、冷却時間もまた、所定の温度に到達できる時間であれば特に制限されないが、通常、３分以上が好ましく、５分以上が好ましく、５～１０分であることが好ましい。また、希釈する場合に使用される希釈剤は、特に制限されず、通常、医療で希釈に使用されるものが同様にして使用できる。具体的には、生理食塩水、滅菌水、リンガー溶液、４．５重量％のブドウ糖等の糖やグリセリン等の細胞に無害な浸透圧保持剤を加えて生体組織細胞と実質的に同じ浸透圧に等張した溶液、脳脊髄手術用洗浄灌流液などが挙げられる。また、希釈倍率は、下記工程（ｉｉｉ）～（ｉｖ）で発光量が測定できる程度であれば特に制限されないが、通常、１，０００～１，０００，０００倍が好ましく、５，０００～１００，０００倍がより好ましい。 In this step, the method for removing the platelet activating substance is not particularly limited, and a known method is used. For example, after allowing the mixed solution of the above step (i) to stand for a predetermined time (for example, 3 minutes or more) to precipitate the platelet activating substance, the supernatant may be obtained. Alternatively, the surgical field washing solution and the platelet activating substance are separated from the mixed solution in the above step (i) using a known separation method such as centrifugation, filtration, suction filtration, porous membrane filtration, and nonwoven fabric filtration. The supernatant or filtrate may be obtained. Here, the supernatant / filtrate thus obtained may be used as it is as a sample to be measured. The separation method may be applied singly or in appropriate combination of two or more, and is appropriately selected depending on the degree of removal of the platelet activating substance. If necessary, the sample to be measured may be used in the following step (ii) after being subjected to treatment such as cooling and dilution. Here, the cooling (sample to be measured) temperature for cooling is not particularly limited, but is usually preferably −5 to 30 ° C., more preferably 0 to 20 ° C. Also, the cooling time is not particularly limited as long as it can reach the predetermined temperature, but it is usually preferably 3 minutes or more, preferably 5 minutes or more, and preferably 5 to 10 minutes. Moreover, the diluent used in the case of dilution is not specifically limited, What is normally used for dilution in medical treatment can be used similarly. Specifically, physiological saline, sterilized water, Ringer's solution, osmotic pressure-retaining agent that is harmless to cells such as 4.5% by weight glucose and sugar, and glycerin are added to substantially the same osmotic pressure as living tissue cells. And an isotonic solution, irrigated perfusate for cerebrospinal surgery, and the like. The dilution factor is not particularly limited as long as the amount of luminescence can be measured in the following steps (iii) to (iv), but is usually preferably 1,000 to 1,000,000 times, and preferably 5,000 to 100 times. 1,000 times is more preferable.
　３．工程（ｉｉｉ）
　本工程は、上記工程（ｉｉ）で得られた被測定試料を、当該被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬（以下、単に「Ｃ因子含有試薬」とも称する）および発光合成基質と反応させて、前記発光合成基質から前記発光基質を遊離させる工程である。なお、本工程において、被測定試料は、工程（ｉｉ）で得られた被測定試料全量を使用する必要はなく、通常、その一部が使用され、その量は、工程（ｉｖ）で発光量が測定できる量であればよく、適宜選択できる。 3. Step (iii)
In this step, the sample to be measured obtained in the above step (ii) is activated by binding to the endotoxin in the sample to be measured (hereinafter also simply referred to as “factor C-containing reagent”). And a reaction with a luminescent synthetic substrate to release the luminescent substrate from the luminescent synthetic substrate. In this step, the sample to be measured does not need to use the entire amount of the sample to be measured obtained in step (ii), and a part of the sample is usually used, and the amount is the amount of luminescence in step (iv). As long as it is an amount that can be measured, it can be appropriately selected.
　本明細書中、「エンドトキシン(endotoxin)」とは、細菌、特にグラム陰性菌の外膜を構成する成分の１つであり、リポ多糖（ＬＰＳ）がＣ因子の活性化に寄与する。エンドトキシンは、細菌、特にグラム陰性菌の表層に外膜の一部として存在する。また、エンドトキシンは、通常、菌の死後、血流中に遊離して存在している。このため、被測定試料中のエンドトキシン濃度を測定することによって、被測定試料中の菌の存在を検出することが可能である。 In the present specification, “endotoxin” is one of the components constituting the outer membrane of bacteria, particularly gram-negative bacteria, and lipopolysaccharide (LPS) contributes to the activation of factor C. Endotoxins are present as part of the outer membrane on the surface of bacteria, particularly gram-negative bacteria. Endotoxin is usually present in the bloodstream after the death of the bacterium. For this reason, it is possible to detect the presence of bacteria in the sample to be measured by measuring the endotoxin concentration in the sample to be measured.
　本工程では、エンドトキシンを含む被測定試料とＣ因子含有試薬（例えば、カブトガニの血球抽出液）とを混合・接触させると、被測定試料中のエンドトキシンにより反応系（例えば、リムルス反応系）が活性化する。より具体的には、Ｃ因子含有試薬がカブトガニの血球抽出液（ＬＡＬ：Limulus Amebocyte Lysate）である場合の、エンドトキシンによる反応系（リムルス反応系）の活性化メカニズムは下記のとおりである。すなわち、エンドトキシンは、Ｃ因子(Factor C)と結合してＣ因子を活性化し、活性化されたＣ因子（活性型Ｃ因子）はＢ因子(Factor B)をさらに活性化する。続いて、活性化されたＢ因子（活性型Ｂ因子）は前凝固酵素(Preclotting Enzyme)を活性化し、凝固酵素(Clotting Enzyme)が生成する。この凝固酵素はコアギュローゲン(Coagulogen)を基質として部分水解し、凝固タンパク質であるコアギュリン(Coagulin)を生成して、ゲル化する[T. Miyata, M. Hiranaga et al., Amino Acid Sequence of the Coagulogen from Limulus polyphemus Hemocytes, The Journal of Biological Chemistry, 259, 8924-8933 (1984)参照]。次に、当該反応系に発光合成基質が存在すると、発光合成基質のＣ末端のペプチド部分（例えば、Ａｒｇ）と発光基質との結合が切断して、発光合成基質から発光基質が遊離する。 In this step, when a sample to be measured containing endotoxin and a factor C-containing reagent (for example, a blood cell extract of horseshoe crab) are mixed and contacted, a reaction system (for example, a Limulus reaction system) is activated by endotoxin in the sample to be measured. Turn into. More specifically, the activation mechanism of the endotoxin reaction system (Limulus reaction system) when the C-factor-containing reagent is a horseshoe crab blood cell extract (LAL: Limulus 下 記 Amebocyte Lysate) is as follows. That is, endotoxin binds to factor C (Factor C) and activates factor C, and activated factor C (active factor C) further activates factor B (Factor B). Subsequently, the activated factor B (active factor B) activates a precoagulase (Preclotting Enzyme) to generate a coagulation enzyme (Clotting Enzyme). This coagulation enzyme is partially hydrolyzed using coagulogen as a substrate to produce coagulin, a coagulation protein, which gels [T. Miyata, M. Hiranaga et al., Amino Acid Sequence of the Coagulogen from Limulus polyphemus Hemocytes, The Journal of Biological Chemistry, 259, 8924-8933 (1984)]. Next, when a luminescent synthetic substrate is present in the reaction system, the bond between the C-terminal peptide portion (eg, Arg) of the luminescent synthetic substrate and the luminescent substrate is cleaved to release the luminescent substrate from the luminescent synthetic substrate.
　本工程で使用されうるＣ因子含有試薬は、エンドトキシンとの反応により凝固酵素が生成されるものであれば、特に制限されない。例えば、従来リムルステストに使用されているカブトガニ血球抽出液(amebocyte lysate)の成分を好適に用いることができる。また、カブトガニ血球抽出液も特に制限されず、例えば、リムルス(Limulus)属、タキプレウス(Tachypleus)属、およびカルシノスコルピウス(Carcinoscorpius)属に属するカブトガニの血球から得られたものが使用できる。また、Ｃ因子含有試薬は、市販品を使用してもよく、例えば、リムルス試薬（ＬＡＬ(Limulus Amebocyte Lysate)試薬）として市販されているもの、エンドトキシン測定用のキットに付属のリムルス試薬（ＬＡＬ試薬）を用いることができる。エンドトキシン測定用のキットとしては、具体的には、Ｋｉｎｅｔｉｃ－ＱＣＬ、ＱＣＬ－１０００、パイロジェント５０００、パイロジェント０６プラス、パイロジェント０３プラス（いずれも、ロンザジャパン株式会社製の商品名）；リムルスＪテストワコー、リムルスＪシングルテストワコー、リムルスＪシングルテストワコー、リムルスＨＳ－Ｊシングルテストワコー、リムルスＥＳ－Ｊテストワコー、リムルスＦシングルテストワコー、リムルスＨＳ－Ｆテストワコー、リムルスＨＳ－Ｆシングルテストワコー、リムルスＥＳ－ＩＩテストワコー、リムルスＥＳ－ＩＩシングルテストワコー、リムルスＨＳ－Ｔシングルテストワコー、リムルスカラーＫＹテストワコー、リムルスカラーＫＹシングルテストワコー、リムルスＰＳシングルテストワコー、エンドトキシン－シングルテストワコー（いずれも、和光純薬工業株式会社製の商品名）などが挙げられる。または、カブトガニ血球抽出物Ｊ凍結乾燥品、カブトガニ血球抽出物ＨＳ－Ｊ凍結乾燥品、カブトガニ血球抽出物Ｆ凍結乾燥品、カブトガニ血球抽出物ＨＳ－Ｆ凍結乾燥品、カブトガニ血球抽出物ＥＳ－ＩＩ凍結乾燥品（いずれも、和光純薬工業株式会社製の商品名）などをエンドトキシン測定用専用試薬として使用してもよい。 The C-factor-containing reagent that can be used in this step is not particularly limited as long as a clotting enzyme is generated by reaction with endotoxin. For example, a component of a horseshoe crab blood cell extract (amebocyte lysate) conventionally used in the Limulus test can be suitably used. Further, the horseshoe crab blood cell extract is not particularly limited, and for example, those obtained from blood cells of horseshoe crab belonging to the genus Limulus, Tachypleus, and Carcinoscorpius can be used. In addition, as the factor C-containing reagent, a commercially available product may be used. For example, a commercially available Limulus reagent (LAL (Limulus Amebocyte Lysate) reagent), a Limulus reagent (LAL reagent included in a kit for measuring endotoxin). ) Can be used. Specific examples of kits for measuring endotoxin include Kinetic-QCL, QCL-1000, Pyrogent 5000, Pyrogent 06 plus, Pyrogent 03 plus (all are trade names of Lonza Japan Co., Ltd.); Limulus J Test Wako, Limulus J Single Test Wako, Limulus J Single Test Wako, Limulus HS-J Single Test Wako, Limulus ES-J Test Wako, Limulus F Single Test Wako, Limulus HS-F Test Wako, Limulus HS-F Single Test Wako , Limulus ES-II Test Wako, Limulus ES-II Single Test Wako, Limulus HS-T Single Test Wako, Limulus Color KY Test Wako, Limulus Color KY Single Test Wako, Limulus PS Guru Test Wako, endotoxin - (none, Wako Pure Chemical Industries, K.K.) Single Test Wako and the like. Alternatively, horseshoe crab blood cell extract J freeze-dried product, horseshoe crab blood cell extract HS-J freeze-dried product, horseshoe crab blood cell extract F freeze-dried product, horseshoe crab blood cell extract HS-F freeze-dried product, horseshoe crab blood cell extract ES-II frozen Dry products (both trade names manufactured by Wako Pure Chemical Industries, Ltd.) and the like may be used as endotoxin measurement dedicated reagents.
　このように、Ｃ因子含有試薬として、従来、リムルステストに使用されているカブトガニ血球抽出成分（例えば、市販のリムルス試薬）を使用した場合には、上述したように、エンドトキシンを含む試料との反応により、活性型Ｃ因子、活性型Ｂ因子および凝固酵素が生成する。これらはいずれもプロテアーゼ活性を有するタンパク質であることが知られている。したがって、発光合成基質としては、活性型Ｃ因子の認識配列を有するもの、活性型Ｂ因子の認識配列を有するもの、および凝固酵素の認識配列を有するものが使用できる。ここで、本発明の方法では、活性型Ｃ因子、活性型Ｂ因子および凝固酵素のうちの１種を単独でプロテアーゼ活性を指標としても、または２種以上をプロテアーゼ活性の指標としてもよいが、操作の簡便性などを考慮すると、１種を使用することが好ましい。このため、それぞれの場合に応じて、指標とする酵素に対応する発色合成基質を適宜選択して用いればよい。 As described above, when a horseshoe crab blood cell extract component (for example, a commercially available Limulus reagent) conventionally used in the Limulus test is used as a factor C-containing reagent, as described above, the reaction with a sample containing endotoxin , Active factor C, active factor B and coagulase are produced. These are all known to be proteins having protease activity. Therefore, as the luminescent synthetic substrate, those having an active factor C recognition sequence, those having an active factor B recognition sequence, and those having a coagulation enzyme recognition sequence can be used. Here, in the method of the present invention, one of active factor C, active factor B and clotting enzyme may be used alone as an indicator of protease activity, or two or more may be used as an indicator of protease activity. In consideration of ease of operation, it is preferable to use one type. For this reason, according to each case, the coloring synthetic substrate corresponding to the enzyme used as an index may be appropriately selected and used.
