Rapid antibiotic sensitivity detection and identification method

Abstract

The invention relates to a rapid antibiotic sensitivity detection and identification method, and the identification method can carry out antibiotic sensitivity detection and identification detection in a single process, and rapidly provide antibiotic information suitable for microorganisms. The invention provides a rapid antibiotic sensitivity detection and identification method which comprises the following steps: (a) transferring a sample extracted from a patient to a first reaction space in which a sample separation device exists; (b) capturing pathogens in a sample from a patient by a separation device within the first reaction space to separate and concentrate them; and (c) performing the identification of said pathogens or the detection of antibiotic sensitivity.

Description

Technical Field

[0001] The present invention relates to a rapid antibiotic sensitivity detection and identification method. More specifically, the present invention relates to a rapid antibiotic sensitivity detection and identification method that can more quickly provide antibiotic information applicable to microorganisms in whole blood or patient-derived samples including urine by eliminating the lengthy blood culture process or minimizing the fungal culture process that is considered necessary for antibiotic sensitivity detection and identification. Background Art

[0002] In order to prescribe appropriate antibiotics for infections including bacteremia or sepsis, it is necessary to identify the pathogen and test the antibiotic sensitivity to determine whether the causative pathogen is resistant to a specific antibiotic. Many attempts have been made to shorten the time required for antibiotic sensitivity testing, as shortening the time required to administer the appropriate antibiotic significantly improves patient survival, inhibits the development of antibiotic resistance, and reduces healthcare costs by shortening treatment cycles. Initial attempts were made to shorten the time required by improving methods for determining the growth of AST pathogens, and several methods such as microscopy, absorbance measurement, fluorescence molecular measurement, electrochemical measurement, and genomic analysis have produced significant results.

[0003] However, while this testing method can shorten the time required for antibiotic sensitivity testing, it still suffers from the limitation of requiring a long time to culture the pathogens in samples extracted from patients for antibiotic sensitivity testing. Previously, blood cultures from patient samples were performed for one day, followed by another day of pure culture, which takes at least two days. To overcome this limitation, attempts have been made to omit the pure culture required for antibiotic sensitivity testing. The advantages of pure culture include filtering out substances from the human body with antibiotic activity and a wide range of antibiotics present in patient samples when administered, and allowing antibiotic sensitivity testing while maintaining the integrity of the pathogen. Furthermore, it improves test reliability by allowing the concentration of the pathogen, which is crucial for the reproducibility of antibiotic sensitivity testing, to be adjusted to a certain level before injection into the test. Therefore, using blood culture for testing instead of pure culture has the disadvantage of having to account for errors caused by these factors. However, due to the significant advantages of shortening the testing time by more than one day and the increasing demand in emergency situations, continued product development efforts are underway. For this product, various technologies are being introduced to minimize the shortcomings of using blood samples, such as detection methods that are less affected by pathogen concentration and deriving results through machine learning.

[0004] The present invention provides a method for minimizing the preparation time required for antibiotic sensitivity testing while preventing the aforementioned negative effects by performing the process of isolating, purifying, concentrating, and efficiently culturing pathogens immediately after collecting patient samples (without the need for blood culture or pure culture procedures). In other words, the present invention provides a procedure and method that includes the entire sample-to-answer process from patient sample to very rapid, highly sensitive identification and derivation of antibiotic sensitivity test results.

[0005] The reasons why it takes a long time to culture pathogens from patient samples are as follows: 1. The growth of pathogens is inhibited by blood components and residual antibiotic components administered in the body. 2. Starting from an initial concentration of 10 CFU / ml or lower to 10 8 To achieve a final concentration of CFU / ml or higher, it must undergo approximately 23 or more divisions. Because each pathogen's division cycle varies, and the time required to reach the desired number is predetermined, shortening this time is difficult. According to current diagnostic laboratory protocols, blood and pure cultures are cultured for a sufficient period of time to allow the pathogen to reach saturation. Therefore, if impurities that interfere with culture are removed early, culture is performed in a solution optimized for pathogen growth, and the culture sample, after being cultured to the minimum concentration required for antibiotic sensitivity, is used directly for testing, the detection time can be significantly shortened. To achieve this goal, a method is needed that can selectively isolate only bacteria, and this selection process is expected to have the effect of concentrating and purifying the bacteria in the sample. Currently, many methods are available, such as centrifugation, filtration, and methods using various probes attached to solid matrices, but they have limitations in sensitivity and specificity, making their effectiveness difficult to predict, especially in whole blood samples containing a variety of complex substances.

