Methods and kits for determining antibiotic sensitivity

Abstract

The present disclosure relates to a method of determining antibiotic sensitivity of a microorganism in a sample, the method comprising: receiving a sample comprising the microorganism; incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample without antibiotics; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with microorganisms present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids from the at least one first portion and the at least one second portion of the sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority or the benefit of International Patent Application No. 202241031205 filed on May 31, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of sample analysis and more particularly to the field of determining the antibiotic sensitivity of microorganisms. Background Art

[0004] The rate of antibiotic resistance in microorganisms is steadily increasing. This makes it necessary to administer appropriate and adequate antibiotic treatment to patients. Current methods for determining antibiotic resistance are slow and do not provide complete information on antibiotic susceptibility.

[0005] Traditionally, methods based on microbial culture are used to determine antibiotic sensitivity or resistance, and it takes about 3 to 4 days to obtain clinically feasible results. Methods based on polymerase chain reaction (PCR) can also be used to determine antimicrobial resistance information. However, the turnaround time of such genotypic methods is about 2 to 3 hours, and the amount of antimicrobial resistance information obtained is limited. These tests need to be continuously developed and improved to include evolutionary genetic characteristics from resistant microorganisms.

[0006] Interesting prior art includes: U.S. Patent Publication No. 20170058314 to Zhang et al., U.S. Patent Publication No. 20230054472 to Varma et al., entitled Compositions, devices and methods for diagnosing and treating infectious disease, and U.S. Patent No. 9,677,109 (the entire contents of which are incorporated herein by reference in their entirety).

[0007] Therefore, there is a continuing need for an effective method to determine the antibiotic susceptibility of a microorganism that is rapid and provides complete information related to antimicrobial resistance to help physicians make timely clinical decisions. Summary of the invention

[0008] Provided herein are methods, kits, and devices for determining antibiotic sensitivity of one or more microorganisms. In some embodiments, methods for determining antibiotic sensitivity of one or more bacterial microorganisms in a sample, such as a biological sample, are disclosed.

[0009] In some embodiments, the method of the present disclosure includes receiving a sample comprising one or more microorganisms. In addition, the method includes incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic. The method also includes extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganisms present in the sample. In some embodiments, the extracted nucleic acids are amplified, and antibiotic sensitivity information associated with the microorganisms is obtained from the observed differences in the results of the amplified nucleic acids with and without the antibiotic response.

[0010] In some embodiments, the present disclosure includes a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: receiving a sample comprising a microorganism; incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; contacting at least one first portion and / or at least one second portion with one or more intercalating dyes; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with the microorganism present in the sample; amplifying the extracted nucleic acid from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid from at least one first portion and at least one second portion of the sample. In some embodiments, the intercalating dye can be ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, and the like.

[0011] In some embodiments, the methods of the present disclosure include incubating or culturing at least one first portion of a biological sample containing one or more bacterial microorganisms with at least one antibiotic, and incubating or culturing at least one second portion of the sample in the absence of the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganisms present in the sample; and amplifying the extracted nucleic acids so that antibiotic sensitivity information associated with the microorganisms is obtained from the differences observed in the results of the amplified nucleic acids with and without the antibiotic response.

[0012] In some embodiments, the methods of the present disclosure include incubating or culturing at least one first portion of a biological sample containing one or more bacterial microorganisms with at least one antibiotic, and incubating or culturing at least one second portion of the sample without the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganisms present in the sample; contacting the extracted nucleic acids with one or more intercalating dyes; and amplifying the extracted nucleic acids so that antibiotic sensitivity information associated with the microorganisms is obtained from the differences observed in the results of amplified nucleic acids with and without antibiotic reactions. In some embodiments, contacting the extracted nucleic acids with one or more intercalating dyes also includes applying light (e.g., UV light) under conditions sufficient to associate the extracted nucleic acids with the intercalating dyes or form a nucleic acid / intercalating dye complex. For example, the intercalating dye can be ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, and the like.

[0013] In some embodiments, the present disclosure includes a kit for determining the antibiotic sensitivity of a microorganism in a sample. In some embodiments, the kit includes one or more antibiotics. The antibiotic can be provided in a limited amount so as to achieve an effective result. The kit may also include a reaction volume specific to the growth conditions of the microorganism that may be present in the sample. In addition, the kit may include a culture medium for the growth of microorganisms present in the sample. The volume of the growth culture medium may be limited to achieve the best results for the growth of microorganisms. In addition, in some embodiments, the kit includes reagents based on nucleic acid extraction and reagents based on nucleic acid amplification. Specifically, the nucleic acid amplification reagent may include one or more primers for 16srRNA and / or 16srDNA of the microorganism in the target sample. In addition, the nucleic acid amplification kit may also include one or more TaqMan probes. In another embodiment, the kit includes a melt analysis software based on polymerase chain reaction. In yet another embodiment, the kit includes at least one intercalating dye. For example, the dye may be ethidium monoazide, ethidium monoazide bromide, propidium monoazide, isomers thereof, etc.

[0014] In some embodiments, the present disclosure includes a kit for determining the antibiotic sensitivity of a microorganism in a sample, the kit comprising: one or more antibiotics; a growth medium for the microorganism; reagents associated with nucleic acid extraction; reagents associated with nucleic acid amplification; and polymerase chain reaction-based melting analysis software. In some embodiments, the kit comprises instructions for using the kit and / or the software. In some embodiments, the kit comprises one or more intercalating dyes.

[0015] In some embodiments, the present disclosure includes an article, such as a system or component thereof, comprising a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method of determining antibiotic sensitivity of a microorganism in a sample, the method comprising: incubating at least one first portion of the sample containing the microorganism with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with the microorganism present in the sample; amplifying the extracted nucleic acid from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid from at least one first portion and at least one second portion of the sample. In some embodiments, the method further comprises contacting the sample, portion of the sample, or the extracted nucleic acid with one or more intercalating dyes prior to amplification.

[0016] In some embodiments, the present disclosure includes a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: incubating at least one first portion of a sample containing a microorganism with at least one antibiotic, and incubating at least one second portion of the sample in the absence of the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganism present in the sample; contacting the nucleic acids with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids, the amplified nucleic acids being from at least one first portion and at least one second portion of the sample.

[0017] This summary is provided to introduce in simplified form selected concepts that will be further described below in the following description. It is not intended to identify features or essential features of the claimed subject matter. Furthermore, the claimed subject matter is not limited to implementations that address any or all of the disadvantages noted in any part of this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The embodiments of the present disclosure, briefly summarized above and discussed in more detail below, may be understood by reference to the illustrative embodiments of the present disclosure shown in the accompanying drawings. However, the drawings illustrate only typical embodiments of the present disclosure and are therefore not to be considered limiting of scope, as the present disclosure may admit to other equally effective embodiments.

[0019] Figure 1A method of determining antibiotic susceptibility of a microorganism according to one embodiment is shown.

[0020] Figure 2 A method of determining antibiotic susceptibility of a microorganism according to another embodiment is shown.

[0021] Figure 3 A method of determining antibiotic sensitivity of a microorganism according to yet another embodiment is shown.

[0022] Figure 4A and 4B Shown are graphs of cycle threshold values ​​associated with samples exposed to antibiotic only and samples exposed to a combination of antibiotic and intercalating dye.

[0023] Figure 5 A system for determining bacterial sensitivity according to one embodiment is shown.

[0024] Figure 6 A method of determining antibiotic susceptibility of a microorganism according to yet another embodiment including EMA is shown.

[0025] Figure 7 A method of determining antibiotic sensitivity of a microorganism according to yet another embodiment is shown.

[0026] Figure 8 A method of determining antibiotic sensitivity of a microorganism according to yet another embodiment is shown.

[0027] Fig. 9 A method of determining antibiotic sensitivity of a microorganism according to yet another embodiment is shown.

[0028] Fig.10 Data related to Example 1 are shown.

[0029] Fig.11 Data related to Escherichia coli (E. coli) and ciprofloxacin obtained according to one embodiment of the present disclosure are shown.

[0030] Fig.12 Data related to E. coli obtained according to one embodiment of the present disclosure are shown.

[0031] Fig.13 Data related to Pseudomonas and ciprofloxacin and chloramphenicol obtained according to one embodiment of the present disclosure are shown.

[0032] Fig.14 Data related to Example 2 are shown.

[0033] Fig.15 Antibiotic incubation times according to some embodiments of the present disclosure are shown.

