KR102365852B1 - Kit for detecting carbapenemase-producing enterobacteriaceae comprising fluorescent probe and method for detecting carbapenemase-producing enterobacteriaceae using the same - Google Patents

Abstract

The present invention provides a kit for detecting carbapenemase-producing enterobacteriaceae containing a lysis buffer, a reaction buffer, and a fluorescent probe substrate, and a kit using the kit The present invention relates to a method for detecting carbapenem-degrading enzyme-producing enterobacteria.

Description

Kit for detecting carbapenemase-producing enterobacteriaceae comprising fluorescent probe and method for detecting carbapenemase-producing enterobacteriaceae using the same

The present invention relates to a kit for detecting carbapenem-degrading enterobacteriaceae containing a fluorescent probe and a method for detecting carbapenem-degrading enterobacteriaceae using the same.

Carbapenem is an antibiotic with a broad antibacterial spectrum, and is an antibiotic used to treat gram-negative bacterial infections that produce extended spectrum beta-lactamase. With the recent increase in antibiotic resistance and the increase in the use of carbapenem antibacterial agents, carbapenem-resistant enterobacteriaceae (CRE) have emerged and are rapidly increasing worldwide. In particular, it is known that CRE-induced infections have a higher mortality rate than those of Enterobacteriaceae that are not resistant to antibiotics.

Enterobacteriaceae is defined as a case that has a carbapenem-degrading enzyme directly or is resistant to any one or more antibacterial agents among ertapenem, imipenem, meropenem, and doripenem. Enterobacteriaceae species have a carbapenem-degrading enzyme-producing gene according to the carbapenem resistance mechanism, and thus form a carbapenemase-producing enterobacteriaceae (CPE) and are combined with various resistance mechanisms without carbapenem-degrading enzyme production. It can be classified as a case showing resistance to carpabenem (Non-CPE). CPE resistance standards that directly degrade carbapenem can be divided into A, B, and D types according to the Ambler class. In the case of Enterobacteriaceae (Non-CPE), which do not produce carbapenem-degrading enzymes and are resistant to carbapenems, various It shows resistance to carbapenem due to a combination of resistance mechanisms.

CPE, a carbapenem-degrading enzyme-producing bacterium, cannot be differentiated from non-CPE only by antibiotic susceptibility testing, so a carbapenem-degrading enzyme screening test and confirmation test are required. In case of antibiotic susceptibility to carbapenem resistance, it is classified as CRE. Thereafter, to confirm CPE, a phenotypic test using a modified-Hodge test (MHT) and a carbapenemase inhibition test (CIT) or a genotyping test (PCR or DNA sequencing analysis) through gene amplification are performed. However, although the phenotype test is relatively quick to confirm, the judgment may be subjective, the type of resistance gene cannot be confirmed, and it is difficult to exclude the possibility of false positives. In addition, the genotyping test has disadvantages in that it is expensive and the types of genes that can be detected are limited. Accordingly, there is an increasing demand for tests capable of more rapidly and accurately detecting various carbapenem-degrading enzyme-producing bacteria.

Meanwhile, Korean Patent No. 1317263 discloses "a method and kit for testing carbapenem-resistant Enterobacteriaceae using real-time PCR", and Korean Patent No. 19955622 discloses "Carbapenem-resistant Gram-negative bacteria containing ADK protein as an active ingredient" antibacterial composition for ", but the kit for detecting carbapenem-degrading enterobacteriaceae containing the fluorescent probe of the present invention and a method for detecting carbapenem-degrading enterobacteriaceae using the same have not been described.

The present invention was derived from the above needs, and the present inventors have provided a kit and method capable of rapidly and accurately detecting a resistant microorganism that produces carbapenemase, a carbapenem backbone. After preparing a kit containing a novel fluorescent probe to which an ebenzyl alcohol linker and an umbelliferone fluorophore are bound as a substrate, enterobacteriaceae having a carbapenem-degrading enzyme gene ( As a result of analysis of 65 CPE) and 156 Enterobacteriaceae that do not have a carbapenem degrading enzyme gene, it was confirmed that the kit of the present invention has excellent sensitivity and specificity to carbapenem-degrading enterobacteriaceae. The invention was completed.

In order to solve the above problems, the present invention is to detect carbapenemase-producing enterobacteriaceae containing a lysis buffer, a reaction buffer, and a fluorescent probe substrate. kits are provided for

In addition, the present invention comprises the steps of pre-treating a biological sample isolated from a patient suspected of carbapenem-resistant bacterial infection with a lysis buffer; centrifuging the sample pretreated with the lysis buffer to obtain a supernatant; and measuring the fluorescence value after mixing the obtained supernatant with a reaction buffer and a fluorescent probe substrate to react.