[0006] Accordingly, the present invention proposes a method for rapidly performing antibiotic sensitivity testing using small amounts of pathogens (pre-saturation). This method achieves pathogen identification and further pathogen enumeration using small amounts of pathogens by selecting and isolating pathogens, such as bacteria, present in patient samples, optimizing the culture environment, and purifying the sample. Several methods exist for isolating bacteria, but a method utilizing magnetic capture beads coated with specific molecular proteins or peptides capable of binding to bacteria will be described as an example. More specifically, the following steps can be performed: first, collecting a patient sample; treating the patient sample with magnetic capture beads to transfer and concentrate small amounts of pathogens into pure / clarified culture medium; measuring the pathogen concentration in real time during culture in the culture medium; and, if necessary, adding a step to concentrate pathogens bound to the magnetic capture beads for a washing process. Pathogen identification testing can be performed after pathogen isolation or at a desired time after the isolation and culture process. Finally, the present invention proposes a methodology consisting of performing antibiotic sensitivity testing when a certain number of pathogens have been concentrated, and reporting the results of the antibiotic sensitivity testing. This method aims to demonstrate that results can be obtained in a shorter time than previously possible. Summary of the Invention

[0007] Technical issues

[0008] In order to solve the above problems, the present invention aims to provide a rapid antibiotic sensitivity detection and identification method, which can eliminate the traditional blood culture step, shorten the time required for the steps required for pathogen growth including pure culture, and quickly provide information on antibiotics suitable for microorganisms.

[0009] Technical Solution

[0010] In order to solve the above problems, a rapid antibiotic sensitivity detection and identification method is provided, comprising the following steps: (a) transferring a sample extracted from a patient to a first reaction space, wherein a sample separation device is present in the first reaction space; (b) capturing pathogens by the separation device in the first reaction space to separate and concentrate the pathogens in the sample from the patient; and (c) performing identification of the pathogen or antibiotic sensitivity testing.

[0011] In one embodiment, the patient sample is whole blood.

[0012] In one embodiment, the capture of the pathogens can be accomplished using substances that can specifically bind to Gram-positive bacteria, Gram-negative bacteria, and fungi.

[0013] In one embodiment, the substance capable of specific binding may be at least one selected from the group consisting of antibodies, ApoH, aptamers and polymers.

[0014] In one embodiment, the separation device can be a matrix, a filter or magnetic particles with capture molecules.

[0015] In one embodiment, after step (b), the method may further comprise the step (b') of culturing the captured pathogen in a culture medium.

[0016] In one embodiment, step (b') may include the following steps: monitoring the increase in the concentration of the pathogen in the culture solution; and when the concentration of the pathogen in the culture solution is higher than a preset concentration, separating a portion of the culture solution for antibiotic sensitivity testing or a portion of the culture solution for pathogen identification testing from all or at least a portion of the culture solution.

[0017] In one embodiment, the method may include: when the concentration of the pathogen in the culture medium in step (b') is 10 3 When the CFU / ml is higher, a portion of the culture medium for pathogen identification detection is transferred to the identification module.

[0018] In one embodiment, the method may include: 3 CFU / ml or higher, a portion of the culture solution used for antibiotic sensitivity testing is transferred to a pretreatment module for antibiotic sensitivity testing.

[0019] In one embodiment, the following steps can be performed in the pretreatment module for antibiotic sensitivity: (i) transferring a portion of the culture solution for antibiotic sensitivity detection to a second reaction space in which magnetic particles having capture molecules are present; (ii) separating and concentrating pathogens in the second reaction space; (iii) monitoring the increase in the concentration of the pathogen in the portion of the culture solution; (iv) when the concentration of the pathogen in the portion of the culture solution is 10 3 CFU / ml or higher, transferring a portion of the culture solution to an antibiotic sensitivity detection module; and (v) performing the antibiotic sensitivity detection on the pathogen in the portion of the culture solution.

[0020] In one embodiment, it may further comprise the step of diluting a portion of the culture solution before performing the antibiotic sensitivity test.

[0021] In one embodiment, the step (c) may further comprise the step of using the results of the pathogen identification test to select a test drug for antibiotic sensitivity testing.

[0022] In addition, the present invention provides a device for rapid antibiotic sensitivity detection and identification detection, including: a first reaction space, used to separate and concentrate pathogens from patient samples; a pathogen identification detection module, used to perform identification detection on the pathogens separated and concentrated in the first reaction space; an antibiotic sensitivity detection module, used to detect the antibiotic sensitivity of the pathogens separated and concentrated in the first reaction space; and a control module, used to control the first reaction space, the pathogen identification detection module, and the antibiotic sensitivity detection module, and output each progress status and result.

[0023] In one embodiment, the first reaction space can be manufactured as a cassette containing 1 to 10 sub-reaction spaces.

[0024] In one embodiment, the first reaction space may include one or more sub-reaction spaces, and the one or more sub-reaction spaces are loaded with a separation device equipped with a plurality of capture molecules.