[0034] For ease of understanding, the same reference numerals are used, where possible, to represent the same elements common to the drawings. The drawings are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated into other embodiments without further description. DETAILED DESCRIPTION

[0035] Before explaining at least one embodiment of the present disclosure in detail through exemplary language and results, it should be understood that the present disclosure is not limited in its application to the details of the construction and arrangement of the components set forth in the following description. The present disclosure can have other embodiments or can be practiced or executed in a variety of ways. Therefore, the language used herein is intended to give the widest possible scope and meaning; and these embodiments are intended to be exemplary, not exhaustive. In addition, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0036] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0037] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the technical level of those skilled in the art to which the present disclosure pertains. The entire contents of all patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference, to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated herein by reference.

[0038] According to the present disclosure, all compositions, devices, kits and / or methods disclosed herein can be carried out and implemented without excessive experimentation. Although compositions, devices, kits and / or methods have been described according to some specific embodiments, it is obvious to those skilled in the art that, without departing from the concept, spirit and scope of the present disclosure, changes can be applied to the steps or step sequences of the compositions, devices, kits and / or methods and methods described herein. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0039] Some embodiments of the present disclosure beneficially provide robust rapid drug sensitivity testing and drug screening. The various features and advantages that can be achieved by the present disclosure will be understood from the discussion herein, and may include one or more of the following: (1) applicability for prokaryotic cells that can grow poorly in culture; and (2) use of targeted drug concentrations for inhibiting the growth of organisms. In some embodiments, sensitivity includes situations where the antimicrobial has an inhibitory effect on the growth of microorganisms or has a lethal effect on microorganisms. In some embodiments, sensitivity includes situations where the antimicrobial has a lethal effect on microorganisms. Sensitivity also includes the concept of the minimum inhibitory concentration (minimuminhibitory concentration) ("MIC") of an antimicrobial, i.e., the concentration of an antimicrobial that inhibits the growth of a microorganism. For example, using the systems and methods described herein to identify sensitivity or lack of sensitivity can provide clinicians or health care decision makers with information useful for making decisions about antimicrobial treatment for patients in need thereof. In some embodiments, a reduced growth or functional response relative to a control in the presence of an antibiotic agent indicates that the microorganism is sensitive to the tested antibiotic agent.

[0040] definition

[0041] As used in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0042] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term without a quantifier may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Therefore, unless the context clearly indicates otherwise, the term without a quantifier includes plural referents. Thus, for example, reference to a "compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" means "two or more".

[0043] The use of the term "at least one" will be understood to include one and any number more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 should not be considered limiting, as higher limits can also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0044] The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing between two or more items and, unless expressly stated otherwise, is not meant to imply any order or sequence or importance of one item relative to another, or any order of addition, for example.

[0045] The use of the term "or" in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0046] The words "comprise" (and any variation thereof), "have" (and any variation thereof), "include" (and any variation thereof), or "contain" (and any variation thereof) as used in this specification and claims are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent therein.

[0047] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0048] Any reference to "one embodiment" and variations thereof, "some embodiments", "for example", or "an example" and variations thereof as used herein means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the appearance of the phrase "in some embodiments" or "an example" in multiple places in the specification does not necessarily refer to the same embodiment. In addition, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0049] Throughout this application, the term "about" is used to indicate a value that includes the inherent variation of error for the composition / device / apparatus, the method used to determine the value, or the variation that exists in the subject matter. For example, and not as a limitation, when the term "about" is used, the specified value can vary by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent of the particular value as such variations are appropriate for performing the disclosed methods and as understood by one of ordinary skill in the art.

[0050] "Analyte" is a nucleic acid macromolecule that can be recognized by an analyte-specific binding partner. In some embodiments, analyte refers to a nucleic acid macromolecule that can be recognized by an analyte-specific binding partner, such as (but not limited to) a DNA or RNA segment, chain or oligomer or a portion thereof that is complementary or substantially complementary to the analyte so that it can bind thereto.

[0051] The term "antibiotic" used herein refers to a compound with the ability to kill or inhibit bacterial growth. In some embodiments, the term "antibiotic" refers to a compound with the ability to kill or inhibit bacterial growth, particularly selected from the following genus, including but not limited to Borrelia, Staphylococcus, Escherichia, Klebsiella, Acinetobacter and Mycobacterium. In some embodiments, antimicrobial agents are agents that kill microorganisms or inhibit microbial growth. In some embodiments, antimicrobial agents (such as drugs) can be grouped according to the microorganisms they are mainly targeted at. For example, antibiotics are used to resist bacteria. The term "β-lactam" or "β-lactam antibiotics" used herein refers to antibiotics with a β-lactam ring as a part of its core structure, such as penicillin and penicillin derivatives (penicillanes), cephalosporins (cephems), monocyclic β-lactams and carbapenems. Although these antibiotics work by inhibiting bacterial cell wall biosynthesis, other antibiotics such as protein synthesis inhibitors (tetracyclines, aminoglycosides, macrolides, etc.), nucleic acid synthesis inhibitors (fluoroquinolones, rifamycins, etc.), and antimetabolite sulfonamides can also be similarly used in the antibiotic susceptibility testing of the methods described herein.

[0052] As used herein, the term "bacterial infection" refers to the invasion, proliferation and / or presence of bacteria in a cell or a subject.

[0053] The term "cell culture" or "culture" used herein includes reference to a cell group in a culture. In some embodiments, a cell culture or culture may refer to a cell being in a culture medium that is conducive to cell growth, and optionally further allowing the cell to grow in a culture medium. Therefore, in some embodiments, the term "bacterial culture" or "culture" may refer to bacteria grown or incubated in a culture medium that is conducive to the growth of those bacteria. Bacterial cultures may be present in any type of container, such as a flask, tube, microplate, or array, etc. Typically, bacteria have different growth phases. When a bacterial colony first enters a high nutrient environment that allows growth, cells need to adapt to their new environment. The first stage of growth is a lag phase, i.e., a slow growth phase when cells adapt to a high nutrient environment and are ready to grow rapidly. The lag phase has a high biosynthesis rate because proteins necessary for rapid growth are produced. The second stage of growth is a logarithmic (log) phase, also referred to as a logarithmic (logarithmic) phase or an exponential phase, in which bacteria experience rapid exponential growth. During the logarithmic phase, nutrients are metabolized at the fastest rate until one of the nutrients is exhausted and begins to limit growth. The third stage of growth is the stationary phase, and is caused by nutrient depletion. In some embodiments, a bacterial culture in the "stationary phase" means that the bacteria in the culture have approximately equal growth rates and mortality rates. The "growth form" of the term bacteria used herein generally refers to bacteria that are in a lag phase or logarithmic phase rather than in a stationary phase. In some embodiments, the stationary phase bacterial culture has grown for about 7 days. In other embodiments, the stationary phase bacterial culture comprises non-replicating persister cells. "Non-replicating persister cells" means bacterial cells that have entered a state where replication has stopped and the ability to tolerate antibiotics.

[0054] As used herein, the term "contacting" refers to any action that causes at least one compound or component of the presently disclosed subject matter to come into physical contact with at least one cell (e.g., a bacterial cell) or an environment (e.g., a culture medium or a sample) in which at least one cell (e.g., a bacterial cell) exists.

[0055] As used herein, the terms "disease" and "disorder" are used interchangeably and refer to a condition in a subject that includes a deleterious deviation from the normal structural or functional state of an organism. Non-limiting examples of diseases / disorders include subjects suffering from one or more bacterial infections or sepsis.

[0056] Detection agent: The term "detection agent" as used herein refers to any detectable element, molecule, functional group, compound, fragment or part. In some embodiments, a detection agent is provided or used alone. In some embodiments, a detection agent associated with (e.g., combined with) another reagent is provided and / or used. Some examples of detection agents include, but are not limited to: multiple ligands, fluorescent dyes, chemiluminescent agents (e.g., acridinum esters, stable dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antiserum or monoclonal antibodies are available.

[0057] Diagnostic test: As used herein, a "diagnostic test" is a step or series of steps that is performed or has been performed to obtain such information: the information can be used to determine whether a patient has a disease, disorder or condition and / or can be used to classify the disease, disorder or condition into a phenotypic category or any category that is meaningful for the prognosis of the disease, disorder or condition or the possible response to the treatment (general treatment or any specific treatment) of the disease, disorder or condition. Similarly, "diagnosis" refers to providing any type of diagnostic information, including but not limited to whether a subject is likely to have or develop a disease, disorder or condition, such as the state, stage or characteristics of the disease, disorder or condition manifested in the subject, information related to the nature or classification of the tumor, information related to the prognosis and / or information that can be used to select an appropriate treatment or additional diagnostic test. The choice of treatment may include: the choice of a specific therapeutic agent or other treatment modality (e.g., surgery, radiation, etc.), the choice of whether to withhold treatment or deliver treatment, the choice related to the dosing regimen (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents), etc. The choice of additional diagnostic tests may include more specific tests for a given disease, disorder or condition.

