Detection reagents using enzyme cycling reactions

Abstract

To provide a reagent that can be used for drug susceptibility testing of a target or for determining the presence or absence of a target substance, which is versatile, has a simple testing method, and can quickly obtain results, which has not been achieved with conventional technology.SOLUTION: The present invention relates to e.g., a reagent for detecting a target component within a cell in a sample, which comprises a cell treatment agent for removing the target component from the cell, and an enzyme cycling reaction composition for performing an enzyme cycling reaction on the target component, and to a method for determining the sensitivity of microbial cells to a cell treatment agent using the reagent, or for determining the presence or absence of microbial cells in a sample.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a detection reagent using an enzyme cycling reaction, a detection method using the same, and the like.

Background Art

[0002] In the treatment of infectious diseases caused by pathogens such as bacteria, in hospitals and the like, both a test for identifying the pathogen infecting a patient and a test for determining an antibacterial drug or the like effective against the pathogen are performed. In recent years, regarding the identification of bacteria, an extremely rapid bacterial species identification method using MALDI-TOF / MS, which is a mass spectrometer, has been put into practical use (Patent Document 1). On the other hand, regarding the determination of an antibacterial drug effective against bacteria, there is still no rapid test method. For this reason, antibacterial drugs are often administered based on experience, but when the antibacterial drug is inappropriate for the drug susceptibility of bacteria, it has been reported that a significantly high mortality rate is shown (Non-Patent Document 1). In particular, in septic patients, the survival rate when an appropriate antibacterial drug is administered within 2 hours after the onset is about 70%, and it has been shown that the survival rate decreases by 7.6% every hour thereafter (Non-Patent Document 2).

[0003] As general drug susceptibility test methods, the microbroth dilution method and the disk diffusion method are widely used. The microbroth dilution method is a method of inoculating a certain amount of bacteria into a medium containing antibacterial drugs at different concentrations and observing up to what concentration the bacteria grow to determine the susceptibility to the drug. The disk diffusion method is a method of spreading bacteria on an agar medium, placing a disk containing an antibacterial drug thereon, culturing, and determining the susceptibility to the drug from the size of the growth inhibition zone formed around the disk (Non-Patent Document 3). These methods require about 18 hours by the conventional method until the determination, and even when an expensive automatic determination device is introduced, it takes 4 to 16 hours. The time required for the drug susceptibility test has become an obstacle to selecting an appropriate antibacterial drug and starting treatment early.

[0004] As an attempt to speed up drug susceptibility tests, for example, Patent Document 2 describes a method of culturing bacteria to be measured in a chamber containing an antibacterial drug and a chamber not containing it, and measuring the dissolved oxygen concentration to determine drug susceptibility from the difference in the activity (oxygen consumption) of the bacteria in the two chambers. Patent Document 3 also describes a method of observing with an electron microscope or the like and determining drug susceptibility based on the external appearance changes of bacteria caused by an antibacterial drug. There are also methods such as a method of determining the susceptibility of bacteria to a drug by measuring the amount of ATP derived from dead bacteria in a culture solution containing the drug and bacteria as luminescence using luciferase (Patent Document 4). However, even when using these techniques, it takes more than 2 hours to make a determination, which hinders the rapid selection of antibacterial drugs. In addition, large-scale equipment and special devices are essential, so the facilities that can introduce these test methods are limited.

[0005] In Non-Patent Documents 4 and 5, systems using NAD(P)H in bacteria have been reported. This utilizes the reduction action of NAD(P)H in living bacteria to reduce an electron mediator outside the bacterial cell membrane. An electron mediator such as menadione is added to the reaction solution, and the active oxygen generated in the reaction via the electron mediator is subjected to colorimetric measurement by using a chemiluminescence method such as the luminol reaction (Non-Patent Document 4) or a water-soluble formazan dye (Non-Patent Document 5). In this method, it is necessary to use the bacterial solution as the test object after sufficiently reacting, such as culturing the bacteria in the presence of an antibacterial drug for 2 hours or more, and it takes 2 hours or more to make a determination.

[0006] Detecting microorganisms in the environment and food is very important for evaluating the safety and quality of the environment and food. Although various methods are used for such inspections, a common method is the culturing method. In this method, a sample collected from food or the environment is inoculated into a culture medium, and the presence or absence of colony formation and the turbidity of the liquid culture medium are visually confirmed. However, in this method, it is necessary to wait for the microorganisms to grow until the colonies or the turbidity of the culture medium can be visually confirmed, which may take several days (Patent Document 5). In addition, in Patent Document 5, a method of combining image analysis and deep learning image processing software has also been developed for detecting microorganisms from images, but facilities that can introduce these test methods are limited because large-scale equipment and special devices are essential.

[0007] As described above, in the prior art, neither the speed of obtaining results nor the simplicity of the test method is sufficient in the drug sensitivity test of microorganisms or in the determination of the presence or absence of microorganisms. It was necessary to use large-scale equipment and special devices, and the versatility was not sufficient in reality.