　または、カブトガニのＣ因子の遺伝子の一部または全部に基づいて合成された組換遺伝子由来のリコンビナントＣ因子を用いることも可能である。このようなリコンビナントＣ因子は、特に制限されず、例えば、市販のパイロジーンｒＦｃ（ロンザジャパン株式会社製の商品名）に付属のリコンビナントＣ因子などを好適に用いることができる。または、リコンビナントＣ因子は、公知の遺伝子操作を用いて、カブトガニＣ因子の遺伝子を発現ベクターに挿入・スクリーニングし、得られた発現ベクターを適当な宿主細胞に形質転換して、当該細胞で組換タンパク質を発現、精製することによって製造してもよい。 Alternatively, it is also possible to use a recombinant C factor derived from a recombinant gene synthesized based on part or all of the C factor gene of horseshoe crab. Such a recombinant C factor is not particularly limited, and for example, a recombinant C factor attached to a commercially available Pilogin rFc (trade name, manufactured by Lonza Japan Co., Ltd.) can be suitably used. Alternatively, recombinant factor C can be recombined by transforming the obtained expression vector into an appropriate host cell by inserting and screening a horseshoe crab factor C gene into an expression vector using known genetic manipulations. It may be produced by expressing and purifying the protein.
　このように、Ｃ因子含有試薬としてリコンビナントＣ因子を用いた場合には、当該試薬中にＢ因子および前凝固酵素は存在しないので、エンドトキシンを含む試料との反応により生成されるのは活性型のリコンビナントＣ因子のみである。ゆえに、この場合には、発光合成基質には活性型Ｃ因子の認識配列を有するものを使用すればよい。 Thus, when recombinant factor C is used as a factor C-containing reagent, since factor B and precoagulase are not present in the reagent, the active form is produced by the reaction with a sample containing endotoxin. Recombinant factor C only. Therefore, in this case, a luminescent synthetic substrate having an active factor C recognition sequence may be used.
　Ｃ因子含有試薬の使用量は、被測定試料中のエンドトキシンと十分結合する量であれば特に制限されない。具体的には、Ｃ因子含有試薬の使用量（タンパク質濃度換算）は、被測定試料１ｍＬに対して、好ましくは１．５～３．５ｍｇ程度、より好ましくは２．０～３．３ｍｇ程度である。なお、市販品を使用する場合には、通常、製造社の指示に従って、適宜選択できる。 The amount of factor C-containing reagent used is not particularly limited as long as it is sufficient to bind to endotoxin in the sample to be measured. Specifically, the amount of factor C-containing reagent used (protein concentration conversion) is preferably about 1.5 to 3.5 mg, more preferably about 2.0 to 3.3 mg, with respect to 1 mL of the sample to be measured. is there. In addition, when using a commercial item, it can select suitably according to a manufacturer's instruction | indication normally.
　また、本工程で使用されうる発光合成基質は、ペプチドに発光基質が結合してなるものであればよい。本明細書において「発光基質」とは、生物発光で反応の基質となって光を発する物質を意味する。発光基質は、特に制限されず、公知の発光基質が使用できる。例えば、ホタルルシフェリン、下記式で表されるアミノルシフェリン、レニラルシフェリン、ウミホタルルシフェリン、ヴァルグリン、渦鞭毛藻類ルシフェリン、バクテリアルシフェリンなどが挙げられる。これらのうち、アミノルシフェリンを使用する場合には、アミノルシフェリン中のアミノ基が、隣接するアミノ酸のカルボキシル基とアミド結合を形成する。 In addition, the luminescent synthetic substrate that can be used in this step may be any substrate formed by binding a luminescent substrate to a peptide. In the present specification, the “luminescent substrate” means a substance that emits light as a reaction substrate by bioluminescence. The luminescent substrate is not particularly limited, and a known luminescent substrate can be used. Examples include firefly luciferin, aminoluciferin represented by the following formula, Renilla luciferin, Cypridina luciferin, vargulin, dinoflagellate luciferin, bacterial luciferin and the like. Among these, when aminoluciferin is used, the amino group in aminoluciferin forms an amide bond with the carboxyl group of the adjacent amino acid.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　または、発光基質は市販品を使用してもよい。具体的には、Ｄ－ルシフェリン(D-Luciferin)、Ｄ－ルシフェリンナトリウム(D-Luciferin Sodium Salt)、Ｄ－ルシフェリンナトリウム一水和物(D-Luciferin Sodium Salt Monohydrate)、Ｄ－ルシフェリンカリウム(D-Luciferin Potassium Salt)、Luciferase-Luciferin, Lyophilized、Luciferase, recombinant（いずれも、和光純薬工業株式会社製の商品名）、Ｄ（－）－ルシフェリン（Photinus pyralis ルシフェリン、ロシュ・ダイアグノスティックス株式会社製）などが挙げられる。 Alternatively, a commercially available luminescent substrate may be used. Specifically, D-Luciferin (D-Luciferin), D-Luciferin sodium (D-Luciferin ル Sodium Salt), D-LuciferinhydrateSodium Salt Monohydrate, D-Luciferin potassium (D-Luciferin) Luciferin Potassium Salt), Luciferase-Luciferin, Lyophilized, Luciferase, recombinant (all trade names made by Wako Pure Chemical Industries, Ltd.), D (-)-Luciferin (Photinus pyralis Luciferin, Roche Diagnostics Inc.) ) And the like.
　アミノルシフェリンと結合するペプチドは、当該ペプチドのＣ末端におけるアミノルシフェリンとのアミド結合が、活性型Ｃ因子、活性型Ｂ因子および凝固酵素のいずれか１種のプロテアーゼ活性により切断されるアミノ酸配列からなるものであればよい。アミノ酸残基数およびアミノ酸配列は、特に限定されない。特異性、合成コスト、取扱い易さ等の観点を考慮すると、アミノ酸残基数は２個～１０個が好ましい。 The peptide that binds to aminoluciferin consists of an amino acid sequence in which the amide bond with aminoluciferin at the C-terminal of the peptide is cleaved by protease activity of any one of active factor C, active factor B, and clotting enzyme. Anything is acceptable. The number of amino acid residues and the amino acid sequence are not particularly limited. In view of specificity, synthesis cost, ease of handling, etc., the number of amino acid residues is preferably 2 to 10.
　具体的には、凝固酵素の認識配列を有するペプチドとしては、以下に制限されないが、Ｇｌｙ－Ｖａｌ－Ｉｌｅ－Ｇｌｙ－Ａｒｇ－、Ｖａｌ－Ｌｅｕ－Ｇｌｙ－Ａｒｇ－、Ｌｅｕ－Ａｒｇ－Ａｒｇ－、Ｉｌｅ－Ｇｌｕ－Ｇｌｙ－Ａｒｇ－、Ｌｅｕ－Ｇｌｙ－Ａｒｇ－、Ｖａｌ－Ｓｅｒ－Ｇｌｙ－Ａｒｇ－、Ｖａｌ－Ｇｌｙ－Ａｒｇ－などが挙げられる。また、活性型Ｃ因子の認識配列を有するペプチドとしては、以下に制限されないが、Ｉｌｅ－Ｇｌｕ－Ａｌａ－Ａｒｇ－、Ｌｅｕ－Ｇｌｙ－Ａｓｎ－Ｌｙｓ－Ｖａｌ－Ｓｅｒ－Ａｒｇ－、Ｉｌｅ－Ｔｈｒ－Ｔｈｒ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－などが挙げられる。活性型Ｂ因子の認識配列を有するペプチドとしては、以下に制限されないが、Ｔｈｒ－Ｔｈｒ－Ｔｈｒ－Ｔｈｒ－Ａｒｇ－、Ｓｅｒ－Ａｒｇ－Ｇｌｎ－Ａｒｇ－Ａｒｇ－などが挙げられる。上記ペプチドは、Ｎ末端が保護基で保護されていてもよい。保護基としては、通常この分野で用いられるものであれば限定されることなく用いることができる。具体的には、例えば、Ｎ－スクシニル基、ｔｅｒｔ－ブトキシカルボニル基、ベンゾイル基、ｐ－トルエンスルホニル基などが挙げられる。 Specifically, the peptide having a recognition sequence for a clotting enzyme is not limited to the following, but includes Gly-Val-Ile-Gly-Arg-, Val-Leu-Gly-Arg-, Leu-Arg-Arg-, Ile. -Glu-Gly-Arg-, Leu-Gly-Arg-, Val-Ser-Gly-Arg-, Val-Gly-Arg- and the like. Further, peptides having a recognition sequence for active factor C are not limited to the following, but include Ile-Glu-Ala-Arg-, Leu-Gly-Asn-Lys-Val-Ser-Arg-, and Ile-Thr-Thr. -Val-Gly-Arg- and the like. Peptides having an active factor B recognition sequence include, but are not limited to, Thr-Thr-Thr-Thr-Arg-, Ser-Arg-Gln-Arg-Arg-, and the like. The peptide may be protected at the N-terminus with a protecting group. Any protecting group that can be used in this field can be used without limitation. Specific examples include N-succinyl group, tert-butoxycarbonyl group, benzoyl group, p-toluenesulfonyl group and the like.
　または、発光合成基質は、例えば、特表２００５－５３０４８５号公報の実施例６及び実施例７に記載の方法を参照することにより合成することができる。また、Ｐｒｏｍｅｇａ社から市販されている「Ｐｒｏｔｅａｓｏｍｅ－ＧｌｏＴＭ　Ａｓｓａｙ　Ｓｙｓｔｅｍｓ」に付属の発光合成基質（ベンゾイル－Ｌｅｕ－Ａｒｇ－Ａｒｇ－アミノルシフェリン）を使用することができる。発光合成基質中に遊離のアミノルシフェリンが含まれる場合は、これを予め除去しておくことが好ましい。発光合成基質から遊離のアミノルシフェリンを除去することにより、バックグラウンド発光を抑制することができる。遊離のアミノルシフェリンを除去する方法としては、例えば、２０ｍＭ　トリシン、８ｍＭ　Ｍｇ２＋、０．１３ｍＭ　ＥＤＴＡの緩衝液（ｐＨ７．８）中、０．８ｍＭ　補酵素Ａ、１．５ｍＭ　ＡＴＰ、２５０μｇ／ｍｌホタルルシフェラーゼおよび９０ｍＭ　ＤＴＴを含む溶液と混合し、室温（２５℃）で１時間～６時間インキュベートする方法が挙げられる。 Alternatively, the luminescent synthetic substrate can be synthesized by referring to the methods described in Example 6 and Example 7 of JP-T-2005-530485, for example. Further, a luminescent synthetic substrate (benzoyl-Leu-Arg-Arg-aminoluciferin) attached to “Proteasome-Glo ™ Assay Systems” commercially available from Promega can be used. When free aminoluciferin is contained in the luminescent synthetic substrate, it is preferable to remove it beforehand. Background luminescence can be suppressed by removing free aminoluciferin from the luminescent synthetic substrate. As a method for removing free aminoluciferin, for example, 0.8 mM coenzyme A, 1.5 mM ATP, 250 μg / ml firefly in 20 mM Tricine, 8 mM Mg 2+ , 0.13 mM EDTA buffer (pH 7.8) can be used. Examples include a method of mixing with a solution containing luciferase and 90 mM DTT and incubating at room temperature (25 ° C.) for 1 to 6 hours.
　または、発光合成基質は、市販品を使用してもよい。具体的には、エンドトキシン用ペプチドルシフェリン（株式会社バイオエネックス製）などがある。 Alternatively, a commercially available product may be used as the luminescent synthetic substrate. Specifically, there is a peptide luciferin for endotoxin (manufactured by Bio-Enex Co., Ltd.).
　発光合成基質の使用量は、十分量の発光基質を遊離できる量であれば特に制限されない。具体的には、発光合成基質の使用量は、好ましくは５０～１００μＭであり、より好ましくは７０～８０μＭである。なお、市販品を使用する場合には、通常、製造社の指示に従って、適宜選択できる。 The amount of the luminescent synthetic substrate used is not particularly limited as long as it can release a sufficient amount of the luminescent substrate. Specifically, the use amount of the luminescent synthetic substrate is preferably 50 to 100 μM, more preferably 70 to 80 μM. In addition, when using a commercial item, it can select suitably according to a manufacturer's instruction | indication normally.