Kits containing the carbapenem fluorescent probe substrate of the present invention include active on imipenem (IMP), verona integron-encoded metallo-β-lactamasea (VIM), new delhi metallo-β-lactamase (NDM), and Klebsiella pneumoniae carbapenemase (KPC). Since carbapenem-resistant enterobacteriaceae (CREs) that produce various carbapenem-degrading enzymes such as carbapenem-degrading enzymes can be detected simply, quickly and accurately, early diagnosis of carbapenem-resistant enterobacteriaceae and prompt response to CRE-infected diseases · It is expected to be useful for management.

1 is a photograph showing the components of the kit product for detecting carbapenemase-producing enterobacteriaceae of the present invention.
2 is a result of performing a receiver operating characteristic curve (ROC) curve analysis for diagnosing whether carbapenem-resistant enterobacteria are infected by using the kit for detecting carbapenem-degrading enterobacteriaceae of the present invention.

In order to achieve the object of the present invention, the present invention provides carbapenemase-producing enterobacteriaceae comprising a lysis buffer, a reaction buffer, and a fluorescent probe substrate. A kit for detecting is provided.

In the kit of the present invention, the lysis buffer is for extracting protein (enzyme) from carbapenem-degrading enzyme-producing enterobacteria. Preferably, EDTA (Ethylenediaminetetraacetic acid), protease inhibitor, CHAPS (3-[( 3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate hydrate), lysozyme, DNase (deoxyribonuclease) and PBS (phosphate buffered saline), more preferably 0.01 to 0.6 mM EDTA, 3 to 5% (w/v) protease inhibitor, 0.5-2% (w/v) CHAPS, 0.05-1mg/ml lysozyme, 4-6U/ml DNaseI and 9-11mM PBS, most preferably It may consist of 0.012 mM EDTA, 4% (w/v) protease inhibitor, 1% (w/v) CHAPS, 0.1 mg/mL lysozyme, 5 U/mL DNase I and 10 mM PBS, It is not limited thereto.

In the kit of the present invention, the reaction buffer is an enzyme reaction solution that provides an environment in which the fluorescent probe substrate according to the present invention and the carbapenem degrading enzyme can react. Preferably, sodium chloride (NaCl), Na ₂ HPO ₄ ( sodium phosphate dibasic), KCl (potassium chloride) and KH ₂ PO ₄ (potassium phosphate monobasic), more preferably 0.1 to 0.2M NaCl, 1-3mM Na ₂ HPO ₄ , 2 to 4mM of It may consist of KCl and 10-12mM KH ₂ PO ₄ , and most preferably 0.14M NaCl, 1.7 mM Na ₂ HPO ₄ , 2.7 mM KCl and 10.6 mM KH ₂ PO ₄ It may be composed of , but not limited thereto.

In the kit of the present invention, the fluorescent probe substrate is a compound in which a benzyl alcohol linker and an umbelliferone fluorescent substance are bound to a carbapenem backbone, preferably (4S, 5R, 6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy) methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.

The fluorescent probe substrate to which the carbapenem skeleton, the linker, and the fluorophore are sequentially bound is a carbapenem degrading enzyme, such as Klebsiella pneumoniae carbapenemase (KPC), guiana extended-spectrum β-lactamase (GES), active on imipenem (IMP), and VIM (active on imipenem) (VIM). Verona integron-encoded metallo-β-lactamasea), NDM (new delhi metallo-β-lactamase), or OXA (oxacillinase) may cause the phosphor to emit light because the linker portion connecting the carbapenem skeleton and the phosphor is cut. not limited

The present invention also

pre-treating a biological sample isolated from a patient suspected of being infected with carbapenem-resistant bacteria with a lysis buffer;

centrifuging the sample pretreated with the lysis buffer to obtain a supernatant; and

Carbapenemase-producing enterobacteriaceae, comprising the step of measuring the fluorescence value after mixing the obtained supernatant with a reaction buffer and a fluorescent probe substrate A method of detection is provided.

In the method according to an embodiment of the present invention, the dissolution buffer and the reaction buffer are the same as described above.

In the method according to an embodiment of the present invention, the fluorescent probe substrate is preferably (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl represented by the following Chemical Formula 1 -7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2 -ene-2-carboxylic acid [(4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H- chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid].