[0008] We have already established a technique for measuring components such as ammonia in a sample using an enzyme cycling reaction (Patent Document 6). There is no report of conducting a drug sensitivity test of microorganisms or determining the presence or absence of microorganisms using such a technique. The enzyme cycling reaction is also disclosed in Patent Documents 7 to 9 and the like.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

[0010] [Non - Patent Document 1] Paul M, et al., “Systematic Review and Meta - Analysis of the Efficacy of Appropriate Empiric Antibiotic Therapy for Sepsis”, Antimicrob. Agents Chemother., 2010, 54, 4851 - 4863 [Non - Patent Document 2] Kumar A, et al., “Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock”, Crit Care Med., 2006, 34, 1589 - 1596 [Non - Patent Document 3] Ryuichiro Hori et al., “Fundamentals of Microbiological Examinations (2) Antimicrobial Susceptibility Testing”, Japanese Journal of Clinical Anesthesiology, Vol.37 No.5, 684 - 686 [Non - Patent Document 4] Shiro Y, et al., “Menadione-Catalyzed O2- Production by Escherichia coli Cells: Application of Rapid Chemiluminescent Assay to Antimicrobial Susceptibility Testing”, Microbiol. Immunol., 2001,45(5),333-34 [Non-Patent Document 5] Tadayuki Tsukaya, “Development of a microbial detection method using water-soluble tetrazolium salt WST and its application to the food field”, Journal of the Japan Society for Food Science and Technology 62 (7), 321-327, 2015 [Summary of the Invention] [Problems to be Solved by the Invention]

[0011] The problem of the present invention is to provide a reagent that is versatile, has a simple test method, can obtain results quickly, and can be used for drug susceptibility testing of a target or determination of the presence or absence, which could not be achieved by the prior art. [Means for Solving the Problems]

[0012] In order to solve the above problems, the present inventors have conducted intensive research and found that by taking out the components in cells extracellularly and detecting such components using an enzyme cycling reaction, it is possible to determine the drug susceptibility and presence or absence of the cells in a very simple and extremely rapid manner. As a result of further research, the present invention has been completed.

[0013] Therefore, the present invention relates to the following. [1] A reagent for detecting a component to be detected in cells in a sample, the reagent comprising a cell treatment agent for taking out the component to be detected in cells extracellularly and a composition for an enzyme cycling reaction for performing an enzyme cycling reaction on the component to be detected. [2] The reagent according to [1] above, wherein the cell is a microbial cell of bacteria or fungi. [3] The reagent according to [2] above, for determining the sensitivity of microbial cells to a cell treatment agent or for determining the presence or absence of microbial cells in a sample. [4] The reagent according to any one of [1] to [3] above, wherein the cell treatment agent is one or more selected from the group consisting of antibacterial agents, benzalkonium chloride, cetyltrimethylammonium bromide, and cetyltrimethylammonium chloride. [5] The reagent according to [4] above, wherein the antibacterial agent is one or more selected from the group consisting of cephem antibacterial agents, penem antibacterial agents, quinolone antibacterial agents, penicillin antibacterial agents, glycopeptide antibacterial agents, and aminoglycoside antibacterial agents. [6] The reagent according to any one of [1] to [5] above, wherein the component to be detected is one or more selected from the group consisting of NAD+, NADP+, NADH, and NADPH.

[0014] [7] The reagent according to any one of [1] to [6] above, wherein the composition for enzyme cycling reaction contains glucose, glucose dehydrogenase, a substrate for diaphorase, and diaphorase. [8] The reagent according to [7] above, wherein the substrate for diaphorase is a tetrazolium salt. [9] The reagent according to [8] above, wherein the tetrazolium salt is WST-8, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, or tetranitroblue tetrazolium.

[10] The reagent according to any one of [1] to [9] above, further containing a nonionic surfactant.

[11] The reagent according to any one of [2] to

[10] above, for determining the sensitivity of microbial cells to a cell treatment agent, further containing a cell culture medium component that does not contain the component to be detected or contains only a concentration of 5 μg / L or less.

[12] The reagent according to

[11] above, wherein the cell culture medium component is peptone and chloride.

[0015]

[13] A method for detecting a component to be detected inside cells in a sample, which comprises detecting, in the reagent according to any one of [1] to

[12] above, the component to be detected that has been released outside the cells by a cell treatment agent using an enzyme cycling reaction.

[14] The method according to

[13] above, which is a method for determining the susceptibility of bacteria to a cell treatment agent or for determining the presence or absence of bacteria in a sample, wherein the cell is a bacterial cell.

[15] A method for detecting a component to be detected inside cells in a sample, which is a method for determining the susceptibility of bacteria to a bactericidal component, and which comprises detecting, in the reagent according to

[11] or

[12] above, the component to be detected that has been released outside the cells by a cell treatment agent using an enzyme cycling reaction. [Effect of the Invention]

[0016] By using the reagent of the present invention, it is possible to simply and rapidly detect that cells such as bacteria and fungi have been destroyed or lysed by a cell treatment agent. By using the reagent of the present invention, it is possible to determine the drug susceptibility of microorganisms such as bacteria and the presence or absence of microorganisms in an extremely short time of about 1 hour and in a simple manner, which was not possible with the prior art. In addition, large-scale equipment and special devices are not required, and the versatility is high. When the cell treatment agent is an antibacterial agent or the like, the reagent of the present invention can be used in a test method for determining whether microorganisms such as bacteria and fungi to be tested are susceptible to a certain antibacterial agent. When the cell treatment agent is a bactericidal agent or the like, the reagent of the present invention can be used in a test method for investigating whether microorganisms such as bacteria and fungi to be tested are present.