[0025] In one embodiment, the antibiotic sensitivity detection module may include a pretreatment module for antibiotic sensitivity detection; and the pretreatment module for antibiotic sensitivity detection may include a second reaction space for secondary separation and concentration of the pathogens separated and concentrated in the first reaction space.

[0026] In one embodiment, the second reaction space can be manufactured as a cassette containing 1 to 10 sub-reaction spaces.

[0027] In one embodiment, the second reaction space may include at least one sub-reaction space, and the at least one sub-reaction space is loaded with a separation device equipped with a plurality of capture molecules.

[0028] Beneficial effects

[0029] The rapid antibiotic sensitivity detection and identification method of the present invention eliminates the blood culture process and allows for highly sensitive identification of pathogens isolated and purified from samples. Furthermore, by promoting and monitoring the culture process under optimal conditions, antibiotic sensitivity testing can be performed using the minimum number of pathogens required for sensitive detection. Consequently, it provides information on antibiotics suitable for microorganisms, enabling rapid and accurate antibiotic administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the overall structure and flow chart of a rapid antibiotic sensitivity detection and identification method according to one embodiment of the present invention.

[0031] Figure 2 A flow chart of pathogen identification and antibiotic sensitivity testing according to one embodiment of the present invention is shown.

[0032] Figure 3 A method and results of separating and purifying pathogens using magnetic particles according to one embodiment of the present invention are shown.

[0033] Figure 4 Shown are a method for identifying and detecting a resistance gene of an isolated pathogen and the results thereof according to one embodiment of the present invention.

[0034] Figure 5 The method and results of antibiotic sensitivity detection according to one embodiment of the present invention are shown.

[0035] Figure 6 The method and results of antibiotic sensitivity detection according to one embodiment of the present invention are shown. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention will be described in detail below. When describing the present invention, if it is determined that a detailed description of related known technologies may obscure the main purpose of the present invention, the detailed description will be omitted. Throughout the specification, when a part is described as "including" certain components, this does not mean that other components are excluded, but rather that other components may be included, unless otherwise specifically stated.

[0037] The present invention can be modified in various ways and has various embodiments, and specific embodiments have been explained and described in detail in the detailed description. However, this is not intended to limit the present invention to the specific embodiments, but should be understood to include all modifications, equivalents or substitutes within the spirit and technical scope of the present invention.

[0038] The technology disclosed in this specification is not limited to the embodiments described herein and may be embodied in other forms. However, the embodiments introduced herein are provided to ensure the comprehensiveness and completeness of the disclosure and to ensure that the technical ideas and concepts of the technology can be fully conveyed to those skilled in the art. When describing the overall drawings, they are described from the perspective of the observer. When referring to a component being located above another component, the meaning included is that the component is directly located above the other component, or an additional component can be inserted between the components. In addition, those skilled in the art will be able to implement the concept of the present invention in various other forms without departing from the technical concept of the present invention.

[0039] The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. Unless the context clearly indicates otherwise, singular expressions should be understood to include plural expressions. And terms such as "including" or "having" are intended to specify the presence of features, numbers, steps, operations, components, parts, or a combination thereof described in the specification, and should be understood as not further excluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or a combination thereof. In addition, when performing the method or manufacturing method, unless the context clearly indicates a specific order, the process constituting the method can be performed in an order different from the order described. That is, each process can be performed in the same order as the order described, can be performed substantially simultaneously, or can be performed in the opposite order.

[0040] Meanwhile, the meanings of the terms used in this specification should be understood as follows. Terms such as "first" or "second" are intended to distinguish one component from another, and the scope of rights should not be limited by these terms. For example, a first component can be named a second component, and similarly, a second component can also be named a first component.

[0041] As used herein, the term "and / or" includes any combination of a plurality of listed items or any one of the plurality of listed items. As used herein, "A or B" can include "A," "B," or "A and B."

[0042] The present invention relates to a rapid antibiotic sensitivity detection and identification method, comprising the following steps: (a) transferring a sample extracted from a patient to a first reaction space, wherein a sample separation device is present in the first reaction space; (b) capturing pathogens by the separation device in the first reaction space to separate and concentrate the pathogens in the sample from the patient; and (c) identifying the pathogens or performing antibiotic sensitivity testing.

[0043] Step (a) is a step of collecting a patient sample, i.e., collecting body fluids or secretions to confirm whether the patient is infected and the type of microorganisms. In this case, the body fluids or secretions may include blood, lymph, tears, nasal mucus, saliva, sweat, semen, vaginal secretions, urine, feces, pus, etc., and may not only be simply collected body fluids or secretions, but also a portion of separated body fluids or secretions. As an example, the patient's sample may be blood, preferably whole blood, or may be plasma, white blood cells, red blood cells, or platelets separated from whole blood.