As used herein, the term "biological sample" means cells, tissues, blood, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, paraffin tissue, urine, saliva, sweat, intracellular body fluids and bacterial expression systems, and the like, and the blood may include, but is not limited to, whole blood, plasma, serum, or blood, plasma, and serum that has been subjected to a predetermined treatment (eg, to prevent coagulation). The presence or absence of infection with carbapenem-degrading enzyme-producing enterobacteria can be checked by reacting these biological samples with the kit of the present invention in a manipulated or non-manipulated state.

In the method according to one embodiment of the present invention, the biological sample isolated from the patient suspected of being infected with carbapenem-resistant bacteria is inoculated with the biological sample isolated from the patient to enrich the microorganisms in the sample, and then a part of the microorganisms proliferated in the medium It may be collected, but is not limited thereto.

In the method according to an embodiment of the present invention, the method for detecting carbapenem-degrading enzyme-producing enterobacteria comprises:

pretreatment by placing 0.5-12 μl of a biological sample isolated from a patient suspected of being infected with carbapenem-resistant bacteria in 140-160 μl of lysis buffer, vortexing for 0.5-2 minutes, and reacting at room temperature for 20-40 minutes;

centrifuging the sample pretreated with the lysis buffer at 9,000 to 11,000 rpm for 4 to 6 minutes to obtain a supernatant;

and measuring the fluorescence value after mixing 25-35 μl of the obtained supernatant with 90-110 μl of reaction buffer and 11-14 μl of fluorescent probe substrate;

More specifically,

A biological sample isolated from a patient suspected of being infected with carbapenem-resistant bacteria is inoculated into the medium, the microorganisms in the sample are enriched, and centrifuged. pretreatment by reacting at room temperature;

centrifuging the sample pretreated with the lysis buffer at 10,000 rpm for 5 minutes to obtain a supernatant;

30 μl of the obtained supernatant is mixed with 100 μl of a reaction buffer and 13 μl of a fluorescent probe substrate and reacted, followed by measuring the fluorescence value at an excitation wavelength of 260 nm and an emission wavelength of 465 nm; but is not limited thereto.

In the method according to one embodiment of the present invention, the supernatant obtained by centrifuging the sample pretreated with the lysis buffer contains carbapenem-degrading enzyme contained in a biological sample isolated from a patient suspected of being infected with carbapenem-resistant bacteria. there is.

In the method according to an embodiment of the present invention, the fluorescence value was measured at an excitation wavelength of 250 to 270 nm and an emission wavelength of 455 to 475 nm, preferably, an excitation wavelength of 260 nm and an emission wavelength of 465 nm Therefore, after setting the gain value to 50, the fluorescence value may be measured at 5 minute intervals for a total of 50 minutes, but is not limited thereto. In the method of the present invention, the determination of positive or negative for CRE is positive when the difference between the fluorescence value measured at 50 minutes and the fluorescence value measured at 0 minutes is greater than the cut-off value, and negative when smaller. It can be judged by the reaction, and the cut-off value was 3,188, which was determined through a receiver-operating characteristic (ROC) curve.

Hereinafter, an embodiment of the present invention will be described in detail. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Preparation Example 1. Kit for detecting carbapenem-degrading enzyme-producing Enterobacteriaceae (CPE)

The components of the kit for detecting carbapenem-degrading enterobacteriaceae of the present invention are shown in Table 1 and FIG. prepared.

First, the lysis buffer is 4.8 ml of 2.5 mM EDTA (SAMCHUN, Cat No. E0060) based on a total volume of 1 L, protease inhibitor (1 tablet/10 ml; Roche, Cat No. 4693132001) 200 ml, 1X CHAPS (Sigma, Cat) No. 19899) 10 ml, 50 mg/ml of lysozyme (Sigma, Cat No. L-6876) 2 ml, 2,500 U/ml of DNase I (Thermo, Cat No. 90083) 2 ml, 30 mM PBS (Sigma, Cat No. No. 90083) P3813) was prepared by mixing 333 ml of distilled water and 448.2 ml of distilled water and then filtering (pore size 0.22 μm) (Table 2).

In addition, the reaction buffer is sodium chloride (NaCl; DAEJUNG, Cat No. 7548-4100) 8 g, disodium phosphate dibasic (Na ₂ HPO ₄ ; DAEJUNG, Cat No. 7613-4400) 0.24 g, potassium chloride ( Potassium chloride, KCl; YAKURI, Cat No. 28514) 0.2 g and potassium phosphate monobasic (KH ₂ PO ₄ ; JUNSEI, Cat No. 84185A1250) 1.44 g were mixed and distilled water was added so that the total volume was 1L. After filtering (pore size 0.22㎛), it was prepared by autoclaving at 121 ℃ for 15 minutes (Table 3).