　また、本工程において、被測定試料とＣ因子含有試薬と発光合成基質との反応条件は、これらが反応して発光合成基質から発光基質が遊離する条件であれば特に制限されない。具体的には、反応温度は、好ましくは１５～４５℃であり、より好ましくは２０～４０℃である。また、反応時間は、好ましくは０．５～２０分であり、より好ましくは１～１５分であり、特に好ましくは３～１０分である。また、被測定試料とＣ因子含有試薬と発光合成基質との反応順序は、上記３成分を同時に反応させても、あるいは、被測定試料とＣ因子含有試薬とを反応させた後、さらに発光合成基質を加えるなど、いずれの反応順序でもよいが、後者が好ましい。その際の反応条件もまた、これらが反応して発光合成基質から発光基質が遊離する条件であれば特に制限されない。好ましくは、被測定試料とＣ因子含有試薬とをまず混合して、１５～４５℃であり、より好ましくは２０～４０℃で、０．５～２０分間、より好ましくは１～１５分間、特に好ましくは３～１０分間程度反応（インキュベート）した後、発光合成基質を添加・混合して、１５～４５℃であり、より好ましくは２０～４０℃で、０．５～２０分間、より好ましくは１～１０分間、特に好ましくは３～７分間程度反応（インキュベート）する。 In this step, the reaction conditions of the sample to be measured, the C-factor-containing reagent, and the luminescent synthetic substrate are not particularly limited as long as they react to release the luminescent substrate from the luminescent synthetic substrate. Specifically, the reaction temperature is preferably 15 to 45 ° C, more preferably 20 to 40 ° C. The reaction time is preferably 0.5 to 20 minutes, more preferably 1 to 15 minutes, and particularly preferably 3 to 10 minutes. In addition, the reaction sequence of the sample to be measured, the C-factor-containing reagent, and the luminescent synthesis substrate may be the same as the reaction of the above three components or after the sample to be measured and the C-factor-containing reagent are reacted. Any reaction sequence may be used, such as adding a substrate, but the latter is preferred. The reaction conditions at that time are not particularly limited as long as they react to release the luminescent substrate from the luminescent synthetic substrate. Preferably, the sample to be measured and the C-factor-containing reagent are first mixed and the temperature is 15 to 45 ° C., more preferably 20 to 40 ° C., 0.5 to 20 minutes, more preferably 1 to 15 minutes, particularly Preferably, after reacting (incubating) for about 3 to 10 minutes, a luminescent synthetic substrate is added and mixed, and the temperature is 15 to 45 ° C., more preferably 20 to 40 ° C. and 0.5 to 20 minutes, more preferably The reaction (incubation) is performed for 1 to 10 minutes, particularly preferably about 3 to 7 minutes.
　４．工程（ｉｖ）
　本工程は、上記工程（ｉｉｉ）で遊離した発光基質に発光酵素を作用させて、発光量を測定する。本工程では、発光基質（例えば、アミノルシフェリン、ルシフェリン）に発光酵素（例えば、ルシフェラーゼ）を作用させることにより、光が発生し、その発光量を測定する。 4). Step (iv)
In this step, the amount of luminescence is measured by allowing a luminescent enzyme to act on the luminescent substrate released in the above step (iii). In this step, light is generated by causing a luminescent enzyme (eg, luciferase) to act on a luminescent substrate (eg, aminoluciferin, luciferin), and the amount of luminescence is measured.
　本工程で使用できる発光酵素は、特に制限されず、発光合成基質から遊離した発光基質の発光を触媒して、光を発生させるものであればいずれの発光酵素も使用できる。例えば、発光バクテリア(例えば、Vibrio fischeri)、ホタルイカ(Watasenia scintillans)、ウミホタル、昆虫等の生物の発光器官から精製した天然型ルシフェラーゼ、遺伝子工学的手法により調製した組み換え型ルシフェラーゼ、および天然型ルシフェラーゼのアミノ酸配列中の１または複数のアミノ酸に付加、欠失または置換等の変異を導入した変異型ルシフェラーゼなどを使用することができる。ここで、昆虫由来のルシフェラーゼとしては、北米ホタル(Photinus pyralis；受託番号 M15077)、ゲンジボタル(Luciola cruciata；受託番号 M26194)、ヘイケボタル(Luciola lateralis；受託番号 Z49891, X66919)、ツチボタル(Arachnocampa luminosa)、ヒメボタル(Hotaria parvula；受託番号 L39929)、ヤエヤマヒメボタル(Yaeyama)、マドボタル(Pyrocoelia miyako；受託番号 L39928, Pyrocoelia pygidialis；受託番号 EU826678, Pyrocoelia pectoralis；受託番号 EF155570, Pyrocoelia rufa；受託番号 AY447203)、オバボタル(Lucidina biplagiata)、光コメツキムシ(Pyrearinus termitilluminar；受託番号 AF116843)、鉄道虫(Phrixothrix vivianii；受託番号 AF139644, Phrixothrix hirtus；受託番号 AF139645)、アキマドボタル(Pyrocoelia rufa)、イリオモテボタル(Rhagophthalmus ohbai)などの甲虫由来のルシフェラーゼを好適に用いることができる。これらの甲虫由来のルシフェラーゼのアミノ酸配列およびそれをコードする遺伝子の塩基配列は、公知のデータベース（例えば、ＥＭＢＬ　Ｎｕｃｌｅｏｔｉｄｅ　Ｓｅｑｕｅｎｃｅ　Ｄａｔａｂａｓｅ（ｈｔｔｐ：／／ｗｗｗ．ｅｂｉ．ａｃ．ｕｋ／ｅｍｂｌ／））に登録されており、一例として上記に受託番号を示す。 The luminescent enzyme that can be used in this step is not particularly limited, and any luminescent enzyme can be used as long as it catalyzes the luminescence of the luminescent substrate released from the luminescent synthetic substrate to generate light. For example, natural luciferase purified from luminescent organs of organisms such as luminescent bacteria (e.g., Vibrio fischeri), firefly squid (Watasenia scintillans), sea fireflies, insects, etc., recombinant luciferase prepared by genetic engineering techniques, and amino acids of natural luciferase Mutant luciferase in which mutation such as addition, deletion or substitution is introduced into one or a plurality of amino acids in the sequence can be used. Here, as insect-derived luciferases, fireflies of North America (Photinus pyralis; accession number M15077), Genji firefly (Luciola cruciata; accession number M26194), Heike firefly (Luciola lateralis; accession number 498Z49891, X66919), acupuncture firefly (Arachnocampa lumina) (Hotaria parvula; accession number L39929), Yaeyama Himebotaru (Yaeyama), madbotaru (Pyrocoelia miyako; accession number L39928, Pyrocoelia pygidialis; accession number EU826678, Pyrocoelia pectoralis; accession number EF155570, Pinaco ), Light beetle (Pyrearinus termitilluminar; accession number AF116843), railway insect (Phrixothrix vivianii; accession number AF139644, Phrixothrix hirtus; accession number AF139645), aquimad firefly (Pyrocoelia rufa), baiha Can be used suitably . The amino acid sequences of these beetle-derived luciferases and the base sequences of the genes encoding them are registered in a known database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)). As an example, the accession number is shown above.
　また、ルシフェラーゼは、上記したような天然型（野生型）ルシフェラーゼのアミノ酸配列を有するものに限定されず、発光基質の生物発光を触媒する機能を有する限り、これらのアミノ酸配列と異なるアミノ酸配列を有する変異型ルシフェラーゼであってもよい。野生型のアミノ酸配列と異なるアミノ酸配列としては、例えば、野生型のアミノ酸配列において１もしくは数個のアミノ酸が欠失、挿入、置換または付加されたアミノ酸配列が挙げられる。ここで、「１もしくは数個のアミノ酸が欠失、挿入、置換または付加された」とは、部位特異的突然変異誘発法等の公知の変異ポリペプチド作製法により欠失、挿入、置換もしくは付加できる程度の数（好ましくは１０個以下、より好ましくは７個以下、最も好ましくは５個以下（下限は１個））のアミノ酸が欠失、挿入、置換または付加されることを意味する。 The luciferase is not limited to those having the amino acid sequence of the natural (wild type) luciferase as described above, and has an amino acid sequence different from these amino acid sequences as long as it has a function of catalyzing the bioluminescence of the luminescent substrate. It may be a mutant luciferase. Examples of the amino acid sequence different from the wild type amino acid sequence include an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the wild type amino acid sequence. Here, “one or several amino acids have been deleted, inserted, substituted or added” means deletion, insertion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. It means that as many amino acids as possible (preferably 10 or less, more preferably 7 or less, most preferably 5 or less (lower limit is 1)) are deleted, inserted, substituted or added.
　変異型ルシフェラーゼを使用する場合には、発光強度を増大するように修飾された変異型ルシフェラーゼを用いることが好ましい。このような変異型ルシフェラーゼを用いると、微量のエンドトキシンであっても高感度で測定できる。発光強度を増大するように修飾された変異型ルシフェラーゼは、公知であり、例えば、特開２００９－７７６６０号公報や特開２００７－９７５７７号公報などに記載される。より具体的には、以下の（ア）～（オ）の変異型ルシフェラーゼが挙げられる。 When a mutant luciferase is used, it is preferable to use a mutant luciferase modified so as to increase the luminescence intensity. When such a mutant luciferase is used, even a trace amount of endotoxin can be measured with high sensitivity. Mutant luciferases modified to increase the luminescence intensity are known, and are described in, for example, Japanese Patent Application Laid-Open Nos. 2009-77660 and 2007-97577. More specifically, the following mutant luciferases (A) to (E) are mentioned.
　（ア）野生型北米ホタルルシフェラーゼのアミノ酸配列(GenBank Accession No. M15077)において、４２３位のイソロイシン（Ｉｌｅ）がロイシン（Ｌｅｕ）に置換され、４３６位のアスパラギン酸（Ａｓｐ）がグリシン（Ｇｌｙ）に置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ（野生型北米ホタルルシフェラーゼの発光強度と比較して約１８倍）；
　（イ）野生型北米ホタルルシフェラーゼのアミノ酸配列において、４２３位のイソロイシン（Ｉｌｅ）がロイシン（Ｌｅｕ）に置換され、５３０位のロイシン（Ｌｅｕ）がアルギニン（Ａｒｇ）に置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ（野生型北米ホタルルシフェラーゼの発光強度と比較して約１８倍）；
　（ウ）野生型北米ホタルルシフェラーゼのアミノ酸配列において、４３６位のアスパラギン酸（Ａｓｐ）がグリシン（Ｇｌｙ）に置換され、５３０位のロイシン（Ｌｅｕ）がアルギニン（Ａｒｇ）に置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ（野生型北米ホタルルシフェラーゼの発光強度と比較して約８倍）；
　（エ）野生型北米ホタルルシフェラーゼのアミノ酸配列において、４２３位のイソロイシン（Ｉｌｅ）がロイシン（Ｌｅｕ）に置換され、４３６位のアスパラギン酸（Ａｓｐ）がグリシン（Ｇｌｙ）に置換され、５３０位のロイシン（Ｌｅｕ）がアルギニン（Ａｒｇ）に置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ（野生型北米ホタルルシフェラーゼの発光強度と比較して約２０倍）；および
　（オ）野生型北米ホタルルシフェラーゼのアミノ酸配列において、４２３位のイソロイシン（Ｉｌｅ）がロイシン（Ｌｅｕ）に、５３０位のロイシン（Ｌｅｕ）がアルギニン（Ａｒｇ）に置換され、さらに、４７位のイソロイシン（Ｉｌｅ）がスレオニン（Ｔｈｒ）に、５０位のアスパラギン（Ａｓｎ）がセリン（Ｓｅｒ）に、５９位のメチオニン（Ｍｅｔ）がスレオニン（Ｔｈｒ）に、２５２位のスレオニン（Ｔｈｒ）がセリン（Ｓｅｒ）に置換されたアミノ酸配列からなる変異型ホタルルシフェラーゼ（野生型北米ホタルルシフェラーゼの発光強度と比較して約２１倍）。 (A) In the amino acid sequence of wild type North American firefly luciferase (GenBank Accession No. M15077), isoleucine (Ile) at position 423 was replaced with leucine (Leu), and aspartic acid (Asp) at position 436 was replaced with glycine (Gly). A mutant firefly luciferase consisting of a substituted amino acid sequence (approximately 18 times the luminescence intensity of wild-type North American firefly luciferase);
(A) A mutation comprising an amino acid sequence of wild-type North American firefly luciferase in which isoleucine (Ile) at position 423 is replaced with leucine (Leu) and leucine (Leu) at position 530 is replaced with arginine (Arg) Type firefly luciferase (approximately 18 times the luminescence intensity of wild type North American firefly luciferase);
(C) The amino acid sequence of wild-type North American firefly luciferase consists of an amino acid sequence in which aspartic acid (Asp) at position 436 is replaced with glycine (Gly) and leucine (Leu) at position 530 is replaced with arginine (Arg). Mutant firefly luciferase (approximately 8 times the luminescence intensity of wild-type North American firefly luciferase);
(D) In the amino acid sequence of wild-type North American firefly luciferase, isoleucine (Ile) at position 423 is replaced with leucine (Leu), and aspartic acid (Asp) at position 436 is replaced with glycine (Gly), and leucine at position 530 (Leu) mutated firefly luciferase consisting of an amino acid sequence substituted with arginine (Arg) (approximately 20 times the luminescence intensity of wild-type North American firefly luciferase); and (e) amino acid sequence of wild-type North American firefly luciferase , Leucine (Leu) at position 423 is replaced by leucine (Leu), leucine (Leu) at position 530 is replaced by arginine (Arg), and isoleucine (Ile) at position 47 is replaced by threonine (Thr) at position 50. Asparagine (Asn) in Serine (Ser) Compared to the luminescence intensity of a mutant firefly luciferase consisting of an amino acid sequence in which the methionine (Met) at the position is replaced with threonine (Thr) and the threonine (Thr) at the position 252 is replaced with serine (Ser) About 21 times).