[0058] Hybridization: The term "hybridization" refers to the physical property of a single-stranded nucleic acid molecule (e.g., DNA or RNA) annealing to a complementary nucleic acid molecule. Hybridization can generally be evaluated in a variety of situations - including in the context of studying interacting nucleic acid molecules in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or in a biological system or cell). Hybridization techniques and methods for evaluating hybridization are well known in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity are stably hybridized, while those with lower complementarity are not stably hybridized. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, NJ.

[0059] The terms "labeled" and "labeled with a detectable agent (or moiety)" are used interchangeably herein to indicate that an entity (e.g., a target sequence) can be visualized, for example, directly or after hybridization with another entity comprising a detectable agent or moiety. In some embodiments, the detectable agent or moiety is selected so that it produces a signal that can be measured, and the signal intensity is related to (e.g., proportional to) the amount of the entity of interest (e.g., target sequence). Methods for labeling nucleic acid molecules are well known in the art. In some embodiments, labeled nucleic acids can be prepared by incorporating or conjugating with a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0060] In some embodiments, the term "oligonucleotide" is used herein to refer to a polynucleotide comprising about 5 to about 150 nucleotides, for example, about 10 to about 100 nucleotides, about 15 to about 75 nucleotides, or about 15 to about 50 nucleotides. Throughout the specification, whenever an oligonucleotide is represented by a sequence of letters (e.g., selected from four base letters representing adenosine, cytidine, guanosine, and thymidine: A, C, G, and T), the nucleotides are presented in a 5' to 3' order from left to right. "Polynucleotide sequence" refers to the sequence of nucleotide monomers along a polymer. Unless otherwise indicated, whenever a polynucleotide sequence is represented, it should be understood that the nucleotides are in a 5' to 3' direction from left to right.

[0061] The term "nucleic acid" as used herein means a core base polymer with a backbone of alternating sugar and phosphate units in DNA and RNA. In some embodiments, "nucleic acid" and "polynucleotide" are considered to be equivalent and interchangeable. Nucleic acids are generally in the form of DNA or RNA. In some embodiments, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide" or "oligonucleotide" are used interchangeably herein. They refer to polymers of nucleotide monomers or their analogs, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleotide source can be genomic, synthetic or semisynthetic. Unless otherwise specified, the term encompasses nucleic acid-like structures and amplification products with synthetic backbones. As will be appreciated by those skilled in the art, the lengths of these polymers (i.e., the number of nucleotides contained in the polymer) can vary widely, generally depending on their intended function or use. Polynucleotides can be linear, branched linear or circular molecules. In some embodiments, polynucleotides also have associated counterions, such as H + NH4 + , trialkylammonium, Mg 2+ 、Na + etc. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. A polynucleotide may be composed of internucleotide nucleobases and sugar analogs.

[0062] The term "substantially" as used herein means that the subsequently described event or situation occurs completely, or that the subsequently described event or situation occurs to a great extent or degree. For example, when associated with a particular event or situation, the term "substantially" means that the subsequently described event or situation occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0063] As used herein, the phrase "associated with" includes both direct association of two moieties with each other and indirect association of two moieties with each other. Non-limiting examples of association include covalent binding of one moiety to another moiety through a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety directly or through a specific binding pair member bound to the moiety, incorporation of one moiety into another moiety, such as by dissolving one moiety in the other moiety or by synthesis, and coating one moiety on another moiety.

[0064] The term "biological fluid sample" as used herein will be understood to include any liquid test sample that can be obtained from a patient and used in accordance with the present disclosure. Examples of biological fluid samples that can be used include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), saliva, sputum, mucus, nasal fluid, nasopharyngeal fluid, anterior nasal fluid, oropharyngeal fluid, tracheal fluid, bronchial alveolar fluid, cerebrospinal fluid (CSF), intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, tears, combinations thereof, and the like.

[0065] The term "volume" as used herein when it relates to a liquid test sample used in accordance with the present disclosure generally refers to a volume of the liquid test sample in the range of about 0.1 μl to about 100 μl, or in the range of about 1 μl to about 75 μl, or in the range of about 2 μl to about 60 μl, or a value less than or equal to about 50 μl, etc.

[0066] The term "patient" as used herein includes both human and veterinary subjects. In certain non-limiting embodiments, the patient is a mammal. In certain other non-limiting embodiments, the patient is a human. The term "mammal" for diagnostic / therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc.

[0067] A "health care provider" or "health care decision maker" includes any individual who is authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient. In the context of identifying a useful new drug for treating a disease, a health care provider may be an individual who is not authorized to diagnose or treat a patient, or to assist in the diagnosis or treatment of a patient.

[0068] As used herein, the term "isolated" means a target, sample, polynucleotide, complex, nucleic acid or oligonucleotide that is separated, by reason of its origin or manipulation, from at least some of the components with which it is naturally associated or with which it was originally obtained.

[0069] "Point of care testing" refers to real-time diagnostic tests that can be completed within a rapid time frame so that the resulting test is performed faster than a comparable test without the use of the system. Point of care testing can be performed quickly and on-site, such as in a doctor's office, at the bedside, in a statistical laboratory, an emergency room, or other such venues, particularly where rapid and accurate results are desired. The patient may be present, but such presence is not required. Points of care include, but are not limited to, emergency rooms, operating rooms, hospital laboratories and other clinical laboratories, doctors' offices, field locations, or any location where rapid and accurate results are desired.

[0070] The term "specific binding partner", as used herein, specifically (but not limited to) in the term "target analyte specific binding partner", will be understood to refer to any molecule capable of specifically associating with the target analyte. For example, but not limited to, the binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), a combination or derivative thereof, and any other molecule capable of specifically binding to the target analyte.

[0071] As used herein, the term "inhibit" and its variations or "significantly reduce" means that the growth and / or survival of cells (e.g., bacteria) in, for example, an object or culture is reduced, inhibited, weakened, reduced or prevented by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% compared to an untreated control culture or object. Inhibiting the "survival" of cells (e.g., bacteria) herein refers to killing cells or reducing the count of viable cells. In some embodiments, the growth of cells is inhibited by more than about 50%. In other embodiments, the percentage of viable cells in the culture after treatment with the test compound is less than about 50% compared to the percentage of viable cells in the control under the same conditions but without the test compound. In other embodiments, the stationary phase culture comprises non-growing cells such as non-replicating persister cells. Furthermore, the term "significantly increased" as used herein means an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100%.

[0072] Sample: The term "sample" as used herein refers to a biological sample obtained or derived from a human subject as described herein. In some embodiments, a biological sample includes a biological tissue or fluid. In some embodiments, a biological sample may include blood; blood cells; tissue or fine needle biopsy samples; body fluids containing cells; free floating nucleic acids; cerebrospinal fluid; lymph; tissue biopsy specimens; surgical specimens; other body fluids, secretions and / or excretions; and / or cells therefrom. In some embodiments, a biological sample includes cells obtained from an individual (e.g., from a human or animal subject). In some embodiments, the obtained cells are cells of an individual from which the sample is obtained or contain cells of an individual from which the sample is obtained. In some embodiments, the sample is a "raw sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, the original biological sample is obtained by a method selected from: biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, body fluid (e.g., blood) collection. In some embodiments, the sample is cardiac tissue obtained from an object. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the original sample (e.g., by removing one or more components and / or by adding one or more reagents). For example, filtration is performed using a semipermeable membrane. As another example of sample processing, the sample can be a plasma sample treated with an anticoagulant selected from EDTA, heparin and citrate. As another example of sample processing, the sample can be processed to separate one or more proteins (e.g., by capturing proteins with one or more antibodies). A "processed sample" may include, for example, nucleic acids or polypeptides extracted from the sample or nucleic acids or polypeptides obtained by performing techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components on the original sample.

