Technology for detecting target nucleic acid sequence by using argonaute protein and artificial nucleic acid circuit

Abstract

The present invention relates to a technology for detecting a target nucleic acid by using an argonaute protein and an artificial nucleic acid circuit. The system according to the present invention can specifically detect target pathogens added to a human sample with 100% clinical sensitivity without nucleic acid amplification and separate extraction steps, and can directly identify target pathogens collected from the surface of an object by using the system together with a three-dimensional nanopillar array structure. In addition, since a trigger sequence cleaved by an Ago protein is designed to be used as a guide, a single protein is used, and thus manufacturing is simpler than that of a conventional nucleic acid detection circuit, and cost are reduced in that target pathogens can be detected with high sensitivity even with trace amounts of target nucleic acid sequences as long as the Ago protein, a reporter, and a reporter complement according to the system of the present invention are not depleted. In addition, the system according to the present invention proceeds in a single step, and can be easily applied to the detection of various target nucleic acid sequences by appropriately changing a nucleic acid recognition site to match a target pathogen.

Description

Target nucleic acid sequence detection technology using Argonaute protein and artificial nucleic acid circuit

[0001] The present invention relates to a technology for detecting a target nucleic acid sequence using an agonist protein and an artificial nucleic acid circuit.

[0002] Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a significant global public health problem due to their rapid spread, high mortality, and limited treatment options. CPE is a group of Gram-negative strains, including Klebsiella pneumoniaecarbapenemase (KPC), Imipenemase (IMP), New-Delhi metallo-beta-lactamase (NDM), Verona Integron-encoded metallo-beta-lactamase (VIM), and Oxacillinase-48 (OXA-48), which exhibit hydrolytic activity against carbapenem antibiotics, which are used as a last resort for infections with multidrug-resistant strains. With the increasing prevalence of CPE infections in healthcare settings and the community, there is a critical need for rapid, accurate, and cost-effective diagnostic methods to identify these resistant strains.

[0003] Existing CPE detection methods, such as culture-based detection and polymerase chain reaction (PCR), are limited by long processing times, labor-intensiveness, and the need for specialized equipment and skilled personnel. Consequently, isothermal nucleic acid amplification (AAL) is gaining attention as an alternative diagnostic technique to overcome these challenges and enable timely detection of CPE infection and appropriate treatment. However, the composition of the isothermal amplification mixture is relatively complex, often making it incompatible with clinical samples. To overcome this, most methods employ additional purification processes, equipment, and reagents. This increased cost and complexity ultimately limits the practical applicability of isothermal nucleic acid diagnostics.

[0004] Recently, endonucleases have attracted attention as useful tools for target nucleic acid identification due to their sequence-specific catalytic activity. In particular, the CRISPR / Cas system has attracted significant attention for its potential in molecular diagnostics. Among these, the CRISPR / Cas12 and CRISPR / Cas13 systems have demonstrated exceptional diagnostic potential due to their secondary cleavage activity. Upon recognition and binding to a target sequence, Cas12 and Cas13 possess the activity of non-specifically cleaving nearby single-stranded DNA and RNA molecules, respectively. This secondary cleavage activity can be utilized for signal amplification, enabling highly sensitive detection of target nucleic acids. Despite this potential, diagnostic applications of the CRISPR / Cas system face several challenges, including the increased cost and instability associated with the use of guide RNAs, and the requirement that the preferred cleavage site of the Cas protein be adjacent to the target sequence. To address these limitations, active research is underway on strategies such as the development of alternative endonucleases and the use of chemically modified guide RNAs.

[0005] Accordingly, the inventor of the present invention has developed a technology that solves the above-mentioned problem and exhibits an excellent target nucleic acid sequence detection effect by using an agonist protein and an artificial nucleic acid circuit, thereby completing the present invention.

[0006] One object of the present invention is to provide a composition for detecting a target nucleic acid, comprising: an agonist protein; a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are bound; and a reporter complement.

[0007] Another object of the present invention is to provide a kit comprising the composition for detecting the above-described target nucleic acid and a method for detecting a target nucleic acid sequence using the same.