　上記変異型ホタルルシフェラーゼは、野生型ホタルルシフェラーゼの遺伝子を修飾して得られた変異型ホタルルシフェラーゼ遺伝子を、公知の方法により発現ベクターに挿入し、適当な宿主細胞に導入して、組換えタンパク質として発現・精製することにより得ることができる。遺伝子の修飾は、部位特異的変異導入、ランダム変異導入、有機合成等の、当業者に周知の方法により行うことができる。なお、北米ホタルルシフェラーゼ遺伝子（ｃＤＮＡ）の塩基配列は、Accession No. M15077としてデータベース（例えば、EMBL Nucleotide Sequence Database(http://www.ebi.ac.uk/embl/)）に登録されている。また、上記（ア）～（オ）に記載の変異型ホタルルシフェラーゼは、特開２００７－９７５７７号公報の実施例を参照することにより作製することができる。 The above-mentioned mutant firefly luciferase is a recombinant protein obtained by inserting a mutant firefly luciferase gene obtained by modifying a wild-type firefly luciferase gene into an expression vector by a known method and introducing it into an appropriate host cell. It can be obtained by expression and purification. The gene can be modified by methods well known to those skilled in the art, such as site-directed mutagenesis, random mutagenesis, and organic synthesis. The base sequence of the North American firefly luciferase gene (cDNA) is registered in a database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)) as Accession No. M15077. In addition, the mutant firefly luciferase described in (a) to (e) above can be produced by referring to the examples in JP-A-2007-97577.
　また、ゲンジボタル、ヘイケボタル、ヒメボタル、マドボタル、光コメツキムシおよび鉄道虫のルシフェラーゼについても、上記北米ホタルのルシフェラーゼの場合と同様、公知のデータベースに登録されたルシフェラーゼ遺伝子の塩基配列に基づいて、公知の方法により変異型ルシフェラーゼを容易に取得することができる。さらに、上記北米ホタル由来の変異型ホタルルシフェラーゼの置換アミノ酸を参照することによって、他の甲虫由来のルシフェラーゼにおける同等の位置のアミノ酸を置換することにより、当業者は発光強度が増大した変異型ルシフェラーゼを容易に取得することができる。 In addition, as for the luciferase of Genji firefly, Heike firefly, Hime firefly, Japanese firefly, light beetle and railroad insect, as well as the above-mentioned North American firefly luciferase, based on the base sequence of the luciferase gene registered in a known database, by a known method Mutant luciferase can be easily obtained. Furthermore, by referring to the substituted amino acid of the mutant firefly luciferase derived from the above-mentioned North American firefly, the person skilled in the art can identify the mutant luciferase with increased luminescence intensity by substituting the amino acid at the equivalent position in the luciferase derived from other beetles. Can be easily obtained.
　または、発光酵素は、市販品を使用してもよい。具体的には、エンドトキシン用ペプチドルシフェラーゼ（株式会社バイオエネックス製）、ルシフェラーゼ（北米ホタルルシフェラーゼ、ロシュ・ダイアグノスティックス株式会社製）、ルシフェラーゼレポータージーンアッセイキット，高感度（ロシュ・ダイアグノスティックス株式会社製）などがある。 Alternatively, a commercially available luminescent enzyme may be used. Specifically, peptide luciferase for endotoxin (manufactured by Bio-Enex Co., Ltd.), luciferase (North American firefly luciferase, manufactured by Roche Diagnostics Inc.), luciferase reporter gene assay kit, high sensitivity (Roche Diagnostics Inc.) Etc.).
　上記工程（ｉｉｉ）で遊離した発光基質への発光酵素の作用形式は、特に制限されず、公知と同様の方法が使用できる。また、市販品を使用する場合には、製造社の指示に従って反応（作用）および測定を行えばよい。通常、上記工程（ｉｉｉ）で得られた術野洗浄液に、発光酵素（ルシフェラーゼ）を添加して、発光基質に発光酵素を作用させるが、この際、ルシフェリン／ルシフェラーゼの発光反応には、ＡＴＰおよび２価金属イオンが必要である。このため、例えば、ＡＴＰおよびマグネシウムイオンを含む緩衝液にルシフェラーゼを溶解し、このルシフェラーゼ溶液を添加することが好ましい。具体的には、例えば、２０～４０℃、好ましくは約３７℃で反応を行い、ルシフェラーゼ溶液を添加後２秒から１０秒の発光量を計測する方法が挙げられる。ここで、発光量の測定方法は、特に制限されず、公知の方法が使用できる。例えば、発光量の測定には、市販のルミノメーター（発光測定装置）、蛍光光度計を用いることができる。メーカーおよび性能については特に限定されないが、相対光量測定値が広範（例えば、０～１０，０００，０００）に測定できる装置が好ましく使用される。具体的には、キッコーマン食品株式会社製のルミテスターＣ－１１０、キッコーマン食品株式会社製のルミテスターＣ１０００やパーキンエルマー社製のＡＲＶＯ　Ｌｉｇｈｔ等の仕様が好適である。測定は、使用する装置の説明書に従って行えばよい。 The mode of action of the luminescent enzyme on the luminescent substrate released in the above step (iii) is not particularly limited, and a known method can be used. Moreover, when using a commercial item, what is necessary is just to perform reaction (action) and a measurement according to a manufacturer's instruction | indication. Usually, a luminescent enzyme (luciferase) is added to the surgical field washing solution obtained in the above step (iii) to cause the luminescent enzyme to act on the luminescent substrate. At this time, the luminescent reaction of luciferin / luciferase includes ATP and Divalent metal ions are required. For this reason, for example, it is preferable to dissolve luciferase in a buffer containing ATP and magnesium ions and add this luciferase solution. Specifically, for example, there is a method in which the reaction is performed at 20 to 40 ° C., preferably about 37 ° C., and the luminescence amount is measured for 2 to 10 seconds after the luciferase solution is added. Here, the measuring method of the light emission amount is not particularly limited, and a known method can be used. For example, a commercially available luminometer (luminescence measuring device) or fluorometer can be used for measuring the amount of luminescence. The manufacturer and performance are not particularly limited, but an apparatus capable of measuring a relative light quantity measurement value in a wide range (for example, 0 to 10,000,000) is preferably used. Specifically, specifications such as Lumitester C-110 manufactured by Kikkoman Foods Co., Ltd., Lumitester C1000 manufactured by Kikkoman Foods Co., Ltd., and ARVO Light manufactured by PerkinElmer are suitable. The measurement may be performed according to the instruction manual of the device to be used.
　また、本発明では、エンドトキシンの濃度測定用キットを使用してもよい。ここで、エンドトキシンの濃度測定用キットは、Ｃ因子含有試薬、発光合成基質および発光酵素を構成成分として含有し、必要であれば、上記に加えて、必要な試薬や器具等を適宜選択してキットの構成としてもよい。キットを使用することにより、エンドトキシンの濃度（発光量）を簡便かつ迅速に測定することができる。具体的には、ルシフェラーゼＦＭプラス（ＡＴＰ検出キット）（株式会社バイオエネックス製）、細菌検査用試薬ＣＷＢ－ＧＦＰ（株式会社バイオエネックス製）、エンドトキシン用ペプチドルシフェリン（株式会社バイオエネックス製）、ルシフェラーゼレポータージーンアッセイキット，高感度（ロシュ・ダイアグノスティックス株式会社製）など使用できる。 In the present invention, an endotoxin concentration measurement kit may be used. Here, the kit for measuring the concentration of endotoxin contains a C-factor-containing reagent, a luminescent synthetic substrate, and a luminescent enzyme as constituent components. If necessary, in addition to the above, necessary reagents and instruments are appropriately selected. It is good also as a structure of a kit. By using the kit, the endotoxin concentration (the amount of luminescence) can be measured easily and rapidly. Specifically, luciferase FM plus (ATP detection kit) (manufactured by Bio-Enex Co., Ltd.), reagent for bacterial testing CWB-GFP (manufactured by Bio-Enex Co., Ltd.), peptide luciferin for endotoxin (manufactured by Bio-Enex Co., Ltd.), luciferase reporter gene Assay kits, high sensitivity (Roche Diagnostics Inc.) can be used.
　上記工程（ｉ）～（ｉｖ）は、別々の工程として行っても、または連続して行ってもよい。このうち、操作の容易性、取扱い性等を考慮すると、各工程を連続して行うことができるシステム（デバイス）を用いることが好ましい。この際、使用できるシステム（デバイス）は、特に制限されないが、下記システム（デバイス）を使用することが好ましい。すなわち、本発明はまた、血小板活性化物質（６）、前記血小板活性化物質（６）が配置されかつ術野洗浄液と混合可能になるように区画される血小板活性化物質載置部（５）及び前記血小板活性化物質載置部（５）から流出した術野洗浄液及び血小板活性化物質（６）を分離するための血小板活性化物質除去部（４）を有する第１の中空の管状体（２）；前記血小板活性化物質除去部を通過した被測定試料を採取するための試料採取部（３）を有する第２の中空の管状体（２’）；前記被測定試料中の菌を検出するための容器（１１）；前記被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬；前記Ｃ因子含有試薬との反応により発光基質を遊離する発光合成基質；ならびに前記発光基質の発光量を測定するための発光酵素を有する術野洗浄液中の菌の検出システム（デバイス）に関する。なお、本明細書において、検出システム（システム）は、検出デバイス（デバイス）とほとんど同じ意味で使用される。 The above steps (i) to (iv) may be performed as separate steps or continuously. Among these, it is preferable to use a system (device) capable of performing each process continuously in consideration of ease of operation, handling, and the like. At this time, the system (device) that can be used is not particularly limited, but the following system (device) is preferably used. That is, the present invention also provides a platelet activating substance (6), a platelet activating substance placement section (5) that is partitioned so that the platelet activating substance (6) is disposed and can be mixed with the surgical field washing solution. And a first hollow tubular body having a platelet activating substance removing section (4) for separating the surgical field washing liquid and the platelet activating substance (6) flowing out from the platelet activating substance placing section (5) 2); a second hollow tubular body (2 ′) having a sample collection unit (3) for collecting the sample to be measured that has passed through the platelet activating substance removing unit; detecting bacteria in the sample to be measured A container (11) for carrying out the reaction; a factor C-containing reagent activated by binding to endotoxin in the sample to be measured; a luminescent synthetic substrate that liberates a luminescent substrate by reaction with the factor C-containing reagent; and the luminescent substrate For measuring the amount of luminescence of Of bacteria surgical field in the cleaning liquid having a light enzyme for the detection system (device). In this specification, the detection system (system) is used in almost the same meaning as the detection device (device).
　本発明の検出システム（デバイス）の構造は、上記部材を有するものであれば特に制限されないが、例えば、図４に示されるデバイスを用いることによって、上記工程（ｉ）～（ｉｖ）を連続的に行うことができる。以下、当該形態を図４を参照しながら具体的に説明するが、本発明は下記形態に限定されない。 The structure of the detection system (device) of the present invention is not particularly limited as long as it has the above-mentioned members. For example, by using the device shown in FIG. 4, the above steps (i) to (iv) are continuously performed. Can be done. Hereinafter, although the said form is demonstrated concretely, referring FIG. 4, this invention is not limited to the following form.