In addition, the fluorescent probe substrate is (4S,5R,6S)-6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2) -oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid was provided by Professor Seon-Jun Min of Hanyang University and used. (Table 4).

Example 1. Analysis of carbapenem-degrading enzyme-producing enterobacteriaceae using the kit of the present invention

1-1. Using a conventional kit for detecting carbapenem-degrading enzyme-producing enterobacteria

Experiments were performed on CPE in Table 5 below using a kit (bioMerieux, Inc., France) that has been previously used to detect enterobacteriaceae that produce carbapenem degrading enzymes. As a result of judging a positive reaction up to light orange (refer to 2019 CLSI GUIDE LINE (29 ^th )), as shown in Table 6 below, the sensitivity was 67.7%, and the specificity was 100%. did.

1-2. Use of the kit for detecting carbapenem-degrading enzyme-producing enterobacteria of the present invention

Carbapenem-degrading enzyme-producing enterobacteria were detected using the kit of the present invention. Specifically, a clinical sample is collected, cultured in BAP (Blood Agar Plate) medium, centrifuged, and approximately 1 μl of the sample is collected using a cotton swab or loop, which is a tool for sample collection, and dissolved in a tube containing 150 μl of lysis buffer. After vortexing for 1 minute, the reaction was carried out at room temperature for 30 minutes. After the reaction was completed, the residue was precipitated by centrifugation at 10,000 rpm for 5 minutes to obtain a supernatant containing carbapenem degrading enzyme. Put 30 μl of the obtained supernatant, 100 μl of reaction buffer, and 13 μl of fluorescent probe substrate in a 96-well plate, and measure the fluorescence value with an excitation wavelength of 260 nm and an emission wavelength of 465 nm using a fluorescence meter (Infinite F200 Pro, Tecan coup Ltd.). measured. If the fluorescence value was greater than the cut-off value (cut-off) 3,188, it was determined as a positive reaction, and if the cut-off value was less than 3,188, it was determined as a negative reaction, and the cut-off value (3,188) was confirmed by analyzing the ROC (receiver-operating characteristic) curve. (Fig. 2).

As a result of analysis of 65 enterobacteriaceae (CPE) having a carbapenem degrading enzyme gene and 156 enterobacteriaceae not having a carbapenem degrading enzyme gene using the kit of the present invention, as shown in Table 7 below, the sensitivity It was confirmed that the sensitivity was 75.4% and the specificity was 99.3%.

1-3. Reproducibility analysis

In order to analyze the reproducibility of the kit for detecting carbapenem-degrading enterobacteriaceae of the present invention, a total of 10 enterobacteriaceae having a carbapenem-degrading enzyme gene and 10 enterobacteriaceae not having a carbapenem-degrading enzyme gene were treated in 3 batches. carried out. 10 enterobacteriaceae having the carbapenem degrading enzyme gene were Class A 3 (2 KPC, 1 GES), 4 Class B (1 IMP, 2 VIM, 1 NDM), 2 Class D ( OXA) and one enterobacteriaceae having two or more carbapenem-degrading enzyme genes, of which five (IMP, VIM, KPC, NDM, OXA, one each) are strains that were identified as positive in the previous test, The remaining five (VIM, KPC, GES, OXA, enterobacteriaceae having two or more carbapenem-degrading enzyme genes, one each) were strains that were identified as negative. In addition, 10 enterobacteriaceae that do not have the carbapenem degrading enzyme gene include 3 carbapenem-resistant enterobacteriaceae (CRE), 4 broad-spectrum beta-lactam-producing enterobacteriaceae (ESBL), and enterobacteriaceae sensitive to all antibiotics (Non- ESBL), of which one ESBL is a strain that was identified as negative in the previous test.

First, as a result of analyzing the reproducibility between batches, the reproducibility of batches 1 and 2 and batches 2 and 3 was 95%, and the reproducibility of batches 1 and 3 was 100%. One KPC strain, which was identified as positive in batch 1, was identified as negative in batch 2, and showed 95% reproducibility between batches, and one KPC strain, which was identified as negative in batch 2, was identified as positive in batch 3, resulting in 95% of batch reproducibility. Batch-to-batch reproducibility was demonstrated.

In addition, as a result of analyzing the reproducibility within a batch, batch 1 showed a reproducibility of 95%, and batches 2 and 3 showed a reproducibility of 100%. In the case of batch 1, one ESBL, which had previously been identified as positive, was identified as negative, showing 95% intra-batch reproducibility.