[0044] Furthermore, when whole blood is used as a patient sample, 1 to 10 ml can be used. In the case of a typical blood donation, 300 to 500 ml of blood is collected, but in the case of patients undergoing the above-mentioned tests, since their physical strength will be significantly reduced, the amount of blood collected must be minimized as described above. Furthermore, since whole blood collected from the patient can be used for various tests and identification tests and sensitivity tests such as those in the present invention, it is more desirable to use less whole blood in the present invention to reduce the burden on the patient. However, it is known that in typical bacteremia cases, approximately 1 to 10 pathogens are present in 1 ml of blood, so a certain amount of whole blood is required to stably preserve the pathogens. Therefore, if the whole blood used for the above-mentioned analysis is less than 1 ml, it may be difficult to stably preserve the pathogens; if it exceeds 10 ml, it may place a burden on the patient.

[0045] In step (b), the patient sample collected as described above can be transferred to the first reaction space. The first reaction space is a space in which the magnetic particles having the capture molecules are present. At this time, the reaction space can be simply a container capable of storing liquids, such as a vial, a beaker or a flask, or a well plate provided with a plurality of holes. In addition, the first reaction space is in the form of a cartridge, so that after completing a test, the reaction space can be removed and a new first reaction space cartridge can be inserted for detection. This can not only minimize the false positives caused by cross-contamination, but also allow for rapid preparation for the next test. In addition, in the case of the above-mentioned cartridge, not only the reaction space is included, but also the magnetic particles and chemicals required for separating pathogen cells are included, so that a separate preparation process can be ignored. In other words, the user of the present invention can simply prepare the required magnetic particles and cell separation agent by replacing the cartridge after completing a test.

[0046] In addition, by further expanding this process, the cartridge can include not only a first reaction space, but also a space for separating pathogens and a space for cultivating pathogens, which will be described later. In this case, it is even possible to cultivate pathogens within one cartridge. In addition, when the cultivation space for the pathogen is included in the cartridge, it is more preferred that the cartridge not only include a separation agent, but also a culture medium and a culture agent for cultivating the pathogen. In addition, the above-mentioned cartridge may include 1 to 10 sub-reaction spaces. In the case of separation and concentration of pathogens, it can be carried out within one reaction space or across multiple reaction spaces. Accordingly, in the case of the cartridge, 1 to 10 sub-reaction spaces can be set, and accordingly, the separation and concentration of the pathogen in each reaction space can be carried out smoothly. In addition, since the sub-reaction space is located within one cartridge, new sub-reaction spaces can be installed and used simultaneously by replacing the cartridge.

[0047] As described above, the sample transferred to the first reaction space can separate pathogens from the patient sample.

[0048] At this time, a separation device to which a substance capable of specifically binding to the pathogen is attached may be used, and a matrix, filter, or magnetic particles equipped with capture molecules may be used as the separation device to facilitate the analysis described below.

[0049] Capturing these pathogens can be accomplished using a substance that specifically binds to Gram-positive bacteria, Gram-negative bacteria, and fungi. The substance capable of specific binding can be at least one selected from the group consisting of antibodies, ApoH, aptamers, and polymers. The following explanation will focus on the use of magnetic particles as a separation device and ApoH as the substance capable of specific binding.

[0050] At this time, as explained above, the first reaction space contains magnetic particles inside, and in the case of the present invention, pathogens can be separated from the sample using these magnetic particles.

[0051] From a more detailed perspective, magnetic particles refer to particles coated with probe materials, which include proteins, peptides, antibodies and genes that can specifically bind to pathogens on the surface made of magnetic material. At this time, in addition to the above-mentioned pathogen binding materials, a coating process that can prevent non-specific material defects can be added. In this way, pathogens in the blood can be separated. Magnetic particles can be made into various shapes, such as spherical or cylindrical. In addition, pathogen separation can also be carried out by using various magnetic particles coated with a combination of various pathogen binding substances to achieve the separation of multiple pathogens, rather than just one type of magnetic particle. In addition to the pathogen separation method using magnetic particles as an example, physical methods using filters can also be appropriately applied according to the type of sample. In one embodiment, the magnetic particles perform a pathogen separation process by attaching apolipoprotein H (ApoH) protein or an ApoH peptide synthesized from its functional group to a magnetic core.

[0052] At this time, the ApoH method is as follows.