[0008] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0009] In addition, the terminology used in this specification is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. It should be understood that the terms "comprise" or "have" in this specification are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0010] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0011] Furthermore, to avoid confusion due to overlapping content, the description of overlapping content has been omitted. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0012] Endonucleases are attracting attention as useful tools for identifying target nucleic acids due to their sequence-specific catalytic activity. In particular, the CRISPR / Cas system has attracted significant attention for its potential in molecular diagnostics. Among these, the CRISPR / Cas12 and CRISPR / Cas13 systems have demonstrated outstanding diagnostic potential due to their secondary cleavage activity. However, despite this potential, diagnostic applications of the CRISPR / Cas system face several challenges, including the increased cost and instability associated with the use of guide RNA, and the requirement that the preferred cleavage site of the Cas protein be adjacent to the target sequence.

[0013] Meanwhile, the Argonaute protein, named after its homology with the Argonaute family of proteins in eukaryotes, exhibits unique properties that could be applied to molecular diagnostics. One such feature is its ability to cleave target nucleic acids without the need for a specialized sequence, such as the protospacer adjacent motif (PAM) used by Cas proteins for target cleavage. Instead, the Argonaute protein precisely cleaves target nucleic acids by base pairing with a short nucleic acid strand located between positions 10 and 11 of the guide DNA, facilitating the design of relatively cost-effective and versatile molecular diagnostic techniques.

[0014] More specifically, the "Agonaute protein" may be derived from agonite, which exists in various organisms, including prokaryotes, eukaryotes, and archaea, and may be derived from agonite, which exists in various organisms, including bacteria, animals, plants, and humans, without limitation. Preferably, the protein is derived from a thermophilic microorganism. That is, a thermophilic agonite protein is a protein derived from a thermophilic microorganism and exhibits high stability in high-temperature environments, thereby enhancing the robustness of an agonite protein-based nucleic acid detection method.

[0015] For example, the argonaute protein may be derived from at least one selected from the group consisting of hAgo2 (from Human) Q9UKV8, TtAgo (from Thermus thermophilus) Q746M7, PfAgo (from Pyrococcus furiosus) Q8U3D2, and CbAgo (from Clostridium butyricum). Preferably, the thermophilic argonaute protein may be TtAgo (from Thermus thermophilus).

[0016] The present invention proposes a novel DNA detection technology that combines an artificial nucleic acid circuit with the highly systematic sequence-guided cleavage activity of the Argonaute protein.

[0017] Accordingly, the present invention provides a composition for detecting a target nucleic acid, comprising: an agonist protein (Ago); a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are bound; and a reporter complement; wherein the reporter complement is composed of the same sequence as the reporter sequence to which the quencher and the fluorescent probe are not bound, and wherein the first guide nucleic acid, the second guide nucleic acid, or both of them have a phosphate attached to the 5' end.

[0018] At this time, since the agonist protein cannot utilize the first guide nucleic acid or the second guide nucleic acid, or both, as a guide nucleic acid if a phosphate is not attached to them, attachment of a phosphate to the 5' end of the first guide nucleic acid and / or the second guide nucleic acid is an essential component.

[0019] At this time, it is preferable that the above-mentioned first guide nucleic acid and / or second guide nucleic acid have one or more of the following conditions when designing:

[0020] 1) Trigger 1 (T1) and trigger 2 (T2), which are reaction products cleaved by the agonist protein, will be designed to function as separate guide nucleic acids.

[0021] 2) It will consist of 16 to 18 bases.

[0022] 3) It should have a CG content of between 10 and 30%;

[0023] 4) Thymine will be located at the first base sequence of the nucleic acid, and

[0024] 5) The 12th base sequence of the nucleic acid must not be adenine.

[0025] As described above, in order for the first guide nucleic acid and / or the second guide nucleic acid to be utilized as a guide nucleic acid, the condition as a guide nucleic acid is satisfied if a phosphate is attached to the 5' end.