[0073] Subject: The term "subject" as used herein refers to an organism, e.g., a mammal (e.g., a human). In some embodiments, a human subject is an adult, adolescent, or child subject. In some embodiments, the subject is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. In some embodiments, the subject suffers from a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is susceptible to and / or exhibits an increased risk of developing the disease, disorder, or condition (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical manifestations of the disease) or features of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is being and / or has been administered diagnosis and / or treatment.

[0074] Threshold value: The term "threshold value" as used herein refers to one (or more) values ​​used as a reference to obtain information about a measurement result (e.g., a measurement result obtained in an assay) and / or to classify a measurement result. The threshold value can be determined based on one or more control samples. The threshold value can be determined before, simultaneously with, or after the measurement of interest is performed. In some embodiments, the threshold value can be a range of values. In some embodiments, the threshold value can be a value (or range of values) reported in the relevant field (e.g., a value found in a standard table).

[0075] The term "therapeutically effective amount" as used herein means that when a subject is administered to treat or prevent a specific disorder, disease or condition, the amount of the compound sufficient to affect such treatment or prevention of the disorder, disease or condition. Dosage and therapeutically effective amount can vary, for example, depending on a variety of factors including the activity of the specific agent used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination (if applicable), the effect that the practitioner expects the compound to have on the subject and the characteristics of the compound (e.g., bioavailability, stability, efficacy, toxicity, etc.), and the specific condition that the subject suffers from. In addition, the therapeutically effective amount administered intravenously can depend on the blood parameters of the subject, for example, lipid profile, insulin level, blood sugar or liver metabolism. The therapeutically effective amount will also vary according to the severity of the disease state, organ function or potential disease or complication. Any available assay can be used to determine such an appropriate dose. When one or more compounds or therapeutic agents are administered to a person, for example, a physician can first prescribe a relatively low dose, then increase the dose until an appropriate response is obtained.

[0076] The term "treatment" of a subject and its variations include applying or administering a compound to a subject for the purpose of delaying, slowing down, stabilizing, curing, healing, alleviating, mitigating, altering, remedying, mitigating, ameliorating, improving or affecting a disease or condition, a symptom of a disease or condition, or the risk (or sensitivity) of a disease or condition. The term "treatment" refers to any successful indication in treating or improving an injury, lesion or condition, including any objective or subjective parameter such as mitigation, alleviation, reducing the rate of deterioration, reducing the severity of the disease, stabilizing, alleviating symptoms or making the injury, lesion or condition more tolerable for the subject, slowing the rate of degeneration or decline, making the endpoint of degeneration less debilitating, or improving the physical or mental health of the subject. In some embodiments, the term "treatment" means reducing or improving the course, severity and / or duration of an infection. In some embodiments, the term "treatment" means reducing or improving the course, severity and / or duration of a bacterial infection, or improving one or more symptoms in a bacterial infection caused by the administration of one or more treatments (e.g., one or more therapeutic agents). In a specific embodiment, the term "treating" means improving a measurable physical parameter in a bacterial infection. In some embodiments, the term "treating" means reducing or ameliorating the course, severity, and / or duration of sepsis, or ameliorating one or more symptoms of sepsis caused by the administration of one or more treatments (e.g., one or more therapeutic agents). In a specific embodiment, the term "treating" means improving a measurable physical parameter of sepsis. In some embodiments, "treating" changes the natural or current state of a subject.

[0077] Embodiments for implementing the present invention are described in detail below. Various embodiments are described with reference to the accompanying drawings, wherein the same reference numerals are used to refer to the same elements throughout. In the following description, for the purpose of explanation, many specific details are listed to provide a comprehensive understanding of one or more embodiments. It is obvious that such embodiments can be implemented without these specific details. In other cases, well-known materials or methods are not described in detail to avoid unnecessarily obscuring the embodiments of the present disclosure. Although the present disclosure allows for a variety of modifications and alternative forms, its specific embodiments are shown in the accompanying drawings by way of example and will be described in detail herein. However, it should be understood that it is not intended to limit the present disclosure to the disclosed specific form, but on the contrary, the present disclosure will cover all modifications, equivalents and alternatives that fall within the spirit and scope of the present disclosure. The disclosed embodiments provide methods and kits for determining the antibiotic sensitivity of microorganisms.

[0078] Figure 1 Shown is a flow chart of a method 100 for determining antibiotic sensitivity associated with a microorganism according to a first embodiment. In some embodiments, suitable microorganisms include methods for assessing drug sensitivity of bacterial cells, which may optionally be pathogenic bacteria. The selected organism may be selected genus bacteria such as Staphylococcus, Escherichia, Klebsiella, Acinetobacter or Mycobacterium tuberculosis, and the selected organism may be, for example, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Mycobacterium tuberculosis. In some embodiments, the bacterium may be a bacterial isolate or subspecies (e.g., from one of the above species). Optionally, when the cell is a bacterium or other microorganism or source of infection, the cell may be derived from (including derived from) a single individual or host or from multiple individuals or hosts. In some embodiments, bacterial cells can be analyzed or characterized individually or collectively as a colony or population of bacterial cells.

[0079] In some embodiments, a cell culture including the cells to be tested is obtained or established. The cells can be isolated cells, for example, isolated cells from a selected organism (e.g., bacteria), optionally from a selected species or genus. Thus, in one embodiment, the method is a method for assessing the drug sensitivity of cells from a selected organism, wherein the method comprises obtaining or establishing a culture containing isolated cells from a selected organism (e.g., bacterial cells such as those of the above-mentioned bacterial species and genera, or as otherwise mentioned herein, etc.).

[0080] In some embodiments, a sample containing microorganisms is obtained in step 101. The sample can be, for example, a biological fluid sample, blood, sputum, urine, cerebrospinal fluid, etc. The sample can be distributed so as to produce a major portion of the sample and a minor portion of the sample. In step 102, the major portion of the sample is processed to identify the type of microorganism present in the sample. In one embodiment, in identifying the type of microorganism, the microorganism in the sample can be cracked. Once cracked, nucleic acid is extracted from the cracked microorganism. The nucleic acid can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In another embodiment, the extracted nucleic acid can be amplified using polymerase chain reaction (PCR). The polymerase chain reaction can be symmetrical or asymmetrical. TaqManPCR can be performed after symmetric PCR. For example, TaqManPCR can involve the use of TaqMan probes (oligonucleotide probes), which improve the specificity of quantitative PCR. The TaqMan probe is coupled or associated with a fluorophore at the 5' end of the oligonucleotide probe and coupled or associated with a quencher at the 3' end of the oligonucleotide probe. The fluorophore can be, for example, 6-carboxyfluorescein or tetrachlorofluorescein. The quencher can be, for example, tetramethylrhodamine. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the light source. During the amplification process, Taq polymerase degrades the probe annealed with the nucleic acid associated with the microorganism. When degraded, the fluorophore is released, thereby increasing its distance from the quencher. This results in fluorescence generation.

[0081] In an alternative embodiment, asymmetric PCR can be performed to amplify nucleic acids associated with microorganisms. Asymmetric PCR can be followed by PCR melting curve analysis. The temperature at which 50% of the nucleic acid is denatured is determined as the melting temperature (T m ). T of different microorganisms m are different, thus enabling effective identification of the type of microorganism present in the sample.

[0082] In step 103, the minor part of the sample is further divided into at least one first part and at least one second part. In step 104, at least one first part of the sample is hatched with different antibiotics, and at least one second part of the sample is hatched without any antibiotic. The part size with and without antibiotic hatching is the same. Therefore, at least one second part of the sample can serve as the standard of the antibiotic sensitivity of measuring microorganisms. In one embodiment, at least one first part of each sample can be hatched with different antibiotics. In an alternative embodiment, the concentration of antibiotic can change for different samples. The microorganism in the sample needs to grow / cultivate the shortest possible time, and the discernible nucleic acid difference in this time can be measured in subsequent amplification reactions. For example, the scope of microorganism cultivation at least one to three, one to four or one to five doubling cycles.

[0083] Once at least one first portion and second portion of the sample meets the doubling period range, then in step 105, the incubated sample is lysed. Based on the information obtained from step 102, only those portions incubated with antibiotics associated with the identified pathogens continue for further processing. For example, an enzymatic lysis method or a chemical lysis method can be used to lyse the microorganisms in the sample. In one embodiment, lysis of the microorganism can be performed only on at least one first portion of the selected sample. This can be determined based on the type of microorganism determined in step 102. Once lysed, nucleic acids associated with the microorganism are extracted in step 106. The nucleic acid can be DNA or RNA.