[0017] Regarding the drug susceptibility test, when using conventional methods such as the micro - liquid dilution method or the disk diffusion method, it takes 18 hours until determination using the conventional method, and even when introducing an expensive automated machine, 4 - 16 hours are required. On the other hand, when using the reagent of the present invention, the drug susceptibility can be determined in about 1 hour. Further, for the reagent of the present invention, after inoculating bacteria or the like to be a sample into the reaction solution, since the presence or absence of color development of the reaction solution can be visually determined after incubation, complicated or special operations are not required, and large - scale devices and special equipment are also not required. Since the reagent of the present invention can determine that the target bacteria are sensitive to the drug in about 1 hour by simple operation without using large - scale devices or special equipment, it can be expected to contribute to the rapid selection of antibacterial drugs.

[0018] Regarding the detection of microorganisms, in the general culture method, a culture time of 1 - 3 days is required until colonies large enough to be visually recognized are formed. Also, there is a fluorescence detection method in which a liquid sample or the like is filtered through a membrane filter, this membrane filter is attached to a plate medium and cultured, and the resulting minute micro - colonies are fluorescently stained and fluorescence observation is performed. Even when introducing such an expensive device, 3 - 24 hours are required. On the other hand, when using the reagent of the present invention, the presence or absence of microorganisms can be determined in about 1 hour. In addition, for the reagent of the present invention, after inoculating a sample into the reaction solution, since the change in the color tone of the reaction solution can be visually determined, special operations and special devices are not required.

[0019] Also, for example, in the detection using the conventionally performed luminol reaction, since the luminescence by luminol ends in several tens of seconds, if a cell treatment agent is put in the reagent in advance, the luminescence ends before the timing when the drug susceptibility can be determined, and detection (luminescence detection) cannot be performed. In contrast, in the present invention, since the dye (color tone) generated by the enzyme cycling reaction accumulates without attenuation, a cell treatment agent can be put in the reagent in advance, there is no need to add additional reagents or the like at the time of detection, and detection can be performed by a simple operation. Since the reagent of the present invention can determine the presence or absence of microorganisms in a sample in about one hour with a simple operation without using a special device, for example, it is expected to shorten the time from after production to shipment in a soft drink factory and contribute to hygiene management in a small-scale factory.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0021] The detection principle using the reagent of the present invention will be described below using bacteria as an example with reference to FIG. 1. In the cells of living bacteria, there is a coenzyme (NAD+) essential for life activities. When an antibacterial agent acts on a certain bacterium, an antibacterial agent effective against that bacterium can rupture the bacterial cells and kill them. The NAD+ released from the cells of the ruptured bacteria is converted into a formazan dye and detected by utilizing an enzyme cycling reaction (FIG. 1).

[0022] Specifically, the released NAD+ is converted into NADH by glucose and glucose dehydrogenase (hereinafter referred to as GlucDH). NADH becomes NAD+ again in the presence of a substrate such as a tetrazolium salt and diaphorase, and a formazan dye is produced at that time. Furthermore, NAD+ becomes NADH again by the enzyme reaction of GlucDH, and the reaction in which NADH also returns to NAD+ by the enzyme reaction of diaphorase is repeated, so that the formazan dye accumulates and the color of the reaction solution changes. Therefore, when the color of the reaction solution changes, it can be determined that the bacteria are sensitive (effective) to the antibacterial agent, and if the color of the medium does not change, it can be determined that the bacteria are resistant (ineffective) to the antibacterial agent.

[0023] Also, by using a bactericide (such as benzalkonium chloride) that destroys the cell membranes of most microorganisms instead of an antibacterial agent, it becomes a reagent for detecting microorganisms. Specifically, when microorganisms are present in the target sample, the microorganisms are ruptured by the bactericide, and formazan dye is generated from the released NAD+ in the same manner as described above, changing the color of the reaction solution. Therefore, when the color of the reaction solution changes, it can be determined that microorganisms are present in the sample, and when the color of the reaction solution does not change, it can be determined that there are no microorganisms.

[0024] In one aspect, the present invention relates to a reagent for detecting a component to be detected inside cells in a sample, the reagent including a cell treatment agent for releasing the component to be detected inside the cell to the outside of the cell, and a composition for an enzyme cycling reaction for performing an enzyme cycling reaction on the component to be detected. The reagent of the present invention contains no component to be detected or contains only a concentration of 5 μg / L or less. At this concentration, since no change in color tone occurs in a 1-hour enzyme cycling reaction, even if it contains the component to be detected, it does not show a false positive result and thus does not affect the detection result. Here, the enzyme cycling reaction, taking an example, is a repetitive reaction by the action of a first enzyme that catalyzes a reaction that consumes NAD+ and a first substrate to produce NADH and an oxidation product of the first substrate when NAD+ is supplied, and a second enzyme that catalyzes a reaction that consumes NADH and a second substrate to produce NAD+ and a reduction product of the second substrate.