[0026] At this time, the first guide nucleic acid and / or the second guide nucleic acid are recognized by the agonist protein as a guide, and the trigger 1 and trigger 2 generated by the cleavage thereof naturally have a form in which a phosphate is attached to the 5' end, so the trigger 1 and trigger 2 can function as separate guide nucleic acids.

[0027] However, since the performance of the guide nucleic acid is improved as many of the conditions 1) to 5) above are satisfied as possible, other than the condition of attaching a phosphate to the 5' end of the first guide nucleic acid and / or the second guide nucleic acid, it is preferable that the first guide nucleic acid and / or the second guide nucleic acid satisfy at least one of the above conditions when designing the first guide nucleic acid and / or the second guide nucleic acid.

[0028] At this time, the quencher binding to the above-mentioned reporter sequence may be any one selected from the group consisting of DABCYL, BHQ, ECLIPSE, and TAMRA, and the fluorescent probe may be any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705.

[0029] According to one embodiment of the present invention, since the detection specificity and sensitivity were significantly higher when the quencher and fluorescent probe were bound only to the reporter nucleic acid when applying the agonist protein and artificial nucleic acid circuit (ANCA) method compared to when they were bound to both the reporter nucleic acid and the reporter complement or to the reporter complement only, the quencher and fluorescent probe were preferably bound only to the reporter nucleic acid in the present invention.

[0030] The reaction sequence of the Argonaute protein and the artificial nucleic acid circuit (ANCA) using the composition according to the present invention is briefly introduced as follows:

[0031] 1) The ANCA reaction begins when the agonist protein (Ago) forms a complex with the first guide nucleic acid (G1) and the second guide nucleic acid (G2), respectively, and recognizes the target nucleic acid. Specifically, the Ago / G1 and Ago / G2 complexes hybridize with complementary target DNA sequences and then cleave the target nucleic acid between the 10th and 11th bases from the 5' end of each guide nucleic acid (indicated by the red symbol in Figure 1). As a result, a short nucleic acid fragment, the first trigger (T1), is generated from the target DNA.

[0032] 2) Since the first trigger (T1) can itself be utilized as another guide nucleic acid, an Ago / T1 complex is formed, and this complex recognizes and cleaves the reporter nucleic acid (R), releasing two short DNA fragments, the reaction product (Output) and the second trigger (T2).

[0033] 3) As the reporter nucleic acid is cleaved by the agonist protein, the reaction product (Output) generates a fluorescent signal (indicated by the dotted box in Figure 1), and the second trigger (T2) binds to the agonist protein again to form the Ago / T2 complex.

[0034] 4) The newly formed Ago / T2 complex recognizes and cleaves the reporter complement (R*) to generate the first trigger (T1), thereby completing a repetitive, cyclic positive feedback loop. In other words, this positive feedback loop repeatedly initiates the cleavage reaction in the presence of the target nucleic acid, and as the reaction progresses, reaction products (Output) are rapidly generated, resulting in a highly specific and exponential signal amplification reaction. Ultimately, the presence of the target nucleic acid in the sample can be confirmed by monitoring changes in the fluorescence signal.

[0035] Because the ANCA method utilizes a single protein, it is simpler to manufacture and less expensive than conventional nucleic acid detection methods. Furthermore, this reaction is performed in a single step, and if the target nucleic acid is known, it can be easily applied to detect various target nucleic acids (substances) by modifying the nucleic acid recognition site.

[0036] In the present invention, the possibility of using the ANCA method according to the present invention is confirmed by applying the ANCA method only to antibiotic-resistant strains (CPE) that produce KPC or IMP as an example of implementation, but since the ANCA method according to the present invention can be easily applied to any nucleic acid, strain or virus whose sequence is known, without limitation to the type, there is no particular limitation on the type of target nucleic acid.

[0037] In particular, according to one specific example of the present invention, by using the composition of the present invention, antibiotic-resistant strains (CPE) producing KPC or IMP were successfully detected, and antibiotic-resistant strains (CPE) added to human samples such as urine and blood could be detected without nucleic acid amplification and extraction steps. In addition, the ANCA method was applied to diagnose patients infected with antibiotic-resistant strains (CPE) using rectal swab samples, recording clinical sensitivity and specificity of 100%, respectively.