　図４に示されるシステム（デバイス）は、上部の第１の中空の管状体２および下部の第２の中空の管状体２’を有する（図４Ａ及び図４Ｂ）。ここで、術野洗浄液（図示せず）は、第１の中空の管状体２の上部から注入される。このうち、第１の中空の管状体２の下部には、血小板活性化物質除去部４が設置され、この血小板活性化物質除去部４の上部に血小板活性化物質載置部５が設置される（図４Ａ）。ここで、血小板活性化物質載置部５により、血小板活性化物質６と術野洗浄液とを混合するための区画が設けられる、即ち、血小板活性化物質載置部５は、工程（ｉ）で術野洗浄液と血小板活性化物質６とを混合・接触させるために設置される。このため、血小板活性化物質載置部５は、血小板活性化物質６及び術野洗浄液を通過させない程度の膜状物でありうる。また、後述するが、術野洗浄液と血小板活性化物質６との混合・接触後は、血小板活性化物質載置部５の少なくとも一部に穴を開けることによって、血小板活性化物質６と術野洗浄液との混合物を血小板活性化物質除去部４に移動させ（血小板活性化物質６と術野洗浄液との混合物を血小板活性化物質載置部５から流出させ）、血小板活性化物質除去部４により血小板活性化物質６を術野洗浄液から除去する（工程（ｉｉ））。このため、血小板活性化物質載置部５は、簡単に穴の開く程度の強度を有する膜状物でありうる。この血小板活性化物質載置部５に血小板活性化物質６が配置される。一方、血小板活性化物質除去部４は、工程（ｉｉ）において、以下で詳述するように、血小板活性化物質載置部５から流出した術野洗浄液及び血小板活性化物質を分離する、即ち、術野洗浄液（図示せず）から血小板活性化物質６を除去するために設置される。このため、血小板活性化物質除去部４は、血小板活性化物質６もしくは採取した術野洗浄液中に含まれる組織片を通過させない程度の網状物でありうる。また、第２の中空の管状体２’の底部には、上記血小板活性化物質除去部４を通過した被測定試料を採取するための試料採取部３が配置される（図４Ｂ）。この試料採取部３は、工程（ｉｉｉ）における被測定試料の体積（図１の形態では、５０μＬ）を有する中空の管状構造を有し、上部は開口し、下部はゴムバルブなどで密閉される。このような試料採取部を設けることによって、所定量の被測定試料を正確にかつ再現性よく採取することができる。 The system (device) shown in Fig. 4 has an upper first hollow tubular body 2 and a lower second hollow tubular body 2 '(Figs. 4A and 4B). Here, a surgical field cleaning liquid (not shown) is injected from the upper part of the first hollow tubular body 2. Among these, a platelet activating substance removing unit 4 is installed at the lower part of the first hollow tubular body 2, and a platelet activating substance placing unit 5 is installed above the platelet activating substance removing unit 4. (FIG. 4A). Here, a section for mixing the platelet activating substance 6 and the surgical field washing solution is provided by the platelet activating substance placing part 5, that is, the platelet activating substance placing part 5 is the step (i). It is installed to mix and contact the surgical field washing solution and the platelet activating substance 6. For this reason, the platelet activating substance placement unit 5 may be a film-like material that does not allow the platelet activating substance 6 and the surgical field washing liquid to pass therethrough. In addition, as will be described later, after mixing / contacting the surgical field washing solution and the platelet activating substance 6, a hole is made in at least a part of the platelet activating substance mounting portion 5, thereby allowing the platelet activating substance 6 and the operative field to be formed. The mixture with the washing solution is moved to the platelet activating substance removing unit 4 (the mixture of the platelet activating substance 6 and the surgical field washing solution is allowed to flow out from the platelet activating substance placing unit 5), and the platelet activating substance removing unit 4 The platelet activating substance 6 is removed from the surgical field washing solution (step (ii)). For this reason, the platelet activating substance mounting part 5 can be a film-like object having a strength that can be easily opened. The platelet activating substance 6 is disposed on the platelet activating substance placing portion 5. On the other hand, in step (ii), the platelet activating substance removing unit 4 separates the surgical field washing liquid and the platelet activating substance that have flowed out from the platelet activating substance mounting unit 5 in step (ii), that is, It is installed to remove the platelet activating substance 6 from the surgical field washing solution (not shown). For this reason, the platelet activating substance removing unit 4 may be a reticulated material that does not allow the platelet activating substance 6 or the tissue pieces contained in the collected surgical field washing liquid to pass through. In addition, a sample collection unit 3 for collecting a sample to be measured that has passed through the platelet activating substance removing unit 4 is disposed at the bottom of the second hollow tubular body 2 '(FIG. 4B). The sample collection unit 3 has a hollow tubular structure having the volume of the sample to be measured in the step (iii) (50 μL in the form of FIG. 1), the upper part is opened, and the lower part is sealed with a rubber valve or the like. By providing such a sample collection unit, a predetermined amount of sample to be measured can be collected accurately and with good reproducibility.
　なお、図４では、第１の中空の管状体２は、一体構造を有しているが、複数の部材に分かれていてもよい。例えば、図５に示されるように、第１の中空の管状体２が、血小板活性化物質６が配置される血小板活性化物質載置部５を有する中空の管状体１２、及び血小板活性化物質除去部４を有する中空の管状体１３とから構成されるものであってもよい。 In FIG. 4, the first hollow tubular body 2 has an integral structure, but may be divided into a plurality of members. For example, as shown in FIG. 5, the first hollow tubular body 2 includes a hollow tubular body 12 having a platelet activating substance mounting portion 5 on which the platelet activating substance 6 is disposed, and a platelet activating substance. It may be composed of a hollow tubular body 13 having the removing portion 4.
　ここで、本発明の検出システム（デバイス）は、上記に加えて、血小板活性化物質除去用部材８をさらに有していてもよい（図４Ｃ）。当該血小板活性化物質除去用部材８は、上述したように、血小板活性化物質載置部５に穴を開けるために使用されうる。このように血小板活性化物質除去用部材８で血小板活性化物質載置部５に穴を開けると、術野洗浄液及び血小板活性化物質６が血小板活性化物質除去部４に移動する。この際、血小板活性化物質６や採取した術野洗浄液中に含まれる組織片は血小板活性化物質除去部４上に残り、残りの術野洗浄液のみが下部の第２の中空の管状体２’に移動する。第２の中空の管状体２’中に得られた液が被測定試料となる。 Here, in addition to the above, the detection system (device) of the present invention may further include a platelet activating substance removing member 8 (FIG. 4C). The platelet activating substance removing member 8 can be used to make a hole in the platelet activating substance mounting portion 5 as described above. In this way, when the platelet activating substance mounting member 5 is pierced by the platelet activating substance removing member 8, the surgical field washing solution and the platelet activating substance 6 move to the platelet activating substance removing unit 4. At this time, the platelet activating substance 6 and the tissue pieces contained in the collected surgical field washing liquid remain on the platelet activating substance removing unit 4, and only the remaining surgical field washing liquid is the second hollow tubular body 2 ′ in the lower part. Move to. The liquid obtained in the second hollow tubular body 2 'becomes the sample to be measured.
　本発明の検出システム（デバイス）は、被測定試料中の菌を検出するための容器１１をさらに有する（図４Ｆ）。ここでは、下記で詳述するように、当該容器に上記で得られた被測定試料を入れて、当該被測定試料に対して工程（ｉｉｉ）～（ｉｖ）の操作を行い、発光量を測定する。ゆえに、本発明の検出システム（デバイス）は、図示しないが、当該発光量を測定するための、被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬、前記Ｃ因子含有試薬との反応により発光基質を遊離する発光合成基質、および前記発光基質の発光量を測定するための発光酵素を有する。 The detection system (device) of the present invention further includes a container 11 for detecting bacteria in the sample to be measured (FIG. 4F). Here, as will be described in detail below, the sample to be measured obtained above is placed in the container, and steps (iii) to (iv) are performed on the sample to be measured to measure the amount of luminescence. To do. Therefore, although not shown, the detection system (device) of the present invention is a factor C-containing reagent activated by binding to endotoxin in a sample to be measured, the factor C-containing reagent, A luminescent synthetic substrate that liberates the luminescent substrate by the above reaction, and a luminescent enzyme for measuring the amount of luminescence of the luminescent substrate.
　なお、本発明の検出システム（デバイス）は、儒沖構成を有するものであればよいが、密閉部材９をさらに有していてもよい（図４Ｄ）。この密閉部材９は、試料採取部３の開口部（図４Ｂの上部）を密閉するために使用される。このように、試料採取部３の開口部を密閉部材９で密閉することによって、試料採取部３以外の第２の中空の管状体２’中に術野洗浄液が存在していても、試料採取部３中の術野洗浄液のみを採取することができる。このため、所定量の被測定試料をより正確にかつより再現性よく採取することができる。 Note that the detection system (device) of the present invention only needs to have a Minatooki configuration, but may further include a sealing member 9 (FIG. 4D). The sealing member 9 is used to seal the opening (upper part of FIG. 4B) of the sample collection unit 3. In this way, by sealing the opening of the sample collection unit 3 with the sealing member 9, the sample collection is performed even if the surgical field cleaning liquid is present in the second hollow tubular body 2 ′ other than the sample collection unit 3. Only the surgical field washing liquid in part 3 can be collected. For this reason, a predetermined amount of the sample to be measured can be collected more accurately and with better reproducibility.
　本発明の検出システム（デバイス）は、密閉部材９に代えてまたは密閉部材９に加えて、シリンジ１０をさらに有していてもよい（図４Ｅ）。このシリンジ１０は、試料採取部３の下部に設置されたゴムバルブ（図示せず）に穿刺して、試料採取部３内部の術野洗浄液７’を採取するのに使用できる。 The detection system (device) of the present invention may further include a syringe 10 in place of or in addition to the sealing member 9 (FIG. 4E). The syringe 10 can be used to puncture a rubber valve (not shown) installed at the lower part of the sample collection unit 3 to collect the surgical field washing liquid 7 ′ inside the sample collection unit 3.
　次に、図６を参照しながら、図４の検出システム（デバイス）を用いて本発明の方法を実施する方法の好ましい実施形態を説明する。なお、本発明は、下記形態に限定されるものではない。 Next, a preferred embodiment of a method for carrying out the method of the present invention using the detection system (device) of FIG. 4 will be described with reference to FIG. In addition, this invention is not limited to the following form.
　まず、本発明の検出システム（デバイス）１を用意する（図６Ａ）。工程（ｉ）において、所定量の術野洗浄液７を第１の中空の管状体２に添加する（図６Ｂ）。これにより、術野洗浄液７は、血小板活性化物質載置部５上にある血小板活性化物質６と混合・接触する。血小板活性化物質載置部５で術野洗浄液７を血小板活性化物質６と所定時間混合・接触させた後、血小板活性化物質除去用部材８で血小板活性化物質載置部５に穴を開ける（図６Ｃ）。ここで、血小板活性化物質除去用部材８は、血小板活性化物質載置部５の少なくとも一部に穴を開ける構造を有するものであればよく、図６に示されるような棒状（棒状物が複数設置されてもよい）になっているものに限られず、はさみなどで血小板活性化物質載置部５に穴を開けてもよい。これにより、術野洗浄液７は血小板活性化物質除去部４を通過し、第２の中空の管状体２’に貯溜される。一方、血小板活性化物質６は、血小板活性化物質除去部４に保持される。すなわち、術野洗浄液から血小板活性化物質が除去されて、術野洗浄液７’が得られる。次に、第１の中空の管状体２と第２の中空の管状体２’とを分離し（図６Ｄ）、第２の中空の管状体２’の開口部を、密閉部材９で試料採取部３の開口部を密閉し、試料採取部３の第２に設置されたゴムバルブ（図示せず）にシリンジ１０を穿刺して、試料採取部３内部の術野洗浄液７’を採取する（図６Ｅ）。このように採取された試料が工程（ｉｉ）の被測定試料７”となる。さらに、この被測定試料７”を、容器１１に入れ（図６Ｆ）、この容器１１に、Ｃ因子含有試薬および発光合成基質（図６Ｇ中では、一括して「１２」で示す）（図６Ｇ）を順次添加して、被測定試料７”をＣ因子含有試薬及び発光合成基質と反応させて、発光合成基質から発光基質を遊離させる（工程（ｉｉｉ））。なお、容器１１に、Ｃ因子含有試薬および発光合成基質を予め配置し、この容器１１に被測定試料７”を添加してもよい。ここで遊離した発光基質に発光酵素１３を作用させて（図６Ｈ）、発光量を適当な測定装置（例えば、ルミノメーター）１４で測定する（図６Ｉ）。 First, the detection system (device) 1 of the present invention is prepared (FIG. 6A). In step (i), a predetermined amount of surgical field washing solution 7 is added to the first hollow tubular body 2 (FIG. 6B). As a result, the surgical field washing solution 7 is mixed and contacted with the platelet activating substance 6 on the platelet activating substance placing portion 5. After the surgical field washing solution 7 is mixed and contacted with the platelet activating substance 6 for a predetermined time in the platelet activating substance placing part 5, a hole is made in the platelet activating substance placing part 5 with the platelet activating substance removing member 8. (FIG. 6C). Here, the platelet activating substance removing member 8 only needs to have a structure in which a hole is formed in at least a part of the platelet activating substance placing portion 5, and a rod-like (rod-like object as shown in FIG. However, the platelet-activating substance placement unit 5 may be perforated with scissors or the like. As a result, the surgical field washing solution 7 passes through the platelet activating substance removing unit 4 and is stored in the second hollow tubular body 2 '. On the other hand, the platelet activating substance 6 is held in the platelet activating substance removing unit 4. That is, the platelet activating substance is removed from the surgical field washing liquid, and the surgical field washing liquid 7 'is obtained. Next, the first hollow tubular body 2 and the second hollow tubular body 2 ′ are separated (FIG. 6D), and the opening of the second hollow tubular body 2 ′ is sampled by the sealing member 9. The opening of the part 3 is sealed, and a syringe 10 is punctured into a rubber valve (not shown) installed in the second part of the sample collecting part 3 to collect the surgical field washing liquid 7 ′ inside the sample collecting part 3 (FIG. 6E). The sample collected in this way becomes the sample 7 ″ to be measured in step (ii). Further, this sample 7 ″ is put in the container 11 (FIG. 6F), and the container 11 contains the C-factor-containing reagent and A luminescent synthetic substrate (indicated collectively as “12” in FIG. 6G) (FIG. 6G) is sequentially added, and the sample 7 ″ to be measured is reacted with a C-factor-containing reagent and the luminescent synthetic substrate to obtain a luminescent synthetic substrate. (Step (iii)) The C-factor-containing reagent and the luminescent synthetic substrate may be placed in advance in the container 11 and the sample 7 ″ to be measured may be added to the container 11. Here, the luminescent enzyme 13 is allowed to act on the released luminescent substrate (FIG. 6H), and the amount of luminescence is measured with an appropriate measuring device (for example, luminometer) 14 (FIG. 6I).