[0084] In step 107, the nucleic acid is amplified using PCR. In one embodiment, if the extracted nucleic acid is RNA, the primers used in the amplification process can be specific to the 16srRNA portion, or if the extracted nucleic acid is DNA, the primers used in the amplification process can be specific to the 16srDNA portion. Alternatively, the primers can be specific to 18srRNA, 18srDNA, 23srRNA, 23srDNA. Amplification using PCR can determine the cycle threshold associated with the amplified nucleic acid. The cycle threshold is the number of amplification cycles required to reach such a fluorescence level: at this fluorescence level, the PCR result changes from "undetectable" to "detectable". In step 108, a first cycle threshold associated with amplified nucleic acids from at least one first portion of the sample is determined. In step 109, a second cycle threshold associated with amplified nucleic acids from at least one second portion of the sample is determined. In step 110, the difference between the first cycle threshold and the second cycle threshold is calculated. If the first cycle threshold is greater than the second cycle threshold, the antibiotic sensitivity of the microorganism is low. In one embodiment, a second PCR melting analysis can be performed to determine the type of microorganism to give species information and to determine that the pathogen detected from part 102 is a pathogen that grows in the presence of antibiotics, thereby clearly correlating antimicrobial sensitivity with pathogen identity. In some embodiments, steps 101, 102, 103, 104, 105, 106 are performed in a sequential order.

[0085] Figure 2According to another embodiment, a method for determining the antibiotic sensitivity of a microorganism is shown. In step 201, a sample including a microorganism is received. In step 202, at least one first part of the sample is incubated with an antibiotic, and at least one second part of the sample is incubated without an antibiotic. The microorganism in the sample needs to grow / cultivate for as short a time as possible, and the discernible nucleic acid difference between the two parts in this time can be measured. For example, the microorganism is cultivated for at least one to five doubling cycles. For example, the number of doubling cycles can be determined during the development phase and fixed in the actual workflow. Once the doubling cycle of at least one first part and the second part of the sample is completed, the incubated sample is lysed in step 203. The microorganism in the sample can be lysed, for example, using an enzymatic lysis method or a chemical lysis method. Once lysed, in step 204, nucleic acids associated with the microorganism are extracted and amplified. The nucleic acid can be DNA or RNA. In one embodiment, if the extracted nucleic acid is RNA, the primer used in the amplification process can be specific to the 16srRNA part, or if the extracted nucleic acid is DNA, the primer used in the amplification process can be specific to the 16srDNA part. Alternatively, the primers can be specific for 18srRNA, 18srDNA, 23srRNA, 23srDNA.

[0086] In step 205, determine the first cycle threshold value associated with the amplified nucleic acid from at least one first part of the sample (wherein antibiotics have been added). In step 206, determine the second cycle threshold value associated with the amplified nucleic acid from at least one second part of the sample (wherein antibiotics have not been added). In step 207, calculate the difference between the first cycle threshold value and the second cycle threshold value. If the first cycle threshold value is greater than the second cycle threshold value, the microorganism is sensitive to the antibiotic in the first part, and therefore the threshold cycle is less than the second part where the antibiotic is not present. When there is no antibiotic, the microorganism will double in each doubling cycle, and therefore with the increase of the DNA amount, the threshold cycle will be smaller. If the difference between the threshold cycle can be ignored or is less than the threshold value set, the microorganism incubated with the antibiotic in the first part is resistant to the antibiotic. In step 208, PCR melting analysis can be performed to determine the type of microorganism. Pathogen identification can be achieved using different Tm's through different labeled pathogen-specific probes. Each probe will be combined with a unique pathogen. Pathogens are identified by a combination of color and Tm. In some embodiments, steps 201, 202, 203, 204, 205, 206, 207, and 208 are performed in a sequential order.

[0087] Figure 3According to another embodiment, a method for determining the antibiotic sensitivity of a microorganism is shown. In step 301, a sample comprising a microorganism is received. In step 302, at least one first portion of the sample is incubated with an antibiotic, and at least one second portion of the sample is incubated without an antibiotic. The microorganism in the sample needs to grow / cultivate or incubate the shortest possible time, and the discernible nucleic acid difference in the time can be measured. For example, the microorganism is cultivated or incubated in the range of at least one to five doubling cycles. Antibiotics as cell wall synthesis inhibitors (β-lactams) and cell membrane inhibitors (colistin) affect the integrity of the microbial cell wall. This helps to determine antibiotic sensitivity. Once at least one first portion and a second portion of the sample meet the doubling cycle range, in step 303, an intercalation dye is introduced into at least one first portion of the sample and at least one second portion of the sample. After the intercalation dye is introduced, the sample is incubated for at least 5 minutes (e.g., about 5 minutes, about 6 minutes, 5 to 10 minutes). For example, the intercalation dye can be monoazide ethidium, monoazide ethidium bromide, monoazide propidium, its isomers, etc. Intercalating dyes are known to penetrate dead microorganisms and crosslink with the chromosomal nucleic acids of dead microorganisms. Intercalating dyes do not affect living cells.

[0088] In step 304, a sample with an embedded dye is exposed to visible light for a period of time using a light source. In some embodiments, the light source includes UV light. In some embodiments, the duration of applying the light source is enough to make the embedding of the dye and the nucleic acid irreversible. The light can be a strong light, and the light source can emit light with a wavelength within the range of 465nm to 475nm. For example, the light source can be a PMA-lite LED photolysis device. In one embodiment, the sample can be exposed to a duration of 15 to 20 minutes in the light. Advantageously, light exposure also converts any unbound dye into a compound that can no longer be bound to nucleic acid. In an alternative embodiment, the sample can be precipitated to collect microorganisms and remove unbound dye.

[0089] In step 305, the sample is lysed. For example, an enzymatic lysis method or a chemical lysis method can be used to lyse the microorganisms in the sample. Once lysed, in step 306, nucleic acids associated with the microorganisms are extracted and amplified. Quantitative PCR is performed. In one embodiment, if the extracted nucleic acid is RNA, the primers used in the amplification process can be specific to the 16srRNA portion, or if the extracted nucleic acid is DNA, the primers used in the amplification process can be specific to the 16srDNA portion. Alternatively, the primers can be specific to 18srRNA, 18srDNA, 23srRNA, 23srDNA.

[0090] In step 307, determine the first cycle threshold value associated with the amplified nucleic acid from at least one first portion of the sample. In step 308, determine the second cycle threshold value associated with the amplified nucleic acid from at least one second portion of the sample. In step 309, calculate the difference between the first cycle threshold value and the second cycle threshold value. If the first cycle threshold value is greater than the second cycle threshold value, the antibiotic sensitivity of the microorganism is low. Microorganisms exposed to antibiotics either die (if they are sensitive) or continue to grow (if they are resistant). Sensitive microorganisms with damaged cell walls will enable intercalating dyes to penetrate and be bound to chromosomal DNA, thereby making them incompatible with PCR. On the other hand, resistant microorganisms will prevent intercalating dyes from penetrating into the cell wall and will therefore continue to divide.

[0091] Normal human samples contain DNA from dead / dying cells, which can skew the results and make it difficult to distinguish between sensitive and resistant microorganisms in the sample. Advantageously, the intercalating dye also binds to such DNA from dead / dying cells, thereby enabling detection of only DNA from live microorganisms by PCR. Furthermore, this enables the difference between the cycle threshold values ​​associated with at least one first portion of the sample and at least one second portion of the sample to be larger and faster.

[0092] Fig. 4 provides the diagram 401,402 of the difference of the cycle threshold value of following sample: only hatch with antibiotic and hatch with antibiotic and intercalating dye combination.In the present embodiment, the antibiotic used is ampicillin (ampicillin, Amp) and the intercalating dye is ethidium monoazide (ethidiummonoazide, EMA).Figure 401 specifically shows the difference of the cycle threshold value related to the first part of the sample hatched with ampicillin and the second part of the sample hatched under the situation of no ampicillin.Figure 402 specifically shows the difference of the cycle threshold value related to the first part of the sample hatched with ampicillin and EMA and the second part of the sample hatched under the situation of no ampicillin or EMA.It is observed that including EMA in the incubation process improves the difference of the cycle threshold value between the first part and the second part of the sample.EMA penetrates into the cell membrane of the microorganism treated with ampicillin and combines with DNA, and makes it incompatible with PCR.However, the microorganism not treated with ampicillin remains intact and DNA is PCR amplifiable.