[0025] An example of the enzyme cycling reaction is shown in FIG. 1. In FIG. 1, NAD+ is supplied and the enzyme cycling reaction is performed. The first substrate is glucose, the oxidation product of the first substrate is gluconolactone, and the first enzyme is glucose dehydrogenase (GlucDH) (left side of FIG. 1). Also, the second substrate is a substrate such as a tetrazolium salt, the reduction product of the second substrate is a dye such as formazan dye, and the second enzyme is diaphorase (right side of FIG. 1).

[0026] The cells in the present invention are not particularly limited as long as they are cells that can utilize the detection principle using an enzymatic cycling reaction, and can be animal cells, plant cells, or microbial cells such as bacteria and fungi. The reagent of the present invention can determine the drug sensitivity and the presence or absence in a sample for these cells. As the cells, microbial cells such as bacterial cells and fungal cells are preferred.

[0027] Examples of bacteria include Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Morganella morganii, Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Enterobacter aerogenes, (Enterobacter asburiae), Pseudomonas aeruginosa, Pseudomonas putida, Stenotrophomonas multophilia, Acinetobacter baumannii, Aeromonas hydrophila, Moraxella catarrhalis, Hafnia alvei, Sphingomonas paucimobilis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus epidermidis, Enterococcus fecalis, Enterococcus feacium, Enterococcus casseliflavus (EnterococcusExamples include casseliflavus).

[0028] Examples of fungi include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Candida aruris, Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Cryptococcus neoformans, etc.

[0029] The cell treatment agent of the present invention is not particularly limited as long as it can destroy or lyse the target cells, and can be appropriately selected according to the type of cells. For example, when the target cells are bacterial cells, antibacterial drugs (such as cephem antibacterial drugs, penem antibacterial drugs, quinolone antibacterial drugs, penicillin antibacterial drugs, aminoglycoside antibacterial drugs, etc.), benzalkonium chloride, cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, etc. can be mentioned.

[0030] Examples of cephem antibacterial drugs include cefepime, ceftazidime, cefpodoxime, cefotaxime, cefazolin, cephalexin, etc. Examples of penem antibacterial drugs include imipenem, meropenem, ertapenem, faropenem, etc. Examples of quinolone antibacterial drugs include ciprofloxacin (CPFX), levofloxacin, ofloxacin, norfloxacin, etc. Examples of penicillin antibacterial drugs include ampicillin, cloxacillin, etc. Examples of glycopeptide antibacterial drugs include vancomycin, etc. Examples of aminoglycoside antibiotics include amikacin, tobramycin, arbekacin, kanamycin, and the like.

[0031] Examples of the component to be detected in the present invention include NAD+, NADP+, NADH, NADPH, and the like. From the viewpoint that the product after the enzyme cycling reaction is a water-soluble formazan that can be visually observed as a dye, NAD+, NADP+, NADH, and NADPH are preferable as the component to be detected.

[0032] In the present invention, the composition for enzyme cycling reaction refers to a composition containing components necessary for the enzyme cycling reaction. Examples of the components necessary for the enzyme cycling reaction include the above-described first substrate, first enzyme, second substrate, and second enzyme. The combination of the substrate and the enzyme can be appropriately selected by those skilled in the art from the descriptions of prior art documents such as Patent Documents 6 to 9 according to the component to be detected (the substance supplied to the enzyme cycling reaction system).

[0033] For example, when the component to be detected is NAD+, NADP+, NADH, or NADPH, the first substrate, the oxidation product of the first substrate, and the first enzyme of the enzyme cycling reaction are glucose, glucono-δ-lactone, and glucose dehydrogenase, respectively, and the second substrate, the oxidation product of the second substrate, and the second enzyme are WST-8, water-soluble formazan, and diaphorase, respectively. can be. In one aspect, the composition for enzyme cycling reaction contains glucose, glucose dehydrogenase, a substrate for diaphorase, and diaphorase.

[0034] The substrate for diaphorase may be a tetrazolium salt, and it is preferable that the tetrazolium salt is WST-8, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, or tetranitroblue tetrazolium.

[0035] In one aspect, the reagent of the present invention may further contain a nonionic surfactant. Examples of the nonionic surfactant include polyethylene glycol sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyethylene glycol sorbitan monostearate (Tween 60), polyoxyethylene sorbitan oleate (Tween 80), polyethylene glycol tert-octylphenyl ether (Triton TM X-405), and the like. It is known that by including a nonionic surfactant, the cell membrane permeability of microorganisms is affected, and the uptake efficiency of nutrient components is increased. Furthermore, by improving the dispersibility of microorganisms in the reagent, the growth of microorganisms can be promoted. As a result, antibacterial agents having an effect of rupturing microorganisms when the microorganisms divide, such as cephem antibacterial agents, quinolone antibacterial agents, and aminoglycoside antibacterial agents, can act efficiently on the microorganisms.