　５．工程（ｖ）
　本工程では、上記工程（ｉｖ）で得られた発光量を、基準値と比較する。上述したように、術野洗浄液中に特定数以下の菌数が存在していても、自助回復力によってその菌を死滅させることが期待できる。このため、「基準値」は、当該自助回復力によってその菌を死滅させることが期待できる菌数（エンドトキシン濃度）に対応する発光量となりうる。また、自助回復力によってその菌を死滅させることが期待できる菌数は、術式や術式、病気の重篤度、患者の体重などによって異なり、最終的には術者の判断にゆだねられる。通常、自助回復力によってその菌を死滅させることが期待できる菌数は、３０ＣＦＵ／ｍＬ以下（下限＝０ＣＦＵ／ｍＬ）であり、好ましくは２０ＣＦＵ／ｍＬ以下（下限＝０ＣＦＵ／ｍＬ）である。 5. Step (v)
In this step, the light emission amount obtained in the step (iv) is compared with a reference value. As described above, even if the number of bacteria below a specific number is present in the surgical field washing solution, it can be expected that the bacteria will be killed by the self-help recovery force. Therefore, the “reference value” can be the amount of luminescence corresponding to the number of bacteria (endotoxin concentration) that can be expected to kill the bacteria by the self-help recovery ability. In addition, the number of bacteria that can be expected to be killed by self-help resilience varies depending on the procedure, procedure, severity of illness, patient weight, etc., and is ultimately left to the judgment of the operator. Usually, the number of bacteria that can be expected to be killed by self-recovery is 30 CFU / mL or less (lower limit = 0 CFU / mL), preferably 20 CFU / mL or less (lower limit = 0 CFU / mL).
　以下では、自助回復力によってその菌を死滅させることが期待できる菌数が２０ＣＦＵ／ｍＬ以下（下限＝０ＣＦＵ／ｍＬ）である場合の、基準値を設定する方法について具体的に説明するが、本発明は下記方法に限定されるものではない。 In the following, a method for setting a reference value when the number of bacteria that can be expected to be killed by self-help resilience is 20 CFU / mL or less (lower limit = 0 CFU / mL) will be specifically described. The invention is not limited to the following method.
　５－１．方法（１）
　本方法は、菌数が既知な試料を用いる場合の基準値の測定方法を説明する。 5-1. Method (1)
This method describes a method for measuring a reference value when a sample with a known number of bacteria is used.
　すなわち、既知の菌数（本形態の場合には、２０ＣＦＵ／ｍＬ）を含む標準物質を調製し、上記工程（ｉｉｉ）及び（ｉｖ）において、被測定試料の代わりに当該物質を使用する以外は、上記工程（ｉｉｉ）及び（ｉｖ）と同様の操作を行い、得られた発光量を基準値とする。すなわち、基準値は、既知の菌数を含む標準物質を前記被測定試料の代わりに用いて前記工程（ｉｉｉ）および（ｉｖ）を行うことによって得られる。なお、既知の菌数を含む標準物質は、例えば、Bio Ball（登録商標）シリーズ（シスメックス株式会社製）が使用できる。例えば、Bio Ball（登録商標）SingleShot 30 Escherichia coli NCTC12923（シスメックス株式会社製）は、Escherichia coli NCTC12923を約３０ＣＦＵ／個（２８～３３ＣＦＵ／個）含むように作製されたボール状の製品（微生物定量試験用標準菌株）である。このため、当該製品を７５μＬの生理食塩水または蒸留水に添加し、均一に混合した混合液を５０μＬ採取することによって、２０ＣＦＵ／ｍＬの菌数を含む標準物質を調製できる。 That is, a standard substance containing a known number of bacteria (in the case of this embodiment, 20 CFU / mL) is prepared, and in the above steps (iii) and (iv), the substance is used in place of the sample to be measured. The same operations as in the above steps (iii) and (iv) are performed, and the obtained light emission amount is set as a reference value. That is, the reference value is obtained by performing the steps (iii) and (iv) using a standard substance containing a known number of bacteria instead of the sample to be measured. In addition, as a standard substance containing a known number of bacteria, for example, Bio Ball (registered trademark) series (manufactured by Sysmex Corporation) can be used. For example, Bio Ball (registered trademark) SingleShot 30 Escherichia coli NCTC12923 (manufactured by Sysmex Corporation) is a ball-shaped product (microbe quantitative test) prepared to contain about 30 CFU / piece (28-33 CFU / piece) of Escherichia coli NCTC12923 Standard strain). Therefore, a standard substance containing 20 CFU / mL can be prepared by adding the product to 75 μL of physiological saline or distilled water and collecting 50 μL of the uniformly mixed solution.
　５－２．方法（２）
　本方法は、菌数が不明な試料を用いる場合の基準値の測定方法を説明する。 5-2. Method (2)
This method explains a method for measuring a reference value when a sample with an unknown number of bacteria is used.
　すなわち、まず、検出すべき菌（手術部位感染（ＳＳＩ）の原因と推定される細菌）を、一般的な培養方法によって、培養する。得られた培養液を遠心分離など公知の方法を用いて、菌体を分離する。この菌体数（菌濃度）が未知の菌試料に、蒸留水　１ｍＬを加え、濁度（ＯＤ６００）を測定する。別途、同じ種に属し、菌数が既知の試料（参考試料）を用いて、２０ＣＦＵ／ｍＬ菌液の濁度（ＯＤ６００）を測定する。この結果をもとに、既知の２０ＣＦＵ／ｍＬ菌液の濁度と同じになるように、上記菌試料を蒸留水で希釈する。なお、本方法（２）において参考試料としては、上記方法（１）に記載されるBio Ball（登録商標）シリーズ（シスメックス株式会社製）が同様にして使用できる。このようにして得られた２０ＣＦＵ／ｍＬ菌液を、上記工程（ｉｉｉ）及び（ｉｖ）において、被測定試料の代わりに使用して、上記工程（ｉｉｉ）及び（ｉｖ）と同様の操作を行うことによって、得られた発光量を基準値とすることができる。 That is, first, the bacteria to be detected (bacteria presumed to cause surgical site infection (SSI)) are cultured by a general culture method. The cells are separated from the obtained culture solution using a known method such as centrifugation. 1 mL of distilled water is added to the bacterial sample whose cell number (bacterial concentration) is unknown, and turbidity (OD600) is measured. Separately, the turbidity (OD600) of 20 CFU / mL bacterial solution is measured using a sample (reference sample) belonging to the same species and having a known number of bacteria. Based on this result, the bacterial sample is diluted with distilled water so that the turbidity of the known 20 CFU / mL bacterial solution is the same. In addition, as a reference sample in this method (2), Bio Ball (registered trademark) series (manufactured by Sysmex Corporation) described in the above method (1) can be used in the same manner. The 20 CFU / mL bacterial solution thus obtained is used in place of the sample to be measured in the above steps (iii) and (iv), and the same operations as in the above steps (iii) and (iv) are performed. Thus, the obtained light emission amount can be used as a reference value.
　このようにして、上記工程（ｉｖ）で得られた発光量を、上記で得られた基準値と比較し、その結果、上記工程（ｉｖ）で得られた発光量が基準値以下である場合には、工程（ｉ）で採取した術野洗浄液中には自助回復力によってその菌を死滅させることが期待できる程度の菌数しか存在しないこととなる。このため、このような場合には、図２の「Ｙｅｓ」で示されるように、術野洗浄を終了して、術野洗浄液を吸引等によって術野から除去・廃液し、表皮を縫合して、手術を終了する。一方、上記工程（ｉｖ）で得られた発光量が基準値を超える場合には、工程（ｉ）で採取した術野洗浄液中には自助回復力によってはその菌を死滅できないほどの菌が存在している可能性がある。このため、このような場合には、図２の「Ｎｏ」で示されるように、再度術野洗浄を行い、得られた術野洗浄液について、再度工程（ｉ）～（ｉｖ）を繰り返し、上記工程（ｉｖ）で得られた発光量が基準値以下になるまで、術野洗浄を繰り返す。このような場合であっても、工程（ｉ）～（ｉｖ）が短時間で行えるため、患者への負担が少なくすむ。また、このような方法を使用することによって、術中にＳＳＩの原因と推定される細菌の存在を確認できるため、手術部位感染症を抑制・防止できる。 In this way, when the light emission amount obtained in the step (iv) is compared with the reference value obtained above, as a result, the light emission amount obtained in the step (iv) is less than the reference value. In the operative field washing solution collected in step (i), there are only the number of bacteria that can be expected to be killed by self-help recovery. Therefore, in such a case, as shown by “Yes” in FIG. 2, the surgical field cleaning is finished, the surgical field cleaning liquid is removed from the surgical field by suction or the like, and the epidermis is sutured. End the surgery. On the other hand, when the amount of luminescence obtained in the above step (iv) exceeds the reference value, the operative field washing solution collected in step (i) contains bacteria that cannot be killed by self-help recovery ability. There is a possibility. Therefore, in such a case, as indicated by “No” in FIG. 2, the surgical field cleaning is performed again, and the steps (i) to (iv) are repeated again for the obtained surgical field cleaning liquid. The operative field cleaning is repeated until the amount of luminescence obtained in step (iv) is below the reference value. Even in such a case, steps (i) to (iv) can be performed in a short time, so that the burden on the patient is reduced. Also, by using such a method, it is possible to confirm the presence of bacteria presumed to be the cause of SSI during the operation, and thus it is possible to suppress / prevent surgical site infections.