[0093] In some embodiments, the present disclosure includes a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: receiving a sample comprising a microorganism; incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; contacting the sample, e.g., at least one first portion and / or at least one second portion, with one or more intercalating dyes; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids from at least one first portion and at least one second portion of the sample. In some embodiments, contacting the intercalating dye is performed under conditions sufficient for the intercalating dye to form a complex with nucleic acids in the sample or in the microorganism.

[0094] Reference Figure 6 , shows an EMA workflow suitable for use in accordance with the present disclosure. Here, intercalating dye EMA can be used to observe early changes in cycle threshold differences in cells exposed to beta-lactam antibiotics such as ampicillin. Figure 6 EMA-ethidium bromide monoazide for improving the differentiation between sensitive and resistant strains of beta lactam antibiotics is provided. For beta lactam antibiotics, the use of EMA results in improved and earlier [Δ]Ct values ​​(cycle threshold) between aliquots with and without antibiotics.

[0095] Reference Figure 5 , a block diagram of a system 1000 is shown in which such an embodiment may be implemented, for example, as a system 1000 for determining antibiotic susceptibility of a microorganism, which is configured to perform a process as described herein. Figure 5 , the system 1000 includes a processing unit 1010 , a memory 1020 , a storage unit 1030 , an input unit 1040 , a bus 1060 , an output unit 1050 , and a network interface 1070 .

[0096] As used herein, processing unit 1010 means any type of computing circuit, such as but not limited to a microprocessor, a microcontroller, a complex instruction set computing microprocessor, a reduced instruction set computing microprocessor, a very long instruction word microprocessor, an explicit parallel instruction computing microprocessor, a graphics processor, a digital signal processor, or any other type of processing circuit. Processing unit 1010 may also include an embedded controller, such as a general or programmable logic device or array, an application specific integrated circuit, a single chip computer, etc.

[0097] The memory 1020 may be a volatile memory and a non-volatile memory. The memory 1020 may be coupled for communication with the processing unit 1010. The processing unit 1010 may execute instructions and / or codes stored in the memory 1020. A variety of computer-readable storage media may be stored in the memory 1020 and may be accessed from the memory 1020. The memory 1020 may include any suitable element for storing data and machine-readable instructions, such as a read-only memory, a random access memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a hard disk drive, a removable media drive for processing compact disks, digital video disks, diskettes, cassettes, memory cards, etc. In the present embodiment, the memory 1020 includes a susceptibility module 1100 stored in the form of machine-readable instructions on any of the above-mentioned storage media, and may communicate with and be executed by the processor 1010. In some embodiments, the method steps performed by the processor 1010 to implement the above-mentioned functions are Figure 1 , 2 and / or as detailed in 3.

[0098] The storage unit 1030 may be a non-transitory storage medium storing a medical database 1120 or other information. The medical database 1120 is a patient data repository including blood parameters maintained by a health care service provider. The input unit 1040 may include an input device capable of receiving input signals such as a keypad, a touch-sensitive display, a camera (e.g., a camera that receives gesture-based input), etc. The bus 106 serves as an interconnection between the processing unit 1010, the memory 1020, the storage unit 1030, the input unit 1040, the output unit 1050, and the network interface 1070.

[0099] Those of ordinary skill in the art will understand that Figure 5 The hardware shown in the example may vary for a particular implementation. For example, other peripheral devices such as optical disk drives, local area network (LAN) / wide area network (WAN) / wireless (e.g., Wi-Fi) adapters, graphics adapters, disk controllers, input / output (I / O) adapters may also be used in addition to or in place of the hardware. The examples shown are provided for explanation purposes only and are not meant to imply architectural limitations regarding the present disclosure.

[0100] The system 1000 according to one embodiment of the present disclosure includes an operating system that employs a graphical user interface. The operating system allows for the simultaneous presentation of multiple display windows in the graphical user interface, wherein each display window provides an interface to different applications or different instances of the same application. The cursor in the graphical user interface can be manipulated by the user via a pointing device. The position of the cursor can be changed and / or an event such as a click of a mouse button can be generated to initiate a desired response. If appropriately modified, one of a variety of commercial operating systems can be used, such as Microsoft Windows 1000, a product of Microsoft Corporation located in Redmond, Washington. TM The operating system is modified or created according to the content of the present disclosure described.

[0101] In addition, a non-transitory computer-readable medium containing executable instructions is provided, which, when executed, can cause a processor to perform operations including methods as provided herein. In some embodiments, the present disclosure includes an article, such as a system or component thereof, comprising a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method including: determining antibiotic susceptibility of a microorganism according to the present disclosure. In some embodiments, the present disclosure includes a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method including: determining antibiotic susceptibility of a microorganism according to the present disclosure.

[0102] In some embodiments, the present disclosure includes an article, such as a system or component thereof, comprising a non-transitory computer-readable medium having encoded thereon instructions configured to cause one or more processors to perform a method comprising: determining antibiotic susceptibility of a microorganism in a sample, the method comprising: a non-transitory computer-readable medium having encoded thereon instructions configured to cause one or more processors to perform a method of determining antibiotic susceptibility of a microorganism (e.g., method 100, 200, or 300).

[0103] In some embodiments, the present disclosure includes an article, such as a system or component thereof, comprising a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: incubating at least one first portion of the sample containing the microorganism with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with the microorganism present in the sample; amplifying the extracted nucleic acid from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid, the amplified nucleic acid being from at least one first portion and at least one second portion of the sample. In some embodiments, the method further comprises contacting the nucleic acid with one or more intercalating dyes prior to amplification or prior to extracting the nucleic acid. In some embodiments, the method further comprises contacting the nucleic acid with one or more intercalating dyes prior to extracting the nucleic acid.

[0104] In some embodiments, the present disclosure includes a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: incubating at least one first portion of a sample containing a microorganism with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with the microorganism present in the sample; contacting the nucleic acid with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex; amplifying the extracted nucleic acid from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid, the amplified nucleic acid being from at least one first portion and at least one second portion of the sample. In some embodiments, prior to extraction, the nucleic acid is contacted with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex. In some embodiments, prior to amplification, the nucleic acid is contacted with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex.

[0105] Reference Figure 7 , a method for determining the antibiotic sensitivity of a microorganism according to yet another embodiment is shown. A culture step is shown here, followed by extraction and amplification according to the present disclosure. AST is shown as resolved, and the pathogen ID is resolved using Tm information and color identification code.

[0106] Reference Figure 8, shows a method for determining the antibiotic sensitivity of a microorganism according to yet another embodiment. Figure 8 , reducing the duration of the workflow by obtaining bacterial identification in advance.

[0107] Fig. 9 According to yet another embodiment, a method of determining the antibiotic sensitivity of a microorganism is shown.

[0108] Fig.15 Antibiotic incubation times according to some embodiments of the present disclosure are shown. Fig.15 It was shown that an incubation time of approximately 30 minutes is sufficient to tune out the antibiotic sensitivity of E. coli.

[0109] deal with

[0110] In some embodiments, the present disclosure includes such a method for treating an object in need thereof: determining that the object is an object in need thereof (e.g., suffering from a pathogenic bacterial infection or sepsis), and then treating the object. In some embodiments, the methods, systems, and kits described herein are suitable for use by a health care provider in a point-of-care environment. For example, the method of the present disclosure can be applied as a diagnostic test to diagnose a patient or object as positive for a pathogenic bacterial infection or sepsis, and then administering a therapeutic agent or compound to an object in need thereof in a therapeutically effective amount. For example, a physician may administer a therapeutically effective amount of any drug, therapeutic agent, or biological agent suitable for treating a bacterial infection or disease state after diagnosis according to the present disclosure. A non-limiting example of a medicament suitable for treating a bacterial infection includes one or more antibiotics according to the present disclosure. The therapeutically effective amount of the provided medicament can be determined by a physician based on the response, comorbidities, etc. of the object. In some embodiments, a therapeutic compound is administered to an object in a disease state in an effective amount. In some embodiments, a therapeutically effective amount of a compound such as an antibiotic is administered to an object in need thereof in an amount sufficient to change the natural or current state of the object.

[0111] In certain embodiments, the disease treated according to the methods described herein is a disease caused by a bacterial infection. Non-limiting examples of pathogenic bacteria include: Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jiroveci, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis, and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, and pestis), Shigella, Escherichia coli, Corynebacterium diphtheriae, and Treponema pallidum.

[0112] In certain embodiments, the composition, e.g., an antibiotic, is administered to a patient who has been diagnosed with a disease caused by a bacterial infection, e.g., the patient has been infected with Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and / or Treponema pallidum.