[0036] In one aspect, the reagent of the present invention may further contain a cell culture medium component for determining the susceptibility of bacteria to a cell treatment agent. Here, the cell culture medium component either does not contain the component to be detected or contains only a concentration of 5 μg / L or less that does not cause a color change in a 1-hour enzyme cycling reaction. For example, when NAD+ is the component to be detected in the cell, the cell culture medium component either does not contain NAD+ or contains only a concentration of 5 μg / L or less that does not cause a color change in a 1-hour enzyme cycling reaction. By including such a culture medium component, cell growth (including processes such as cell division) is promoted, the cell treatment agent can act efficiently, and the susceptibility of bacteria to the cell treatment agent can be easily determined. The cell culture medium component that can be used in the present invention is generally not limited as long as it either does not contain the component to be detected or contains only a concentration of 5 μg / L or less that does not cause a color change in a 1-hour enzyme cycling reaction and is a component known as a culture medium component for the cell. For example, when NAD+ is the component to be detected, tryptone, bactopeptone, glucose, xylose, sodium chloride, potassium chloride, etc. can be used as the cell culture medium component.

[0037] The cell culture medium component is preferably peptone and chloride. Here, peptone includes tryptone (casein peptone), bactopeptone (meat peptone), heart peptone (myocardial peptone), soy peptone, gelatin peptone, etc. In the reagent of the present invention, peptone is added as a nitrogen source (amino acid source) for microorganisms, and without these, microorganisms cannot divide (grow). For example, cephem antibiotics inhibit cell wall synthesis by binding to cell wall synthesis enzymes. When a microorganism attempts to divide in a state where cell wall synthesis is inhibited, the cell wall becomes thinner and the bacterial cell bursts because it cannot withstand the osmotic pressure difference with the external solution. However, in the absence of a nitrogen source, microorganisms do not attempt to divide (grow), so the bacterial cells do not burst. Therefore, in the absence of a nitrogen source, even microorganisms that are sensitive to cephem antibiotics do not burst, resulting in an incorrect determination of resistance. Thus, by including peptone in the reagent of the present invention, this incorrect determination can be prevented.

[0038] In addition, chloride refers to, for example, potassium chloride, sodium chloride, etc. By including this, the osmotic pressure inside and outside the microorganism can be adjusted. When a microorganism divides, it increases the cytoplasm and synthesizes the cell wall. However, when this balance is disrupted and cell division occurs in a state where the cell wall synthesis is incomplete. At that time, in a hypotonic solution state without chloride in the reagent, the bacterial cells will burst due to the osmotic pressure. Therefore, for example, even for microorganisms resistant to cephem antibiotics, the bacterial cells will burst due to the osmotic pressure, resulting in an incorrect determination of sensitivity. Thus, by including chloride in the reagent, this incorrect determination can be prevented.

[0039] The present invention also relates to a method for detecting a component to be detected inside a cell in a sample, which includes detecting, in the reagent of the present invention, the component to be detected that has been released outside the cell by a cell treatment agent using an enzyme cycling reaction.

[0040] In one aspect, the present invention may also be a method for determining the sensitivity of bacteria to a cell treatment agent or for determining the presence or absence of bacteria in a sample when the cell is a bacterial cell. The determination can be confirmed by visually observing the dye generated by the enzyme cycling reaction or by measuring the absorbance.

[0041] In one aspect, the present invention is a method for detecting a component to be detected inside a cell in a sample, which is a method for determining the sensitivity of bacteria to a bactericidal component, and includes detecting, in the reagent of the present invention containing peptone and chloride, the component to be detected that has been released outside the cell by a cell treatment agent using an enzyme cycling reaction.

Example

[0042] [Experiment 1] Bacterial drug sensitivity test 1. Preparation of the reagent of the present invention The composition of the reagent of the present invention tested (hereinafter referred to as "this reagent 1") is as follows. <Composition of this reagent 1> Sodium dihydrogen phosphate dihydrate 3.9 g / L Disodium hydrogen phosphate dodecahydrate 2.5 g / L Sodium chloride 1.0 g / L Potassium chloride 6.0 g / L Tween 20 1.0 g / L Tryptone 0.5 g / L Bacto Peptone 2.0 g / L Xylose 1.0 g / L

[0043] (1) Cell treatment agent: The agents described in Table 1, Table 3, Table 5, Table 7, Table 9, Table 11, Table 13, Table 15 (2) Composition for enzyme cycling reaction: Glucose 5.0 g / L Glucose dehydrogenase 3750 U / L WST-8 270 mg / L Diaphorase 1 KU / L Note that WST-8 is a substrate of diaphorase, which is a tetrazolium salt, and produces yellow water-soluble formazan by the enzyme cycling reaction.

[0044] 2. Test bacteria Using the bacteria shown in Table 1 (20 species, 234 strains), the drug susceptibility was determined using this reagent 1, and a comparison was made with the drug susceptibility determination by the disk diffusion method using the KB disk (registered trademark) 'Eiken' cefpodoxime according to the attached document of this reagent.

Table 1

[0045] The isolated bacteria (20 species, 234 strains) were each spread on a chromo agar orientation agar medium and cultured at 37°C for 24 hours. Then, the colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of about McFarland 2, and this was used as the bacterial suspension.