[0053] Isolation and purification of apolipoprotein H from human serum albumin

[0054] ApoH was purified from a human plasma albumin solution as described. Briefly, a human albumin solution from Cohn plasma supernatant IV was added to a column of cellulose sulfate beads (Chisso, Japan) pre-equilibrated with 0.15 M NaCl. After washing with 10 mM phosphate buffer (pH 7.4) to 0.2 M NaCl, a portion was eluted with a 2 M NaCl gradient, then diluted 10-fold in 10 mM sodium phosphate buffer (pH 6.8) and loaded onto a hydroxyapatite gel (Biorad, USA) pre-equilibrated with the same buffer. The gel was then washed with the same buffer, and ApoH was eluted by increasing the ionic strength with 1 M KCl. The resulting solution was dialyzed against distilled water and freeze-dried. At this stage, the purity of ApoH was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%), which revealed a single band at 50 kDa. The Apoh thus purified was coupled to nanomagnetic beads (ApoH-Technologies, France).

[0055] When a sample is injected into the reaction space, pathogens can adhere to the surface of the magnetic particles. Therefore, the sample mixed with the magnetic particles in the reaction space can be stirred for 15 to 30 minutes in a thermomixer or shaking incubator to promote pathogen attachment. This stirring can also be achieved by applying an external magnetic field to move the magnetic particles up and down or left and right, which can also be implemented using a microfluidic chip. Furthermore, to achieve rapid movement of the magnetic particles, the reaction space is flipped up and down 1 to 5 times to allow the pathogens to mix with the magnetic particles.

[0056] After the mixing is completed as described above, the pathogens adhere to the surface of the magnetic particles, and the magnetic particles can be separated from the sample. At this time, since the magnetic particles are magnetic, they can be attached to the side or bottom of the reaction space using a magnet. In addition, a magnetic field can be formed at the bottom of the reaction space to fix the magnetic particles at the bottom of the reaction space. The remaining sample can then be discharged to separate the magnetic particles. This separation method can be appropriately selected according to the structure of the reaction space and the cartridge. Preferably, when multiple reaction spaces (sub-reaction spaces) are used in the cartridge, the reaction can be carried out by moving the magnetic particles using a magnet or a device or material that generates magnetism; and when there is only one sub-reaction space in the cartridge, the magnetic particles can be separated by forming a magnetic field on the side or bottom of the sub-reaction space.

[0057] The magnetic particles separated from the sample as described above can be washed 1 to 10 times as needed. Even after the magnetic particles are separated, sample components such as white blood cells, red blood cells, and platelets that have nonspecifically attached to the magnetic particles, in addition to pathogens, may remain on the surface. If these components are cultured and detected as is, they may become foreign matter, reducing the reliability of the detection. Therefore, to minimize these sample components, the magnetic particles can be washed 1 to 10 times as needed. If washed more than 10 times, the time and reagents required for detection will increase, and pathogens attached to the surface of the magnetic particles may also be washed away, which may reduce the accuracy of the detection. When using a single reaction space, the above-mentioned cleaning can be performed by repeatedly injecting and draining the cleaning solution into the reaction space. When using multiple reaction spaces, it can be performed by sequentially moving the magnetic particles into the reaction space containing the cleaning solution.

[0058] After step (b), the method may further include a step (b') of culturing the captured pathogen in a culture medium. Although additional cultivation of the pathogen may be performed in a subsequent stage according to the needs of each test, each cultivation may result in a significant waste of culture medium and space, which may increase the cost of the test. Therefore, by isolating the pathogen in the above step (b) and then culturing it to a certain concentration, the overall detection cost and time can be reduced. This additional cultivation is possible even when attached to magnetic particles, and the additional cultivation can also be performed separately after the pathogen is separated from the magnetic particles.

[0059] In addition, step (b') may include the following steps: monitoring the increase in the concentration of the pathogen in the culture solution; and when the concentration of the pathogen in the culture solution is higher than a preset concentration, separating a portion of the culture solution for antibiotic sensitivity testing or a portion of the culture solution for pathogen identification testing from at least a portion or all of the culture solution.

[0060] The step of monitoring the increase in concentration of the pathogen can use an optical method, which is the same as the monitoring method of step (g) below.

[0061] Through the above monitoring, if the pathogen concentration in the culture solution becomes higher than a predetermined concentration, at least a portion or the entire culture solution can be divided into a portion of the culture solution for antibiotic sensitivity testing or a portion of the culture solution for pathogen identification testing. In other words, a portion of the culture solution is divided into a portion of the culture solution for antibiotic sensitivity testing or a portion of the culture solution for pathogen identification testing and used in each step.

[0062] As described above, pathogens attached to the surface of the magnetic particles can be injected into the culture medium without or after cleaning. Subsequently, through step (c), a portion of the culture medium used for identification testing and a portion of the culture medium used for antibiotic sensitivity testing can be separated and transferred to separate devices. For identification testing and sensitivity testing, steps (e) and (fh) can be performed independently and simultaneously in the second and third reaction spaces, respectively.

