WO2013021894A1 - Procédé et système de détection de bactéries dans une solution de nettoyage chirurgicale - Google Patents

Procédé et système de détection de bactéries dans une solution de nettoyage chirurgicale 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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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

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  • 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

L'invention porte sur un procédé et un système, dont chacun peut détecter la présence de bactéries dans une solution de nettoyage chirurgicale contenant un échantillon biologique, d'une manière simple. La présente invention concerne un procédé de détection de bactéries dans une solution de nettoyage chirurgicale, comprenant les étapes consistant à : (i) mélanger la solution de nettoyage chirurgicale avec une substance d'activation plaquettaire pour permettre à la solution de nettoyage chirurgicale et la substance d'activation plaquettaire de venir en contact l'une avec l'autre ; (ii) retirer la substance d'activation plaquettaire de la solution de nettoyage chirurgicale pour produire un échantillon d'intérêt ; (iii) faire réagir l'échantillon d'intérêt avec un réactif à teneur en facteur C qui peut être activé par la liaison à une endotoxine contenue dans l'échantillon d'intérêt et un substrat synthétique luminescent pour produire la libération du substrat luminescent à partir du substrat synthétique luminescent ; (iv) permettre à une enzyme luminescente d'agir sur le substrat luminescent qui a été libéré dans l'étape mentionnée ci-dessus (iii) et mesurer la quantité de lumière émise ; et (v) comparer la quantité de lumière émise obtenue dans l'étape mentionnée ci-dessus (iv) avec une valeur de référence.
PCT/JP2012/069584 2011-08-11 2012-08-01 Procédé et système de détection de bactéries dans une solution de nettoyage chirurgicale WO2013021894A1 (fr)

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JP2019149971A (ja) * 2018-03-02 2019-09-12 キッコーマン株式会社 ジンジパインを産生する微生物又はジンジバリス菌を検出する方法及びキット
JP7083663B2 (ja) 2018-03-02 2022-06-13 キッコーマン株式会社 ジンジパインを産生する微生物又はジンジバリス菌を検出する方法及びキット

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