[0046] 3. Determination of drug susceptibility using this reagent 1 A 100-μL aliquot of the bacterial suspension of each bacterium prepared in step 2 was inoculated into this reagent 1 (1 mL), incubated at 37°C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether this reagent 1 turned yellow or not.

[0047] 4. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in step 2, with the KB disk (registered trademark) 'Eiken' cefpodoxime, according to the disk diffusion method performed in accordance with the attached document of this reagent (cultured at 37°C for 18 hours), the drug susceptibility of each bacterium was determined.

[0048] 5. Results The results are shown in Table 2.

Table 2

[0049] When the determination results of the method using this reagent 1 were compared with the determination results of the disk diffusion method (Table 2), 89% of the bacteria determined to be sensitive were in agreement, and 92% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using this reagent 1 can obtain determination results of drug susceptibility with a high agreement rate with the conventional method in an extremely short time.

[0050] 6. Test bacteria Using the bacteria (11 species, 94 strains) shown in Table 3, a comparison was made between the determination of drug susceptibility using this reagent 1 and the determination of drug susceptibility by the disk diffusion method using the KB disk (registered trademark) 'Eiken' ceftazidime in accordance with the attached document of this reagent.

Table 3

[0051] The isolated bacteria (11 species, 94 strains) were each streaked on blood agar medium and cultured at 37°C for 24 hours. Then, the colonies grown on the medium were picked, suspended in sterilized physiological saline to a turbidity of about McFarland 2, and this was used as the bacterial suspension.

[0052] 7. Determination of drug susceptibility using this reagent 1 100 μL of the bacterial suspension of each bacterium prepared in 6 above was inoculated into this reagent 1 (1 mL), and after incubation at 37°C for 1 hour, the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether this reagent 1 turned yellow or not.

[0053] 8. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in 6 above, with KB disk (registered trademark) 'Eiken' cefotaxime, according to the disk diffusion method performed in accordance with the attached document of this reagent (cultured at 37°C for 18 hours), the drug susceptibility of each bacterium was determined.

[0054] 9. Results The results are shown in Table 4.

Table 4

[0055] When the determination results of the method using this reagent 1 were compared with the determination results of the disk diffusion method (Table 4), 97% of the bacteria determined to be sensitive were in agreement, and 85% of the bacteria determined to be resistant were in agreement. From this result, it was shown that the method using this reagent 1 can obtain determination results of drug susceptibility with a high agreement rate with the conventional method in an extremely short time.

[0056] 10. Test bacteria Using the bacteria (11 species, 94 strains) shown in Table 5, a comparison was made between the determination of drug susceptibility using this reagent 1 and the determination of drug susceptibility by the disk diffusion method using KB disk (registered trademark) 'Eiken' ceftazidime according to the attached document of this reagent.

Table 5

[0057] Isolated bacteria (11 species, 94 strains) were each streaked on ChromoAgar Orientation agar medium and cultured at 37°C for 24 hours. Then, colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of approximately McFarland 2, and this was used as the bacterial suspension.

[0058] 11. Determination of drug susceptibility using Reagent 1 The bacterial suspension of each bacterium prepared in step 10 was inoculated with 100 μL into Reagent 1 (1 mL). After incubation at 37°C for 1 hour, the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether Reagent 1 turned yellow.

[0059] 12. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in step 10, with the KB disk (registered trademark) 'Eiken' ceftazidime, according to the disk diffusion method performed in accordance with the attached document of this reagent (cultured at 37°C for 18 hours), the drug susceptibility of each bacterium was determined.

[0060] 13. Results The results are shown in Table 6.

Table 6

[0061] When the determination results of the method using Reagent 1 were compared with the determination results of the disk diffusion method (Table 6), 88% of the bacteria determined to be sensitive were in agreement, and 95% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using Reagent 1 can obtain determination results of drug susceptibility with a high coincidence rate with the conventional method in an extremely short time.

[0062] 14. Test bacteria Using the bacteria (5 species, 8 strains) shown in Table 7, a comparison was made between the determination of drug susceptibility using Reagent 1 and the determination of drug susceptibility by the disk diffusion method using the KB disk (registered trademark) 'Eiken' levofloxacin in accordance with the attached document of this reagent.

Table 7

[0063] Five species and eight strains of isolated bacteria were each streaked onto Chromo Agar Orientation agar medium and cultured at 37 °C for 24 hours. Then, the colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of approximately McFarland 2, and this was used as the bacterial suspension.

[0064] 15. Determination of drug susceptibility using Reagent 1 100 μL of each bacterial suspension prepared in step 14 was inoculated into 1 mL of Reagent 1, incubated at 37 °C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether Reagent 1 turned yellow.

[0065] 16. Determination of drug susceptibility using the disk diffusion method Using each bacterial suspension prepared in step 14, the disk diffusion method was performed using the KB disk (registered trademark) 'Eiken' levofloxacin according to the attached document of this reagent (cultured at 37 °C for 18 hours), and the drug susceptibility of each bacterium was determined.

[0066] 17. Results The results are shown in Table 8.