[0063] In the pathogen identification and detection module of step (e), the following steps can be performed: i) transferring a portion of the culture medium used for identification and detection to a second reaction space; ii) cleaning the pathogens in the second reaction space; iii) lysing the pathogens in the portion of the culture medium; iv) labeling and amplifying the extracted genes; and v) reacting the pathogen genes with encoded microbead particles coated with complementary probe genes, and performing identification and detection of the pathogens through fluorescent signals.

[0064] In one embodiment, in the case of identification testing, the culture medium used for identification testing separated in step (c) is transferred to a second reaction space, the pathogen is lysed, and the gene region containing the pathogen type and whether it has resistance is labeled and amplified. In this case, depending on the purpose of removing factors that interfere with the PCR process, the pathogen containing magnetic particles can be washed 1 to 10 times in a manner similar to step (b) before amplifying the above sample. In addition, in the case of identification testing, the number of pathogens is 1 to 5 CFU or less, that is, it can be detected. However, in this case, for more stable detection, step (e) can also be performed after additional culture, as in the antibiotic sensitivity test described later. In this case, the number of pathogens required for antibiotic sensitivity testing is not used, so it can be completed in a short time (1 to 2 hours). The above-mentioned amplified genes indicating identification and antibiotic resistance can be mixed and bound to various types of beads loaded with probes for each target gene. By adding fluorescent particles, the presence of the pathogen in the sample, whether the pathogen is Gram-positive or Gram-negative, and the identification and resistance genes of the pathogen can be confirmed. The type of beads capable of detecting pathogen identification and resistance genes can be identified by more than 200 (multiple) codes patterned on the surface. The results of this pathogen identification test are transmitted to the antibiotic sensitivity test module described later so that the test can be carried out using the appropriate reagents and antibiotics.

[0065] In the pretreatment module for antibiotic sensitivity detection in steps (e) and (g), the following steps may be performed: i) transferring a portion of the culture solution for antibiotic sensitivity detection to a third reaction space; ii) culturing pathogens in the third reaction space; iii) monitoring the increase in the concentration and total number of pathogens in the portion of the culture solution; iv) monitoring the concentration of the pathogens in the portion of the culture solution when the concentration of the pathogens in the portion of the culture solution becomes 10 3 CFU / ml or higher, transferring a portion of the culture solution to an antibiotic sensitivity detection module; and v) performing antibiotic sensitivity detection on the pathogens in the portion of the culture solution (h).

[0066] Unlike identification tests that can be performed with small amounts of pathogens, antibiotic sensitivity tests typically require large amounts of pathogens to detect various types and concentrations of antibiotics. Therefore, the pathogens separated from the magnetic particles and transferred to the third reaction space designated above can be cultured in a suitable environment until a certain concentration is reached in step (f) to form a culture solution. At this point, the culture can be carried out in a small amount of culture medium suitable for detection and culture, and additional culture solution can be supplied as needed. At this point, the pathogens can be cultured up to 10 3 CFU / ml or higher concentration. In addition, the cultivation process can include a process for separating defective pathogens in the magnetic particles to promote the growth of pathogens. At this time, it is preferred to use a liquid culture medium as a culture medium for ease of operation, and specifically, the liquid culture medium can include a natural culture medium optimized for the pathogen type, a synthetic culture medium with added nutrients, a restricted culture medium, a complex culture medium, etc. In this process, the effect of concentrating the pathogen can be achieved, and the number and concentration of the pathogen can be increased simultaneously through the cultivation process. Cultivation can be carried out in a culture space configured to allow gas exchange, and the culture space can be included in a cartridge, or installed separately from the cartridge, and can include a heating unit to form a suitable temperature. In addition, for anaerobic strains, the cultivation effect can be optimized by providing separate carbon dioxide.

[0067] As described above, the pathogen separated from the third reaction space is in a purified state, and impurities contained in the sample (including blood cells in the case of whole blood) are removed and present in a transparent culture medium, so it can be optically monitored by step (g). Since the division and growth rates vary depending on the type of isolated pathogen, unlike the conventional blood culture method, which requires the culture of an unnecessary large number of pathogens, regardless of the type of pathogen, when the minimum number of pathogens required for antibiotic sensitivity testing is reached, detection by optical observation can have the effect of shortening the detection time to the shortest possible time. At this time, according to the kit used for antibiotic sensitivity testing described later, the culture medium can be cultured for 103 Pathogen concentrations of CFU / ml or higher. This monitoring step can measure and display the number of pathogens in the culture medium in real time to confirm whether the pathogens are being properly cultured and also enables the separation and transfer of the culture medium at the appropriate time. Pathogen concentration can be measured optically, using lasers, or using ultrasound, and any method capable of measuring pathogens in culture medium can be used without limitation. In one embodiment, holographic imaging is used, but other optical methods can also be used instead. Specifically, part or all of the space containing the culture medium can be made optically transparent. In particular, a flat structure can be mounted within the optically transparent portion to facilitate laser transmission. In the case of culture medium, since the culture medium is regularly mixed and heated for culture efficiency, the culture medium can have a uniform concentration when it is present within the structure. By periodically illuminating the structure with laser light and measuring changes in amplitude or phase, the pathogen concentration in the culture medium can be measured. Furthermore, during monitoring, by measuring the pathogen concentration at the beginning of the culture and then measuring the pathogen concentration at each time point and comparing them, it is possible to determine when the desired pathogen concentration has been reached.