In addition, as a result of analyzing reproducibility between users, batch 1 showed 100% reproducibility, and batch 2 and batch 3 showed 95% reproducibility. In the case of batch 2, one KPC strain, which was identified as negative in user A, was determined as positive in user B, showing 95% reproducibility between users, and in batch 3, two or more carbapenems that were identified as positive in user A One enterobacteriaceae with the enzyme gene was identified as negative in user B, showing 95% reproducibility between users.

From a diagnosis point of view, sensitivity is a value for how well it detects a person with a disease, and specificity is a value for how well it detects a normal person, and is an important item to evaluate the reliability of a test.

While the conventional kit confirms the sensitivity and specificity for detection of enterobacteriaceae with a carbapenem gene simply through color comparison, the kit of the present invention is based on the data on the ROS curve. Since the sensitivity and specificity for the detection of bacteria can be confirmed more accurately and the reproducibility is excellent, it was considered that the diagnosis of antibiotic-resistant enterobacteriaceae infection could be made quickly and accurately compared to the conventional method.

Claims (7)

A kit for detecting carbapenemase-producing enterobacteriaceae comprising a lysis buffer, a reaction buffer and a fluorescent probe substrate, the kit comprising:
The fluorescent probe substrate is (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl-7-oxo-3-((4-(((2) -oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid [(4S,5R,6S)- 6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)- 1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid] A kit for detecting carbapenem-degrading enzyme-producing enterobacteria, characterized in that it is a compound.
[Formula 1]

According to claim 1, wherein the lysis buffer is EDTA (Ethylenediaminetetraacetic acid), a protease inhibitor (Protease inhibitor), CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate hydrate), lysozyme (lysozyme), DNase (deoxyribonuclease) ) and a kit for detecting carbapenem-degrading enzyme-producing enterobacteria, characterized in that it consists of phosphate buffered saline (PBS). The decomposition of carbapenem according to claim 1, wherein the reaction buffer is sodium chloride (NaCl), sodium phosphate dibasic (Na ₂ HPO ₄ ), potassium chloride (KCl) and potassium phosphate monobasic (KH ₂ PO ₄ ). A kit for detecting enzyme-producing enterobacteriaceae. delete According to claim 1, EDTA (Ethylenediaminetetraacetic acid), protease inhibitor (Protease inhibitor), CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate hydrate), lysozyme (lysozyme), DNase (deoxyribonuclease) and PBS ( lysis buffer consisting of phosphate buffered saline); NaCl (sodium chloride), Na ₂ HPO ₄ (sodium phosphate dibasic), KCl (potassium chloride) and KH ₂ PO ₄ (potassium phosphate monobasic) consisting of a reaction buffer; and (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl-7-oxo-3-((4-(((2-oxo-2H) -chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid [(4S,5R,6S)-6-(( R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[ 3.2.0] A kit for detecting carbapenem-degrading enzyme-producing Enterobacteriaceae containing the compound [hept-2-ene-2-carboxylic acid] as a fluorescent probe substrate.
[Formula 1]

pre-treating a biological sample isolated from a patient suspected of being infected with carbapenem-resistant bacteria with a lysis buffer;
centrifuging the sample pretreated with the lysis buffer to obtain a supernatant; and
Carbapenemase-producing enterobacteriaceae, comprising the step of measuring the fluorescence value after mixing the obtained supernatant with a reaction buffer and a fluorescent probe substrate A method of detecting, comprising:
The fluorescent probe substrate is (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl-7-oxo-3-((4-(((2) -oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid [(4S,5R,6S)- 6-((R)-1-Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)- A method for detecting carbapenemase-producing enterobacteriaceae, characterized in that the compound is 1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
[Formula 1]

7. The method of claim 6,
Ethylenediaminetetraacetic acid (EDTA), protease inhibitor, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate), lysozyme, DNase (deoxyribonuclease) and pretreatment with a lysis buffer (lysis buffer) consisting of PBS (phosphate buffered saline);
centrifuging the sample pretreated with the lysis buffer to obtain a supernatant; and
The obtained supernatant is NaCl (sodium chloride), Na ₂ HPO ₄ (sodium phosphate dibasic), KCl (potassium chloride) and KH ₂ PO ₄ (reaction buffer) consisting of (potassium phosphate monobasic) and a reaction buffer (1) Represented (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromene-7) -yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid [(4S,5R,6S)-6-((R)-1- Hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept -2-ene-2-carboxylic acid] After mixing and reacting with a fluorescent probe substrate, the fluorescence value is measured at an excitation wavelength of 250 to 270 nm and an emission wavelength of 455 to 475 nm A method of detecting carbapenemase-producing enterobacteriaceae, comprising the step of:
[Formula 1]

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