Table 8

[0067] When the determination results of the method using Reagent 1 were compared with the determination results of the disk diffusion method (Table 8), 75% of the bacteria determined to be sensitive were in agreement, and 100% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using Reagent 1 can obtain determination results of drug susceptibility with a high coincidence rate with the conventional method in an extremely short time.

[0068] 18. Test bacteria Using the bacteria (five species, eight strains) shown in Table 9, a comparison was made between the determination of drug susceptibility using Reagent 1 and the determination of drug susceptibility by the disk diffusion method using the KB disk (registered trademark) 'Eiken' amikacin according to the attached document of this reagent.

Table 9

[0069] Isolated bacteria (5 species, 8 strains) were each streaked on ChromoAgar Orientation agar medium and cultured at 37°C for 24 hours. Subsequently, colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of approximately McFarland 2, and this was used as the bacterial suspension.

[0070] 19. Determination of drug susceptibility using Reagent 1 100 μL of each bacterial suspension prepared in 18 above was inoculated into Reagent 1 (1 mL), incubated at 37°C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether Reagent 1 turned yellow.

[0071] 20. Determination of drug susceptibility using the disk diffusion method Using each bacterial suspension prepared in 18 above, with the KB disk (registered trademark) 'Eiken' amikacin, according to the disk diffusion method carried out in accordance with the attached document of this reagent (cultured at 37°C for 18 hours), the drug susceptibility of each bacterium was determined.

[0072] 21. Results The results are shown in Table 10.

Table 10

[0073] When the determination results of the method using Reagent 1 were compared with the determination results of the disk diffusion method (Table 10), 50% of the bacteria determined to be sensitive were in agreement, and 100% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using Reagent 1 can obtain determination results of drug susceptibility with a high coincidence rate with the conventional method in an extremely short time.

[0074] 22. Test bacteria Using the bacteria (5 species, 8 strains) shown in Table 11, the drug susceptibility was determined using this Reagent 1, and a comparison was made with the drug susceptibility determination by the disk diffusion method using the KB disk (registered trademark) 'Eiken' meropenem according to the attached document of this reagent.

Table 11

[0075] The isolated bacteria (5 species, 8 strains) were each streaked on a chromoagar orientation agar medium and cultured at 37°C for 24 hours. Then, the colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of about McFarland 2, and this was used as the bacterial suspension.

[0076] 23. Determination of drug susceptibility using this Reagent 1 100 μL of the bacterial suspension of each bacterium prepared in the above 22 was inoculated into this Reagent 1 (1 mL), incubated at 37°C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was carried out by visually observing whether this Reagent 1 turned yellow.

[0077] 24. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in the above 22, the disk diffusion method was carried out using the KB disk (registered trademark) 'Eiken' meropenem according to the attached document of this reagent (cultured at 37°C for 18 hours), and the drug susceptibility of each bacterium was determined.

[0078] 25. Results The results are shown in Table 12.

Table 12

[0079] When the determination results of the method using this Reagent 1 were compared with the determination results of the disk diffusion method (Table 12), 75% of the bacteria determined to be susceptible were in agreement, and 100% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using this Reagent 1 can obtain drug susceptibility determination results with a high coincidence rate with the conventional method in an extremely short time.

[0080] 26. Test bacteria Using the bacteria (2 species, 6 strains) shown in Table 13, the drug susceptibility was determined using this Reagent 1, and a comparison was made with the determination of drug susceptibility by the disk diffusion method using KB Disk (registered trademark) 'Eiken' ampicillin according to the attached document of this reagent.

Table 13

[0081] The isolated bacteria (2 species, 6 strains) were each streaked on soybean casein digest agar medium and cultured at 37°C for 24 hours. Then, the colonies grown on the medium were picked, suspended in sterile physiological saline to a turbidity of about McFarland 2, and this was used as the bacterial suspension.

[0082] 27. Determination of drug susceptibility using Reagent 1 100 μL of the bacterial suspension of each bacterium prepared in the above 22 was inoculated into this Reagent 1 (1 mL), incubated at 37°C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was carried out by visually observing whether this Reagent 1 turned yellow.

[0083] 28. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in the above 22, KB Disk (registered trademark) 'Eiken' ampicillin was used, and according to the disk diffusion method performed according to the attached document of this reagent (cultured at 37°C for 18 hours), the drug susceptibility of each bacterium was determined.

[0084] 29. Results The results are shown in Table 14.

Table 14

[0085] When the determination results of the method using Reagent 1 were compared with those of the disk diffusion method (Table 14), 100% of the bacteria determined to be sensitive and 100% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using Reagent 1 can obtain determination results of drug susceptibility with a high coincidence rate with the conventional method in an extremely short time.

[0086] 30. Test bacteria Using the bacteria shown in Table 15 (4 species, 8 strains), a comparison was made between the determination of drug susceptibility using Reagent 1 and the determination of drug susceptibility by the disk diffusion method using the KB disk (registered trademark) 'Eiken' vancomycin according to the attached document of this reagent.