[0113] In certain embodiments, the disease treated according to the methods described herein is a disease caused by a bacterial infection. Non-limiting examples of pathogenic bacteria include: Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jiroveci, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and Treponema pallidum.

[0114] In certain embodiments, the composition is administered to a patient who has been diagnosed with a disease caused by a bacterial infection, for example, the patient has been infected with: Streptococcus pneumoniae, Mycobacterium tuberculosis, Chlamydia pneumoniae, Bordetella pertussis, Mycoplasma pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella, Pneumocystis jirovecii, Chlamydia psittaci, Chlamydia trachomatis, Bacillus anthracis and Francisella tularensis, Borrelia burgdorferi, Salmonella, Yersinia pestis, Shigella, Escherichia coli, Corynebacterium diphtheriae, and / or Treponema pallidum.

[0115] Exemplary numbered embodiments

[0116] Embodiment 1. A method for determining the antibiotic sensitivity of a microorganism in a sample, the method comprising: receiving a sample containing a microorganism; incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample in the absence of the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids, wherein the amplified nucleic acids are from at least one first portion and at least one second portion of the sample.

[0117] Embodiment 2. The method of embodiment 1, further comprising identifying the type of microorganism present in the sample.

[0118] Embodiment 3. The method described in embodiment 1 or 2 also includes: lysing microorganisms present in the sample; extracting nucleic acids associated with the microorganisms; using polymerase chain reaction to amplify the extracted nucleic acids; and identifying the type of microorganisms present in the sample based on the amplified nucleic acids, wherein the amplified nucleic acids are subjected to polymerase chain reaction-based melting analysis.

[0119] Embodiment 4. The method of any one of embodiments 1 to 3, further comprising: lysing microorganisms present in the sample; extracting nucleic acids associated with the microorganisms; amplifying the extracted nucleic acids using a polymerase chain reaction; and identifying the type of microorganism present in the sample based on the amplified nucleic acids, wherein the nucleic acids are amplified using probes coupled to fluorophores, wherein the probes are complementary to target nucleic acid sequences associated with the microorganisms, and wherein the type of microorganism is identified based on the color emitted by the fluorophore and the Tm of the target with the bound probe. The probes may also have different Tm values.

[0120] Embodiment 5. The method of any one of Embodiments 1 to 4, further comprising: incubating at least one first portion of the sample and at least one second portion of the sample with an intercalating dye; and exposing the incubated samples to visible light using a light source.

[0121] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the intercalating dye is at least one of ethidium monoazide, ethidium bromide monoazide, and propidium monoazide.

[0122] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid comprises: determining a first cycle threshold value associated with the amplified nucleic acid from at least one first portion; determining a second cycle threshold value associated with the amplified nucleic acid from at least one second portion of the sample; calculating the difference between the first cycle threshold value and the second cycle threshold value; and determining the antibiotic sensitivity information based on the cycle threshold value associated with the amplified nucleic acid based on the difference between the first cycle threshold value and the second cycle threshold value, wherein if the cycle threshold value is higher, the antibiotic sensitivity of the microorganism is lower.

[0123] Embodiment 9. The method of any one of Embodiments 1 to 8, further comprising determining the identity of the microorganism after obtaining antibiotic sensitivity information associated with the microorganism.

[0124] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein at least a first portion of the sample is incubated with more than one type of antibiotic.

[0125] Embodiment 11. The method of any one of Embodiments 1 to 9, wherein at least a first portion of the sample is incubated with more than one type of antibiotic, wherein the antibiotics are present in different concentrations.

[0126] Embodiment 12. A kit for determining the antibiotic sensitivity of microorganisms in a sample, comprising the components of embodiments 1 to 11.

[0127] Embodiment 13. A kit for determining the antibiotic sensitivity of microorganisms in a sample, the kit comprising: one or more antibiotics; a growth medium for the microorganisms; reagents related to nucleic acid extraction; reagents related to nucleic acid amplification; and polymerase chain reaction-based melting analysis software.

[0128] Embodiment 14. The kit of embodiment 12 or 13, further comprising one or more intercalating dyes.

[0129] Embodiment 15. A method of treating a subject in need thereof, comprising determining the antibiotic sensitivity of a microorganism according to an embodiment of the present disclosure, and then treating the subject in need thereof. In some embodiments, the treatment comprises administering to the subject a therapeutically effective amount of a composition, such as one or more antibiotics.

[0130] Embodiment 16. A non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform a method for determining antibiotic sensitivity of a microorganism in a sample, the method comprising: incubating at least one first portion of the sample containing the microorganism with at least one antibiotic, and incubating at least one second portion of the sample in the absence of the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganism present in the sample; contacting the nucleic acids with one or more intercalating dyes under conditions that form a nucleic acid / intercalating dye complex; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids, wherein the amplified nucleic acids are from at least one first portion and at least one second portion of the sample.

[0131] Embodiment 17. A method of treating a subject in need thereof, comprising determining the antibiotic sensitivity of a microorganism in a biological sample from the subject, the method comprising: receiving a sample comprising a microorganism; incubating at least one first portion of the sample with at least one antibiotic, and incubating at least one second portion of the sample without the antibiotic; extracting nucleic acids from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acids are associated with the microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; and obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acids from at least one first portion and at least one second portion of the sample, and then treating the subject in need thereof. In some embodiments, the treatment comprises administering a therapeutically effective amount of the composition to the subject. In some embodiments, the disease is characterized as a bacterial disease, etc.

[0132] The term "treatment" of a subject and its variations include applying or administering a compound to a subject for the purpose of delaying, slowing down, stabilizing, curing, healing, alleviating, mitigating, altering, remedying, mitigating, ameliorating, improving, or affecting a disease or condition, a symptom of a disease or condition, or the risk (or sensitivity) of a disease or condition. The term "treatment" refers to any successful indication in treating or improving damage, lesions, or conditions, including any objective or subjective parameters such as mitigation, alleviation, reducing the rate of deterioration, reducing the severity of the disease, stabilizing, alleviating symptoms, or making damage, lesions, or conditions more tolerable for the subject, slowing the rate of degeneration or decline, making the endpoint of degeneration less debilitating, or improving the physical or mental health of the subject. In some embodiments, the term "treatment" means reducing or improving the course, severity, and / or duration of a bacterial infection, or improving one or more symptoms in a bacterial infection caused by the administration of one or more treatments (e.g., one or more therapeutic agents). In a specific embodiment, the term "treatment" means improving measurable physical parameters in a bacterial infection. In some embodiments, the term "treatment" means reducing or improving the course, severity and / or duration of a bacterial infection, or improving one or more symptoms of bacteria caused by the administration of one or more treatments (e.g., one or more therapeutic agents). In a specific embodiment, the term "treatment" means improving a measurable physical parameter in a bacterial infection. In some embodiments, the term "treatment" means reducing or improving the course, severity and / or duration of sepsis, or improving one or more symptoms of sepsis caused by the administration of one or more treatments (e.g., one or more therapeutic agents). In a specific embodiment, the term "treatment" means improving a measurable physical parameter of sepsis. In some embodiments, "treatment" changes the natural or current state of the subject.

[0133] The term "therapeutically effective amount" as used herein means that when a subject is administered to treat or prevent a specific disorder, disease or condition, the amount of the compound sufficient to affect such treatment or prevention of the disorder, disease or condition. Dosage and therapeutically effective amount can vary, for example, depending on a variety of factors including the activity of the specific agent used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination (if applicable), the effect that the practitioner expects the compound to have on the subject and the characteristics of the compound (e.g., bioavailability, stability, efficacy, toxicity, etc.), and the specific condition that the subject suffers from. In addition, the therapeutically effective amount administered intravenously can depend on the blood parameters of the subject, for example, lipid profile, insulin level, blood sugar or liver metabolism. The therapeutically effective amount will also vary according to the severity of the disease state, organ function or potential disease or complication. Any available assay can be used to determine such an appropriate dose. When one or more compounds or therapeutic agents are administered to a person, for example, a physician can first prescribe a relatively low dose, then increase the dose until an appropriate response is obtained.

[0134] Example

[0135] Fig. 9 An experimental process flow is shown outlining the objectives of the experiments described below, the antibiotics tested according to the present disclosure, and the bacterial tests.