[0068] As described above, after the culture is completed in the third reaction space, a portion of the culture fluid can be transferred to the antibiotic sensitivity test module, and the antibiotic sensitivity test can be performed according to step (h). In this case, in the case of antibiotic sensitivity testing, appropriate reagents and antibiotics need to be used based on the results of the pathogen identification test. In one embodiment, for antibiotic sensitivity testing, the cultured pathogens are mixed with a heated and liquefied agar solution and dispensed into a cartridge for antibiotic sensitivity testing, and the test results are derived through time-lapse imaging.

[0069] At this point, the process of separating and washing pathogens from the magnetic particles can be added as needed. To this end, the culture fluid can be washed with a solution or culture medium having a characteristic composition. During this process, the binding force between the substance on the surface of the magnetic particles and the pathogens may be weakened, allowing the pathogens to be separated from the magnetic particles. In order to recover the pathogens lost from the magnetic particles, the same method as the pathogen separation process in the first reaction space is used, but here, the supernatant can be recovered and used to recover the pathogens lost from the magnetic particles. The method of separating pathogens can include applying physical forces such as vibration, and adding chemicals that can cut off the binding sites between the magnetic particles and the pathogens.

[0070] The antibiotic sensitivity test module has a well plate consisting of multiple wells, into which the cultured culture fluid can be injected for antibiotic sensitivity testing. At this time, 1 to 4 sub-wells can be formed inside the well plate, and the culture fluid can be injected into one of these sub-wells. The antibiotic selected based on the results of pathogen identification is injected or solidified in another sub-well, or after the culture fluid is injected into the independent space of the sub-well, the culture fluid is injected and then injected into the antibiotic by injecting physiological saline solution or culture medium, so that the antibiotic can be dissolved and contacted with the culture fluid. At this time, the contact between the culture fluid and the antibiotic can be carried out through a point or surface, and the sensitivity of the antibiotic can be tested by observing the reaction of the pathogen in the culture medium that contacts the antibiotic at this contact point or surface.

[0071] In the case of the present invention, by eliminating the blood culture process that is considered necessary and by optimizing and minimizing the isolation of pathogens in whole blood and the culture steps after isolation, pathogen identification testing and antibiotic sensitivity testing and identification are performed, and the testing can be completed in a shorter time period than existing methods. In addition, in the case of the present invention, culture, pathogen identification testing and antibiotic sensitivity testing can be performed in an integrated solution. In the case of antibiotic sensitivity testing, since the type of reagent and antibiotic must be determined based on the results of the pathogen identification test after the pathogen identification test is performed, in the existing methods, it is performed in a time series manner, but in the case of the present invention, the identification test is performed using some isolated pathogens, and the remaining part is cultured during the time of the pathogen identification test, so the test can be completed at a faster rate than the existing methods. In the existing methods, a portion of the culture fluid after blood culture is collected and transferred and supplied to the pathogen identification tester and the antibiotic sensitivity tester for each test. There is a case where the results are erroneous due to contamination caused by the distribution and injection of the culture fluid. However, in the present invention, culture, pathogen identification testing, and antibiotic sensitivity testing can be performed in a single device. This means that the results of the pathogen identification test and antibiotic sensitivity test can be simply output by injecting a patient sample into the device. Furthermore, in the present invention, a cartridge is used in the culture step, while a cartridge-type device such as a well plate is used for the pathogen identification test and antibiotic sensitivity test. Therefore, after a test is completed, errors due to cross-contamination can be minimized by replacing all components that come into contact with the culture medium with a new cartridge.

[0072] In addition, in the case of the above-mentioned antibiotic sensitivity test and pathogen identification test, they can be performed simultaneously or each part can be performed independently. At the same time, even if they are performed simultaneously, they can also be completed sequentially or simultaneously.