Table 15

[0087] The isolated bacteria (4 species, 8 strains) were each spread on a soybean casein digest agar medium and cultured at 37°C for 24 hours. Thereafter, the colonies grown on the medium were picked up and suspended in sterile physiological saline to a turbidity of about McFarland 2 to obtain a bacterial suspension.

[0088] 31. Determination of drug susceptibility using Reagent 1 100 μL of the bacterial suspension of each bacterium prepared in 18 above was inoculated into Reagent 1 (1 mL), incubated at 37°C for 1 hour, and then the drug susceptibility of each bacterium was determined. The determination was performed by visually observing whether Reagent 1 turned yellow.

[0089] 32. Determination of drug susceptibility using the disk diffusion method Using the bacterial suspension of each bacterium prepared in 22 above, the drug susceptibility of each bacterium was determined according to the disk diffusion method using the KB disk (registered trademark) 'Eiken' vancomycin according to the attached document of this reagent (cultured at 37°C for 18 hours).

[0090] 33. Results The results are shown in Table 16.

Table 16

[0091] When the determination results of the method using the reagent 1 were compared with those of the disk diffusion method (Table 16), 100% of the bacteria determined to be sensitive were in agreement, and 80% of the bacteria determined to be resistant were in agreement. From these results, it was shown that the method using the reagent 1 can obtain determination results of drug susceptibility with a high agreement rate with the conventional method in an extremely short time.

[0092] [Experiment 2] Detection of microorganisms 1. Preparation of the reagent of the present invention The same reagent as that used in Experiment 1 (Reagent 1) was prepared, except that benzalkonium chloride (a bactericide) was used as a cell treatment agent at a concentration of 250 mg / L (Reagent 2). 2. Test microorganisms As microorganisms, Escherichia coli, Staphylococcus aureus, and Candida albicans were used. The isolated microorganisms (Escherichia coli, Staphylococcus aureus, Candida) were each spread on tryptone soy agar medium and cultured at 37°C for 24 hours. Then, the colonies grown on the medium were picked and suspended in sterile physiological saline to a turbidity of about McFarland 2, which was used as a microbial suspension.

[0093] 3. Determination of the presence or absence of microorganisms using Reagent 2 100 μL of each microbial suspension and sterile physiological saline without suspended microorganisms (negative control) were inoculated into 1 mL of the product of the present invention, incubated at 37°C for 1 hour, and then the absorbance at 450 nm was measured with a microplate reader. An absorbance of 0.1 Abs. or more was determined as having yellowing.

[0094] 4. Results The results are shown in Table 17 (absorbance when each microbial suspension and sterile physiological saline were reacted with the developed product).

Table 17

Claims

1. A reagent for detecting a component to be detected inside cells in a sample, the reagent comprising: a cell treatment agent for releasing the component to be detected inside the cell to the outside of the cell; and an enzyme cycling reaction composition for performing an enzyme cycling reaction on the component to be detected.

2. The reagent according to claim 1, wherein the cell is a microbial cell of bacteria or fungi.

3. The reagent according to claim 2, for determining the sensitivity of microbial cells to the cell treatment agent or for determining the presence or absence of microbial cells in a sample.

4. The reagent according to claim 3, wherein the cell treatment agent is one or more selected from the group consisting of antibacterial agents, benzalkonium chloride, cetyltrimethylammonium bromide, and cetyltrimethylammonium chloride.

5. The reagent according to claim 4, wherein the antibacterial agent is one or more selected from the group consisting of cephem antibacterial agents, penem antibacterial agents, quinolone antibacterial agents, penicillin antibacterial agents, glycopeptide antibacterial agents, and aminoglycoside antibacterial agents.

6. The reagent according to claim 1, wherein the component to be detected is one or more selected from the group consisting of NAD+, NADP+, NADH, and NADPH.

7. The reagent according to claim 6, wherein the enzyme cycling reaction composition comprises glucose, glucose dehydrogenase, a substrate for diaphorase, and diaphorase.

8. The reagent according to claim 7, wherein the substrate for diaphorase is a tetrazolium salt.

9. The reagent according to claim 8, wherein the tetrazolium salt is WST-8, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, or tetranitroblue tetrazolium.

10. The reagent according to claim 1, further comprising a nonionic surfactant.

11. The reagent according to claim 3, for determining the sensitivity of microbial cells to the cell treatment agent, further comprising a cell culture medium component that does not contain the component to be detected or contains the component at a concentration of 5 μg / L or less.

12. The reagent according to claim 11, wherein the cell culture medium component is peptone and chloride.

13. A method for detecting a component to be detected inside cells in a sample, the method comprising: detecting, using an enzyme cycling reaction, the component to be detected that has been released to the outside of the cell by a cell treatment agent in the reagent according to any one of claims 1 to 12.

14. The method according to claim 13, wherein the cell is a microbial cell of bacteria, fungi or yeast, and is for determining the sensitivity of the microbial cell to the cell treatment agent or for determining the presence or absence of microbial cells in a sample.

15. A method for detecting a component to be detected inside a cell in a sample, the method being a method for determining the sensitivity of a microbial cell to a cell treatment agent, and in the reagent according to claim 11 or 12, detecting a component to be detected that has been released outside the cell by the cell treatment agent using an enzyme cycling reaction.

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