　または、下記参考例１でも示すが、菌を含まない手術部位感染について工程（ｉ）～（ｉｖ）を行っても、微量であるが発光量（バックグランドの発光量）を検出することがある。このような場合には、予めバックグランドの発光量を測定しておいて、図３および下記に示されるような計算式で得られた値（計算結果）に応じて、術野洗浄終了の可否を判断してもよい。すなわち、上記工程（ｉｖ）で得られた発光量、上記工程（ｖ）で得られた基準値およびバックグランドの発光量を下記計算式にあてはめて、得られた結果（計算結果）が１以下である場合には、工程（ｉ）で採取した術野洗浄液中には自助回復力によってその菌を死滅させることが期待できる程度の菌数しか存在しないこととなる。このため、このような場合には、図３の「Ｙｅｓ」で示されるように、術野洗浄を終了して、術野洗浄液を吸引等によって術野から除去・廃液し、縫合して、手術を終了する。一方、上記工程（ｉｖ）で得られた発光量、上記工程（ｖ）で得られた基準値およびバックグランドの発光量を下記計算式にあてはめて、得られた結果（計算結果）が１を超える場合には、工程（ｉ）で採取した術野洗浄液中には自助回復力によってはその菌を死滅できないほどの菌が存在している可能性がある。このため、このような場合には、図３の「Ｎｏ」で示されるように、再度術野洗浄を行い、得られた術野洗浄液について、再度工程（ｉ）～（ｉｖ）を繰り返し、算出される計算結果が１以下になるまで、術野洗浄を繰り返す。このような場合であっても、工程（ｉ）～（ｉｖ）が短時間で行えるため、患者への負担が少なくすむ。また、このような方法を使用することによって、術中にＳＳＩの原因と推定される細菌の存在を確認できるため、手術部位感染症を抑制・防止できる。 Alternatively, as shown in Reference Example 1 below, even if steps (i) to (iv) are performed for surgical site infections that do not contain bacteria, the amount of luminescence (background luminescence amount) may be detected in a small amount. . In such a case, the amount of luminescence in the background is measured in advance, and whether or not the surgical field cleaning is completed according to the value (calculation result) obtained by the calculation formula as shown in FIG. May be judged. That is, the light emission amount obtained in the step (iv), the reference value obtained in the step (v) and the light emission amount of the background are applied to the following calculation formula, and the result (calculation result) is 1 or less. In this case, the number of bacteria that can be expected to be killed by self-help recovery force is present in the surgical field washing liquid collected in step (i). Therefore, in such a case, as shown by “Yes” in FIG. 3, the surgical field cleaning is finished, the surgical field cleaning liquid is removed from the surgical field by suction or the like, drained, sutured, and operated. Exit. On the other hand, the light emission amount obtained in the step (iv), the reference value obtained in the step (v) and the light emission amount in the background are applied to the following calculation formula, and the obtained result (calculation result) is 1. In the case of exceeding, there may be bacteria in the surgical field washing liquid collected in step (i) so that the bacteria cannot be killed by self-help recovery ability. Therefore, in such a case, as indicated by “No” in FIG. 3, the surgical field cleaning is performed again, and the obtained surgical field cleaning solution is repeated to repeat steps (i) to (iv). The surgical field cleaning is repeated until the calculated result is 1 or less. Even in such a case, steps (i) to (iv) can be performed in a short time, so that the burden on the patient is reduced. Also, by using such a method, it is possible to confirm the presence of bacteria presumed to be the cause of SSI during the operation, and thus it is possible to suppress / prevent surgical site infections.
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　なお、バックグランドの発光量は、例えば、以下のようにして得られる。すなわち、患者の血液を採取して、当該血液を生理食塩水で適当（例えば、１０，０００～１，０００，０００倍）に希釈したサンプルをバックグランドサンプルとし、当該サンプルについて、上記工程（ｉ）～（ｉｖ）を行い、得られた発光量を「バックグランドの発光量」とする。 Note that the amount of light emitted from the background can be obtained, for example, as follows. That is, a blood sample of a patient is collected, and a sample obtained by diluting the blood appropriately with physiological saline (eg, 10,000 to 1,000,000 times) is used as a background sample. ) To (iv), and the obtained light emission amount is defined as “background light emission amount”.
　本発明の方法によると、血液成分などの生体試料を含む術野洗浄液であっても、術野洗浄液の菌の存在が自助回復力によって死滅できるか否かを確認することができる。上記利点に加えて、本発明の方法によると、６０分以内という短時間での検出が可能である。このため、患者への負担を有意に軽減することができ、また、術中に菌を検出できるため、表皮を縫合する前に細菌が検出でき、ＳＳＩを有効に予防することができる。また、本発明の方法による検出時間は、好ましくは３５分以内、より好ましくは３０分以内、さらにより好ましくは２０分以内、さらに好ましくは１５分以内、特に好ましくは１２分以内に短縮できる。 According to the method of the present invention, it is possible to confirm whether or not the presence of bacteria in the surgical field washing liquid can be killed by self-help recovery force even in the surgical field washing liquid containing a biological sample such as a blood component. In addition to the above advantages, the method of the present invention allows detection in a short time of 60 minutes or less. For this reason, the burden on the patient can be significantly reduced, and since bacteria can be detected during the operation, bacteria can be detected before the epidermis is sutured, and SSI can be effectively prevented. The detection time according to the method of the present invention can be shortened to preferably within 35 minutes, more preferably within 30 minutes, even more preferably within 20 minutes, even more preferably within 15 minutes, and particularly preferably within 12 minutes.
　本発明の効果を、以下の実施例および比較例を用いて説明する。ただし、本発明の技術的範囲が以下の実施例のみに制限されるわけではない。 The effect of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.
　参考例１
　ヘパリン処理を施したヒト全血を、生理食塩水で１０，０００倍に希釈し、希釈サンプルを調製した。この希釈サンプルを用いて、全培養した大腸菌(Escherichia coli)菌液を、菌濃度が１３ＣＦＵ／ｍＬ、１３０ＣＦＵ／ｍＬ及び１１７３ＣＦＵ／ｍＬになるように、それぞれ調製した。ここで、各サンプルを、それぞれ、試料Ａ－１３（菌濃度が１３ＣＦＵ／ｍＬ）、試料Ａ－１３０（菌濃度が１３０ＣＦＵ／ｍＬ）、および試料Ａ－１１７３（菌濃度が１１７３ＣＦＵ／ｍＬ）と称する。また、菌を添加しない希釈サンプル自体を、コントロールとして使用し、これを試料Ｃ－１と称する。 Reference example 1
Heparinized human whole blood was diluted 10,000 times with physiological saline to prepare a diluted sample. Using this diluted sample, Escherichia coli liquid cultures that had been cultured in total were prepared so that the bacterial concentrations would be 13 CFU / mL, 130 CFU / mL, and 1173 CFU / mL, respectively. Here, the respective samples are referred to as sample A-13 (bacterial concentration is 13 CFU / mL), sample A-130 (bacterial concentration is 130 CFU / mL), and sample A-1173 (bacterial concentration is 1173 CFU / mL). . In addition, a diluted sample itself to which no bacteria are added is used as a control, and this is referred to as sample C-1.
　また、別途、ヘパリン処理を施したヒト全血に血小板活性化物質としての陽イオン交換樹脂アンバーライトＩＲ－１２０を０．２ｇ／ｍＬとなるように添加し、３７℃の静置条件下で１０分間、混合・接触させた後、上澄み液を回収し、５分以上氷冷した。さらに、氷冷後のヒト全血試料を生理食塩水で１０，０００倍に希釈し、得られた被測定試料をＣ－２と称する。なお、上記操作において、氷冷は省略できる。 Separately, cation exchange resin Amberlite IR-120 as a platelet activating substance was added to human whole blood treated with heparin so as to have a concentration of 0.2 g / mL, and the mixture was incubated at 37 ° C. for 10 minutes. After mixing and contacting for 5 minutes, the supernatant was recovered and ice-cooled for 5 minutes or more. Further, the human whole blood sample after ice cooling is diluted 10,000 times with physiological saline, and the obtained sample to be measured is referred to as C-2. In the above operation, ice cooling can be omitted.
　上記試料Ａ－１３、Ａ－１３０、Ａ－１１７３、Ｃ－１及びＣ－２を、それぞれ、５０μＬずつ採取し、これを、それぞれ、エンドトキシンキット（商品名：エンドトキシン－シングルテストワコー（比濁時間分析法）、和光純薬株式会社製）に添付されるリムルス試薬が５０μＬ入った生物発光測定専用試験管（ルミチューブ、キッコーマン株式会社）に添加し、３７℃、１０分間、インキュベーターで加温した。 Samples A-13, A-130, A-1173, C-1 and C-2 were sampled in 50 μL each, and each sample was taken as an endotoxin kit (trade name: endotoxin-single test wako (turbidimetric time). Analysis method), a Limulus reagent attached to Wako Pure Chemical Industries, Ltd.) was added to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) containing 50 μL, and heated in an incubator at 37 ° C. for 10 minutes. .
　その後、１ｍＭ　ＭｇＳＯ４及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　８．０）に溶解した７５μＭ　エンドトキシン用ペプチドルシフェリン（Ｂｚ－ＬＧＲ－Ｌｕｃ溶液、株式会社バイオエネックス製）（発光合成基質）５０μＬを添加し、３７℃、５分間、インキュベーターで加温した。加温後、反応液に１ｍＭ　ＭｇＳＯ４　及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　７．５）に溶解したエンドトキシン用ルシフェラーゼ（ルシフェラーゼＦＭ、株式会社バイオエネックス製）（発光酵素）５０μＬを添加した後、１ｍＭ　ＭｇＳＯ４及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　８．０）に溶解した１０－５Ｍ　ＡＴＰ溶液５０μＬを添加し、数回チューブをタッピングし攪拌させ、ルミノメーター（商品名：ルミテスター　Ｃ－１１０、キッコーマン食品株式会社製）にて発光量（ＲＬＵ）を測定した。なお、エンドトキシン用ペプチドルシフェリン（Ｂｚ－ＬＧＲ－Ｌｕｃ溶液、株式会社バイオエネックス製）（発光合成基質）、エンドトキシン用ルシフェラーゼ（ルシフェラーゼＦＭ、株式会社バイオエネックス製）（発光酵素）、およびＡＴＰ溶液は、予め、３７℃のインキュベーターで加温した。また、各実験は、Ｎ＝３で行った。 Thereafter, 50 μL of 75 μM peptide luciferin for endotoxin (Bz-LGR-Luc solution, manufactured by Bio-Enex Co., Ltd.) (luminescent synthetic substrate) dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added. The mixture was added and warmed in an incubator at 37 ° C. for 5 minutes. After warming, 50 μL of endotoxin luciferase (Luciferase FM, manufactured by Bio-Enex Inc.) (luminescent enzyme) dissolved in 50 mM Tris-Cl (pH 7.5) containing 1 mM MgSO 4 and 10% trehalose was added to the reaction solution. Thereafter, 50 μL of 10 −5 M ATP solution dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added, and the tube was tapped and stirred several times. A luminometer (trade name: Luminescence tester C-110 (manufactured by Kikkoman Foods Co., Ltd.) was used to measure the luminescence (RLU). In addition, the peptide luciferin for endotoxin (Bz-LGR-Luc solution, manufactured by Bio-Enex Co., Ltd.) (luminescent synthesis substrate), luciferase for endotoxin (luciferase FM, manufactured by Bio-Enex Co., Ltd.) (luminescent enzyme), and ATP solution are prepared in advance. And warmed in a 37 ° C. incubator. Each experiment was performed with N = 3.
　結果を、下記表１および図７に示す。 The results are shown in Table 1 below and FIG.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　上記表１および図７から明らかなように、血小板活性化物質で処理しない試料Ｃ－１は、菌濃度が１３ＣＦＵ／ｍＬの試料Ａ－１３とほぼ同等の発光量を示す。これから、生体試料（血液成分）を含む試料は、血小板活性化物質で処理しない場合には、菌（エンドトキシン）が存在しない場合であっても発光してしまうのに対して、血小板活性化物質で処理することによって、菌（エンドトキシン）が存在しない試料Ｃ－２の発光量を有意に低減して、菌濃度が１３ＣＦＵ／ｍＬの試料Ａ－１３の発光量と有意に識別できる。 As can be seen from Table 1 and FIG. 7, sample C-1 not treated with the platelet activating substance shows a light emission level substantially equivalent to that of sample A-13 having a bacterial concentration of 13 CFU / mL. From this, samples containing biological samples (blood components) emit light even when bacteria (endotoxin) are not present when they are not treated with platelet-activating substances, whereas platelet-activating substances By processing, the amount of luminescence of sample C-2 in which no bacteria (endotoxin) is present is significantly reduced, and can be significantly distinguished from the amount of luminescence of sample A-13 having a bacterium concentration of 13 CFU / mL.
　実施例１
　ヘパリン処理を施したヒト全血をヒト全血試料として調製した。 Example 1
Human whole blood treated with heparin was prepared as a human whole blood sample.
　次に、このヒト全血試料に血小板活性化物質としての陽イオン交換樹脂アンバーライトＩＲ－１２０を０．２ｇ／ｍＬとなるように添加し、３７℃の静置条件下で、それぞれ、１、３、５、１０分間、混合・接触させた。所定時間混合・接触させた後、上澄み液を回収し、５分以上氷冷した。さらに、氷冷後のヒト全血試料を生理食塩水で１０，０００倍に希釈することによって、被測定試料Ｂ－１（１分間混合・接触）、Ｂ－３（３分間混合・接触）、Ｂ－５（５分間混合・接触）、Ｂ－１０（１０分間混合・接触）を調製した。また、血小板活性化物質と接触させていない希釈サンプルを、コントロールとして使用し、これを試料Ｃ－３と称する。なお、上記操作において、氷冷は省略できる。 Next, a cation exchange resin Amberlite IR-120 as a platelet activating substance was added to the human whole blood sample so as to have a concentration of 0.2 g / mL. Mix and contact for 3, 5, 10 minutes. After mixing and contacting for a predetermined time, the supernatant was collected and cooled on ice for 5 minutes or more. Furthermore, by diluting the human whole blood sample after ice cooling 10,000 times with physiological saline, sample B-1 to be measured (mixing / contact for 1 minute), B-3 (mixing / contact for 3 minutes), B-5 (mixing / contact for 5 minutes) and B-10 (mixing / contact for 10 minutes) were prepared. A diluted sample that has not been brought into contact with the platelet activating substance is used as a control, and this is referred to as sample C-3. In the above operation, ice cooling can be omitted.