[0038] Therefore, the ANCA method has significant potential for point-of-care diagnosis of strains, facilitating patient treatment in the early stages of strain infection, ultimately increasing diagnostic accuracy and improving treatment outcomes in clinical settings.

[0039] For example, such target nucleic acid detection may preferably be for the detection of nucleic acids for pathogens. A "pathogen" refers to an organism capable of causing disease by directly damaging host tissues or producing toxins, such as bacteria, viruses, fungi, and other parasites.

[0040] The target nucleic acid detection technology according to the present invention detects nucleic acids present in pathogens with high sensitivity and accuracy, thereby providing specific information on whether the target pathogen is infected or whether the pathogen exists.

[0041] Accordingly, the present invention also provides a composition for detecting a pathogen, a kit, and a method for detecting a pathogen using the same, including an agonist protein; a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are combined; and a reporter complement. Exemplary bacterial genera of pathogens include Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Salmonella, Helicobacter, Neisseria, Campylobacter, Chlamydia, Clostridium, Vibrio, Treponema, Escherichia coli, Mycobacterium, Klebsiella, Actinomyces, Bacterioides, Bordetella, Borrelia, Brucella, Corynebacterium, Diplococcus, Enterobacter, Fusobacterium, Leptospira, Listeria, Pasteurella, Proteus, Rickettsia, Shigella, Sphaerophorus, Acinetobacter, Aeromonas Burkholderia, Campylobacter, Corynebacterium, Enterococcus, Erwinia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycoplasma, Neisseria,Veillonella, Vibrio, Coxiella, IMP (Imipenemase), New-Delhi metallo-beta-lactamase (NDM), VIM (Verona Integron-encoded Metallo-beta-lactamase), OXA-48 (Oxacillinase-48) or Yersinia.

[0042] Meanwhile, the present invention provides a kit for detecting a target nucleic acid comprising the composition described above. Specifically, the kit according to the present invention comprises a composition for detecting a target nucleic acid comprising: an agonist protein (Ago); a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are bound; and a reporter complement; wherein the reporter complement is composed of the same sequence as the reporter sequence to which the quencher and the fluorescent probe are not bound, and wherein the first guide nucleic acid, the second guide nucleic acid, or both have a phosphate attached to the 5' end.

[0043] In one embodiment of the present invention, optimal conditions for the ANCA reaction have been established. Therefore, the kit according to the present invention preferably comprises 150 to 400 nM of argonaute protein; 250 to 1,000 nM of a reporter sequence; and 250 to 1,000 nM of a reporter complement; and most preferably 200 nM of argonaute protein; 500 nM of a reporter sequence; and 500 nM of a reporter complement.

[0044] In addition, it is preferable that the above kit further includes a reaction buffer containing MgCl2, wherein the MgCl2 preferably has a concentration of 5 to 30 mM, and most preferably a concentration of 10 mM.

[0045] Additionally, the kit may further include a collection device for non-invasive sample collection. Such a collection device may be, for example, one or more selected from the group consisting of an absorbent pad, a cotton swab, a dropper, a spoon, a swab, and a toothpick.

[0046] Preferably, it includes a cotton swab. More specifically, it may further include a 3D nanopillar array cotton swab. The 3D nanopillar array cotton swab is a cotton swab specially developed for direct application to a 3D nanopillar array. The 3D nanopillar array cotton swab can be manufactured by a method of replicating a nanopillar array and growing polyaniline nanofibers thereon, and the cotton swab has a thickness of 10 2 10 in 7 It can effectively capture strains in the quantitative range of CFU / ㎖, and the maximum capture range is 75 strains / 100 μm. 2 It was confirmed that this high capture efficiency and wide capture range can improve the sensitivity and accuracy of strain detection.