[0136] Example 1: Detection of bacterial sensitivity to ampicillin

[0137] Overnight cultures of ampicillin-sensitive E. coli were further subcultured for 2 to 3 hours to logarithmic phase. The OD 600nm The culture was diluted to 1 × 10 6 cells / mL and aliquot 10 mL into two 50 mL Erlenmeyer flasks. Add 100 μg / mL of ampicillin to one of the Erlenmeyer flasks. Incubate the two flasks with and without antibiotics at 37°C and remove two aliquots of 50 μL from each flask at fixed intervals (0, 30, 40, and 60 minutes). Nucleic acid (NA) was extracted from all sample aliquots using the Brand Sample Preparation 1.0 Kit. NA samples were then subjected to PCR and RT-PCR (reverse transcription-PCR) using 16S rDNA specific primers (each DNA extraction sample was divided into two replicates for PCR). Fig.10 As shown in Table 1 , RNA levels were able to detect ampicillin sensitivity at 60 min, whereas DNA levels were unable to distinguish between antibiotic and no-antibiotic samples even after 60 min of incubation ( Fig.10Table 2). In this experiment, all RNA samples were treated with DNAse for 60 minutes at 37°C before RT-PCR. Subsequent experiments showed that treatment as little as 5 minutes was sufficient to eliminate DNA from the samples.

[0138] Example 2: Using EMA to accelerate the detection of bacterial sensitivity to ampicillin

[0139] Overnight cultures of ampicillin-sensitive E. coli were further subcultured for 2 to 3 hours to logarithmic phase. The OD 600nm The culture was diluted to 1 × 10 6 cells / mL and 10 mL was aliquoted into two 50 mL Erlenmeyer flasks. 100 μg / mL of ampicillin was added to one of the Erlenmeyer flasks. The two flasks with and without antibiotics were incubated at 37°C and four 50 μL aliquots were taken from each flask at fixed intervals (0, 20 and 30 minutes). 25 μL of 2 μM EMA (ethidium monoazide prepared in 20% DMSO) was added to 2 of the 4 aliquots from each sampling. After EMA was added, the samples were mixed by vortexing, incubated in the dark for 5 minutes, and transferred to 1.5 mL Eppendorf tubes and exposed to blue LED light for 15 minutes for photolysis. Use Sample preparation reagents were used to extract DNA from all sample aliquots. PCR was then performed on the DNA samples using 16S rDNA specific primers (each DNA extraction sample was divided into two replicates). Fig.14 As shown in Table 1, within 30 minutes, the samples exposed to EMA had a large difference in Ct between ampicillin exposure and ampicillin non-exposure. The same samples did not show any difference between ampicillin exposure and ampicillin non-exposed samples when not exposed to EMA + blue light ( Fig.14 , Table 2).

[0140] Other embodiments

[0141] An experiment similar to the one described above was performed.

[0142] Fig.11 Data related to E. coli and ciprofloxacin obtained according to one embodiment of the present disclosure are shown.

[0143] Fig.12 Data related to E. coli and ciprofloxacin obtained according to one embodiment of the present disclosure are shown.

[0144] Fig.13Data obtained for Pseudomonas associated with ciprofloxacin and chloramphenicol according to one embodiment of the present disclosure are shown.

[0145] The foregoing examples are provided for the purpose of explanation only and should in no way be construed as limitations on the invention disclosed herein. Although the present invention has been described with reference to a variety of embodiments, it should be understood that the words used herein are descriptive and illustrative, rather than limiting. In addition, although the present invention has been described herein with reference to specific devices, materials, and embodiments, the present invention is not intended to be limited to the details disclosed herein; on the contrary, the present invention extends to, for example, all functionally equivalent structures, methods, and uses within the scope of the appended claims. Those skilled in the art who benefit from the teachings of this specification may make many modifications to this specification and may make changes in its various aspects without departing from the scope and spirit of the present invention.

Claims

1. A method for determining the antibiotic sensitivity of microorganisms in a sample, the method comprising: receiving a sample comprising the microorganism; incubating at least a first portion of the sample with at least one antibiotic and incubating at least a second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with a microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; as well as Antibiotic sensitivity information associated with the microorganism is obtained from the amplified nucleic acids from at least one first portion and at least one second portion of the sample.

2. The method of claim 1, further comprising identifying the type of microorganism present in the sample.

3. The method according to claim 2, further comprising: lysing microorganisms present in the sample; extracting nucleic acid associated with the microorganism; Amplifying the extracted nucleic acid using polymerase chain reaction; as well as The type of microorganism present in the sample is identified based on the amplified nucleic acid, wherein the amplified nucleic acid is subjected to polymerase chain reaction-based melting analysis.

4. The method according to claim 2, further comprising: lysing microorganisms present in the sample; extracting nucleic acid associated with the microorganism; Amplifying the extracted nucleic acid using polymerase chain reaction; as well as The type of microorganism present in the sample is identified based on amplified nucleic acids, wherein the nucleic acids are amplified using one or more probes coupled to a fluorophore, wherein the one or more probes are complementary to a target nucleic acid sequence associated with the microorganism, and wherein the type of microorganism is identified based on the color emitted by the fluorophore and the Tm of the target with the bound probe. The method of claim 4 , wherein the one or more probes have different Tm values.

6. The method according to claim 1, further comprising: incubating at least one first portion of the sample and at least one second portion of the sample with an intercalating dye; as well as The incubated samples were exposed to visible light using a light source.

7. The method of claim 6, wherein the intercalating dye is at least one of ethidium monoazide, ethidium bromide monoazide, propidium monoazide, and combinations thereof.

8. The method of claim 1, wherein obtaining antibiotic sensitivity information associated with the microorganism from the amplified nucleic acid comprises: determining a first cycle threshold value associated with amplified nucleic acid from the at least one first portion; determining a second cycle threshold value associated with at least a second portion of amplified nucleic acid from the sample; calculating a difference between the first cycle threshold value and the second cycle threshold value; as well as The antibiotic sensitivity information is determined according to a cycle threshold value associated with the amplified nucleic acid based on a difference between the first cycle threshold value and the second cycle threshold value, wherein if the cycle threshold value is higher, the antibiotic sensitivity of the microorganism is lower.

9. The method of claim 1, further comprising determining the identity of the microorganism after obtaining antibiotic sensitivity information associated with the microorganism.

10. The method of claim 1, wherein at least a first portion of the sample is incubated with more than one type of antibiotic.

11. The method of claim 1, wherein at least a first portion of the sample is incubated with more than one type of antibiotic, wherein the antibiotics are present in different concentrations.

12. A kit for determining the antibiotic sensitivity of microorganisms in a sample, the kit comprising: one or more antibiotics; Growth media for microorganisms; Reagents related to nucleic acid extraction; Reagents related to nucleic acid amplification; and Polymerase chain reaction-based melting analysis software.

13. The kit of claim 12, further comprising one or more intercalating dyes.

14. An article of manufacture, such as a system or component thereof, comprising a non-transitory computer readable medium having encoded thereon instructions configured to cause one or more processors to perform a method of determining antibiotic susceptibility of microorganisms in a sample, the method comprising: incubating at least a first portion of a sample comprising a microorganism with at least one antibiotic and incubating at least a second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with a microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; as well as Antibiotic sensitivity information associated with the microorganism is obtained from the amplified nucleic acids from at least one first portion and at least one second portion of the sample.

15. A non-transitory computer readable medium having encoded thereon instructions configured to cause one or more processors to perform a method of determining antibiotic susceptibility of microorganisms in a sample, the method comprising: incubating at least a first portion of a sample comprising a microorganism with at least one antibiotic and incubating at least a second portion of the sample without the antibiotic; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with a microorganism present in the sample; contacting the nucleic acid with one or more intercalating dyes under conditions such that a nucleic acid / intercalating dye complex is formed; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; as well as Antibiotic sensitivity information associated with the microorganism is obtained from the amplified nucleic acids from at least one first portion and at least one second portion of the sample.

16. A non-transitory computer readable medium having encoded thereon instructions configured to cause one or more processors to perform a method of determining antibiotic susceptibility of microorganisms in a sample, the method comprising: incubating at least a first portion of a sample comprising a microorganism with at least one antibiotic and incubating at least a second portion of the sample without the antibiotic; contacting the sample or a portion thereof with one or more intercalating dyes; extracting nucleic acid from at least one first portion of the sample and at least one second portion of the sample, wherein the nucleic acid is associated with a microorganism present in the sample; amplifying the extracted nucleic acids from at least one first portion and at least one second portion of the sample; as well as Antibiotic sensitivity information associated with the microorganism is obtained from the amplified nucleic acids from at least one first portion and at least one second portion of the sample.

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