　上記試料Ｂ－１、Ｂ－３、Ｂ－５、Ｂ－１０及びＣ－３を、それぞれ、５０μＬずつ採取し、これを、それぞれ、エンドトキシンキット（商品名：エンドトキシン－シングルテストワコー（比濁時間分析法）、和光純薬株式会社製）に添付されるリムルス試薬が５０μＬ入った生物発光測定専用試験管（ルミチューブ、キッコーマン株式会社）に添加し、３７℃、１０分間、インキュベーターで加温した。 Samples B-1, B-3, B-5, B-10, and C-3 were each collected at 50 μL, and each sample was taken as an endotoxin kit (trade name: endotoxin-single test Wako (turbidimetric time). Analysis method), a Limulus reagent attached to Wako Pure Chemical Industries, Ltd.) was added to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) containing 50 μL, and heated in an incubator at 37 ° C. for 10 minutes. .
　その後、１ｍＭ　ＭｇＳＯ４及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　８．０）に溶解した７５μＭ　エンドトキシン用ペプチドルシフェリン（Ｂｚ－ＬＧＲ－Ｌｕｃ溶液、株式会社バイオエネックス製）（発光合成基質）５０μＬを添加し、３７℃、５分間、インキュベーターで加温した。加温後、反応液に１ｍＭ　ＭｇＳＯ４　及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　７．５）に溶解したエンドトキシン用ルシフェラーゼ（ルシフェラーゼＦＭ、株式会社バイオエネックス製）（発光酵素）５０μＬを添加した後、１ｍＭ　ＭｇＳＯ４及び１０％トレハロースを含む５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ　８．０）に溶解した１０－５Ｍ　ＡＴＰ溶液５０μＬを添加し、数回チューブをタッピングし攪拌させ、ルミノメーター（商品名：ルミテスター Ｃ－１１０、キッコーマン食品株式会社製）にて発光量（ＲＬＵ）を測定した。なお、エンドトキシン用ペプチドルシフェリン（Ｂｚ－ＬＧＲ－Ｌｕｃ溶液、株式会社バイオエネックス製）（発光合成基質）、エンドトキシン用ルシフェラーゼ（ルシフェラーゼＦＭ、株式会社バイオエネックス製）（発光酵素）、およびＡＴＰ溶液は、予め、３７℃のインキュベーターで加温した。また、各実験は、Ｎ＝３で行った。 Thereafter, 50 μL of 75 μM peptide luciferin for endotoxin (Bz-LGR-Luc solution, manufactured by Bio-Enex Co., Ltd.) (luminescent synthetic substrate) dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added. The mixture was added and warmed in an incubator at 37 ° C. for 5 minutes. After warming, 50 μL of endotoxin luciferase (Luciferase FM, manufactured by Bio-Enex Inc.) (luminescent enzyme) dissolved in 50 mM Tris-Cl (pH 7.5) containing 1 mM MgSO 4 and 10% trehalose was added to the reaction solution. Thereafter, 50 μL of 10 −5 M ATP solution dissolved in 50 mM Tris-Cl (pH 8.0) containing 1 mM MgSO 4 and 10% trehalose was added, and the tube was tapped and stirred several times. A luminometer (trade name: Luminescence tester C-110 (manufactured by Kikkoman Foods Co., Ltd.) was used to measure the luminescence (RLU). In addition, the peptide luciferin for endotoxin (Bz-LGR-Luc solution, manufactured by Bio-Enex Co., Ltd.) (luminescent synthesis substrate), luciferase for endotoxin (luciferase FM, manufactured by Bio-Enex Co., Ltd.) (luminescent enzyme), and ATP solution are prepared in advance. And warmed in a 37 ° C. incubator. Each experiment was performed with N = 3.
　結果を、下記表２および図８に示す。 The results are shown in Table 2 below and FIG.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　上記表２および図８から明らかなように、血小板活性化物質で処理した試料Ｂ－１、Ｂ－３、Ｂ－５、Ｂ－１０は、血小板活性化物質で処理しない試料Ｃ－３の発光量に比して、発光量を低減できる。この結果から、血小板活性化物質で処理しない試料Ｃ－３では、血液中に含まれる成分が試薬と反応して、発光反応が進行してしまうが、血液を血小板活性化物質で処理することによって、この血液中に含まれる成分を減少させ、その結果菌が存在しない際のバックグランドの発光量を減少させることできることが考察される。 As apparent from Table 2 and FIG. 8, samples B-1, B-3, B-5, and B-10 treated with the platelet activating substance are luminescence of sample C-3 not treated with the platelet activating substance. The amount of light emission can be reduced compared to the amount. From this result, in sample C-3 which is not treated with the platelet activating substance, the components contained in the blood react with the reagent and the luminescence reaction proceeds, but by treating the blood with the platelet activating substance, It is considered that the amount of light contained in the blood can be reduced, and as a result, the amount of light emitted from the background when bacteria are not present can be reduced.
　また、特に血小板活性化物質との接触・混合時間を３分以上とすることによって、血小板活性化物質で処理しない試料Ｃ－３の発光量と有意に識別できることが分かる。 In addition, it can be seen that, particularly by setting the contact / mixing time with the platelet activating substance to 3 minutes or more, it can be significantly distinguished from the luminescence amount of the sample C-3 not treated with the platelet activating substance.
 
　さらに、本出願は、２０１１年８月１１日に出願された日本特許出願番号２０１１－１７６３３６号に基づいており、その開示内容は、参照され、全体として、組み入れられている。
Furthermore, this application is based on Japanese Patent Application No. 2011-176336 filed on August 11, 2011, the disclosure of which is incorporated by reference in its entirety.
　　１…デバイス、
　　２…上部の中空の管状体、
　　２’…下部の中空の管状体、
　　３…試料採取部、
　　４…血小板活性化物質除去部、
　　５…血小板活性化物質載置部、
　　６…血小板活性化物質、
　　７，７’…術野洗浄液、
　　７”…被測定試料、
　　８…血小板活性化物質除去用部材、
　　９…密閉部材、
　　１０…シリンジ、
　　１１…容器、
　　１２…Ｃ因子含有試薬および発光合成基質、
　　１３…発光酵素、
　　１４…測定装置。 1 ... Device,
2 ... An upper hollow tubular body,
2 '... lower hollow tubular body,
3 ... Sample collection part,
4 ... Platelet activating substance removing unit,
5 ... Platelet-activating substance placement part,
6 ... Platelet activating substance,
7, 7 '... operative field cleaning solution,
7 "... sample to be measured,
8 ... Platelet activating substance removing member,
9 ... Sealing member,
10 ... syringe,
11 ... container,
12: Factor C-containing reagent and luminescent synthetic substrate,
13 ... Luminescent enzyme,
14: Measuring device.

Claims (5)

  1. 　（ｉ）術野洗浄液を血小板活性化物質と混合・接触させ；
    　（ｉｉ）前記血小板活性化物質を前記術野洗浄液から除去して、被測定試料を得；
    　（ｉｉｉ）前記被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬および発光合成基質を反応させて、前記発光合成基質から前記発光基質を遊離させ；
    　（ｉｖ）前記工程（ｉｉｉ）で遊離した発光基質に発光酵素を作用させて、発光量を測定し；さらに
    　（ｖ）前記工程（ｉｖ）で得られた発光量を、基準値と比較する、
    ことを有する術野洗浄液中の菌の検出方法。     (I) mixing and contacting the surgical field washing solution with the platelet activating substance;
    (Ii) removing the platelet activating substance from the surgical field washing solution to obtain a sample to be measured;
    (Iii) reacting a C-factor-containing reagent activated by binding to endotoxin in the sample to be measured and a luminescent synthetic substrate to release the luminescent substrate from the luminescent synthetic substrate;
    (Iv) A luminescent enzyme is allowed to act on the luminescent substrate released in the step (iii) to measure the luminescence amount; and (v) the luminescence amount obtained in the step (iv) is compared with a reference value.
    A method for detecting bacteria in an operative field washing solution.
  2. 　前記基準値は、既知の菌数を含む標準物質を前記被測定試料の代わりに用いて前記工程（ｉｉｉ）および（ｉｖ）を行うことによって得られる、請求項１に記載の方法。 The method according to claim 1, wherein the reference value is obtained by performing the steps (iii) and (iv) using a standard substance containing a known number of bacteria instead of the sample to be measured.
  3. 　前記血小板活性化物質は、陽イオンがＣａ２＋、Ｃｕ２＋、Ｚｎ２＋、Ｍｇ２＋、Ｋ＋、ＮＨ４ ＋、Ｎａ＋、またはＨ＋である陽イオン交換樹脂；陰イオンがＳＯ４ ２－、Ｉ－、ＮＯ３ －、ＣｒＯ４ ２－、Ｂｒ－、Ｃｌ－、ＯＨ－、またはＦ－である陰イオン交換樹脂；コラーゲン、フィブリン、ＡＤＰ、アラキドン酸、トロンビン、セロトニン、プロタミン、カルシウム塩、ＲＧＤペプチド、若しくは凝固因子から構成されるまたは前記いずれかの物質で被覆されるビーズ；珪砂、結晶シリカ、珪藻土、ガラス微粉末、カオリン、ベントナイト；ならびにポリスチレン、ポリオレフィン、ポリイミド、ポリカーボネート、ポリアリレート、ポリエステル、ポリアクリロニトリル、ポリメタクリル酸メチル、およびポリアクリル酸からなる群より選択される少なくとも一種である、請求項１または２に記載の方法。 The platelet activating substance is a cation exchange resin whose cation is Ca 2+ , Cu 2+ , Zn 2+ , Mg 2+ , K + , NH 4 + , Na + , or H + ; an anion is SO 4 2− , I -, NO 3 -, CrO 4 2-, Br -, Cl -, OH -, or F - a is an anion exchange resin; collagen, fibrin, ADP, arachidonic acid, thrombin, serotonin, protamine, calcium, RGD Beads composed of peptides or coagulation factors or coated with any of the above substances; silica sand, crystalline silica, diatomaceous earth, glass fine powder, kaolin, bentonite; and polystyrene, polyolefin, polyimide, polycarbonate, polyarylate, polyester, Polyacrylonitrile, polymethyl methacrylate, and poly The method according to claim 1, wherein the method is at least one selected from the group consisting of reacrylic acid.
  4. 　前記工程（ｉ）において、術野洗浄液を血小板活性化物質と、３～１０分間、混合・接触する、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein in the step (i), the surgical field washing solution is mixed and contacted with the platelet activating substance for 3 to 10 minutes.
  5. 　血小板活性化物質、前記血小板活性化物質が配置されかつ術野洗浄液と混合可能になるように区画される血小板活性化物質載置部及び前記血小板活性化物質載置部から流出した術野洗浄液及び血小板活性化物質を分離するための血小板活性化物質除去部を有する第１の中空の管状体；
    　前記血小板活性化物質除去部を通過した被測定試料を採取するための試料採取部を有する第２の中空の管状体；
    　前記被測定試料中の菌を検出するための容器；
    　前記被測定試料中のエンドトキシンとの結合により活性化されるＣ因子含有試薬；
    　前記Ｃ因子含有試薬との反応により発光基質を遊離する発光合成基質；ならびに
    　前記発光基質の発光量を測定するための発光酵素
    を有する術野洗浄液中の菌の検出システム。     A platelet activating substance, a platelet activating substance placement section where the platelet activating substance is disposed and partitioned so as to be mixed with the operative field washing liquid, and a surgical field washing liquid that has flowed out of the platelet activating substance placement section; A first hollow tubular body having a platelet activating substance removing unit for separating the platelet activating substance;
    A second hollow tubular body having a sample collection unit for collecting a sample to be measured that has passed through the platelet activating substance removing unit;
    A container for detecting bacteria in the sample to be measured;
    A factor C-containing reagent activated by binding to endotoxin in the sample to be measured;
    A luminescent synthetic substrate that liberates a luminescent substrate by reaction with the factor C-containing reagent; and a system for detecting bacteria in an operative field washing solution having a luminescent enzyme for measuring the amount of luminescence of the luminescent substrate.
PCT/JP2012/069584 2011-08-11 2012-08-01 Method and system for detecting bacteria in surgical cleaning solution WO2013021894A1 (en)

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