[0073] As described above, the specific parts of the present invention have been described in detail, so it will be obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the essential scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A rapid antibiotic sensitivity detection and identification method comprising the following steps: (a) transferring a sample extracted from a patient into a first reaction space, wherein a sample separation device is present in the first reaction space; (b) separating and concentrating pathogens in the sample from the patient by capturing them with a separation device in the first reaction space; and (c) performing identification of the pathogen or antibiotic sensitivity testing.

2. The rapid antibiotic sensitivity detection and identification method according to claim 1, wherein the patient's sample is whole blood.

3. The rapid antibiotic sensitivity detection and identification method according to claim 1, wherein the pathogen is captured using a substance that can specifically bind to Gram-positive bacteria, Gram-negative bacteria and fungi. 4 . The rapid antibiotic sensitivity detection and identification method according to claim 3 , wherein the substance capable of specific binding is at least one selected from the group consisting of antibodies, ApoH, aptamers and polymers.

5. The rapid antibiotic sensitivity detection and identification method according to claim 1, wherein the separation device is a matrix with capture molecules, a filter or a magnetic particle matrix.

6. The rapid antibiotic sensitivity detection and identification method according to claim 1, further comprising a step (b') of culturing the captured pathogen in a culture medium after step (b).

7. The rapid antibiotic sensitivity detection and identification method according to claim 6, wherein the step (b') comprises the following steps: monitoring an increase in the concentration of a pathogen in the culture medium; and When the concentration of the pathogen in the culture fluid is higher than a preset concentration, a portion of the culture fluid for antibiotic sensitivity testing or a portion of the culture fluid for pathogen identification testing is separated from all or at least a portion of the culture fluid.

8. The rapid antibiotic sensitivity detection and identification method according to claim 6, wherein when the concentration of the pathogen in the culture medium in step (b') is 10 3 When the CFU / ml is higher, a portion of the culture medium for pathogen identification detection is transferred to the identification module.

9. The rapid antibiotic sensitivity detection and identification method according to claim 6, wherein when the concentration of the pathogen is 10 3 CFU / ml or higher, a portion of the culture solution used for antibiotic sensitivity testing is transferred to a pretreatment module for antibiotic sensitivity testing.

10. The rapid antibiotic sensitivity detection and identification method according to claim 9, comprising the following steps performed in the pretreatment module for antibiotic sensitivity: (i) a step of transferring a portion of the culture solution for antibiotic sensitivity detection to a second reaction space in which magnetic particles having capture molecules are present; (ii) a step of separating and concentrating the pathogens in the second reaction space; (iii) monitoring the increase in the concentration of the pathogen in a portion of the culture fluid; (iv) when the concentration of the pathogen in a portion of the culture solution is 10 3 CFU / ml or higher, transferring a portion of the culture solution to an antibiotic sensitivity detection module; and (v) performing the antibiotic sensitivity test on the pathogen in a portion of the culture solution.

11. The rapid antibiotic sensitivity detection and identification method according to claim 10, further comprising the step of diluting a portion of the culture solution before performing the antibiotic sensitivity detection.

12. The rapid antibiotic sensitivity detection and identification method according to claim 1, wherein the step (c) further comprises the step of using the identification test results of the pathogen to select a test drug for antibiotic sensitivity detection.

13. A device for rapid antibiotic sensitivity detection and identification, comprising: a first reaction space for separating and concentrating pathogens from patient samples; a pathogen identification and detection module, configured to perform identification and detection of the pathogens separated and concentrated in the first reaction space; an antibiotic sensitivity detection module, configured to detect the antibiotic sensitivity of the pathogens separated and concentrated in the first reaction space; A control module is used to control the first reaction space, the pathogen identification and detection module, and the antibiotic sensitivity detection module, and output respective progress states and results. 14 . The rapid antibiotic sensitivity detection and identification device according to claim 13 , wherein the first reaction space is manufactured as a cartridge containing 1 to 10 sub-reaction spaces. 15 . The rapid antibiotic sensitivity detection and identification device according to claim 14 , wherein the first reaction space comprises one or more sub-reaction spaces, and the one or more sub-reaction spaces are loaded with a separation device equipped with a plurality of capture molecules.

16. The rapid antibiotic sensitivity detection and identification device according to claim 13, wherein the antibiotic sensitivity detection module comprises a pre-processing module for antibiotic sensitivity detection; The pretreatment module for antibiotic sensitivity detection includes a second reaction space for secondary separation and concentration of the pathogens separated and concentrated in the first reaction space. 17 . The rapid antibiotic sensitivity detection and identification device according to claim 16 , wherein the second reaction space is manufactured as a cartridge containing 1 to 10 sub-reaction spaces.

18. The rapid antibiotic sensitivity detection and identification device according to claim 17, wherein the second reaction space comprises at least one sub-reaction space, and the at least one sub-reaction space is loaded with a separation device equipped with a plurality of capture molecules.

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