[0047] According to one embodiment of the present invention, the combination of the ANCA method and a 3D nanofiller array swab according to the present invention can improve the detection efficiency of antibiotic-resistant strains (CPE). The unique properties of the 3D nanostructure irreversibly capture harmful strains, ensuring a safe diagnostic procedure. Therefore, the integrated use of the ANCA method and the 3D nanofiller array swab can lead to the promising development of diagnostic tools for various pathogens (including strains and viruses), including antibiotic-resistant strains.

[0048] According to one embodiment of the present invention, for the first time, the ANCA method was used to directly detect antibiotic CPE from a rectal swab (3D nanofiller array swab) collected from a patient, and these results are very impressive. Furthermore, in one embodiment of the present invention, the diagnostic results using the rectal swab were demonstrated to be more accurate than those obtained from the transport medium in which the swab was soaked. In other words, by directly applying the clinical sample obtained using the 3D nanofiller array swab to the ANCA method, the possibility of sample contamination can be avoided and the spread of strains can be suppressed, so it is expected that pathogens (including strains and viruses), including antibiotic-resistant strains, can be precisely diagnosed without the risk of secondary infection.

[0049] Additionally, in the above kit, the optimal amount of reagents used in a specific reaction can be readily determined by those skilled in the art who have learned the disclosure herein. Typically, the kit of the present invention is manufactured in a separate package or compartment containing the aforementioned components.

[0050] Additionally, the kit may further include instructions for use and other tools or equipment necessary for detection.

[0051] Meanwhile, the present invention provides a method for detecting a target nucleic acid, comprising a step of reacting the above-described target nucleic acid detection composition with a sample. Specifically, the composition is a target nucleic acid detection composition comprising an agonist protein (Ago); a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are bound; and a reporter complement; wherein the reporter complement is composed of the same sequence as the reporter sequence to which the quencher and the fluorescent probe are not bound, and the first guide nucleic acid, the second guide nucleic acid, or both have a phosphate attached to the 5' end.

[0052] In one embodiment of the present invention, optimal conditions for the ANCA reaction have been established. Therefore, the reaction according to the present invention is preferably performed at 65 to 75°C, most preferably at 75°C.

[0053] A sample according to the present invention refers to a biological sample and any genes (nucleic acids) therefrom. The term "biological sample" means any sample containing any DNA, RNA, and / or target DNA, RNA. The biological sample may be any tissue or body fluid obtained from a subject. The biological sample includes, but is not limited to, sputum, blood, serum, plasma, blood cells (e.g., white blood cells), tissue, biopsy sample, smear sample, wash sample, swab sample, cell-containing body fluid, fluid nucleic acid, urine, peritoneal fluid and pleural fluid, cerebrospinal fluid, stool, tears or cells therefrom, or pathogens (including strains and viruses, etc.) containing any nucleic acid. The biological sample may be obtained by a method that does not cause harm to the subject.

[0054] In particular, the composition and / or kit according to the present invention has excellent detection efficacy, and thus can detect (diagnose) pathogens (including strains and viruses) present in human samples as well as objects even at very low concentrations.

[0055] Accordingly, the detection method of the present invention may further include a step of visually confirming a change in fluorescence color development of a reactant; or a step of measuring a change in fluorescence color development of a reactant. The measurement of this change in fluorescence color development may be a conventional fluorescence measurement method in which measurement is performed by fixing the measurement wavelength of a fluorescence device. Specifically, the measurement may be performed by fixing the measurement wavelength of the fluorescence device. For example, in the case of FAM fluorescence, a method in which the fluorescence intensity of a reactant at a wavelength of ex;495 / em;520 is measured and the fluorescence change is observed at intervals of 5 to 10 minutes for 1 to 2 hours may be used.

[0056] The detection method according to the present invention (ANCA method) has several notable advantages, particularly in the detection of pathogen (including strains and viruses) infections. First, unlike conventional methods that require separate nucleic acid amplification and extraction steps, the ANCA method can detect target nucleic acids (DNA) in a single step without an extraction step, simplifying the diagnostic process. Second, the reaction is performed isothermally. This feature reduces the need for complex and expensive thermal cycling equipment, making it easier and more cost-effective to use in a variety of environments. Third, it exhibits high sensitivity and specificity when detecting target nucleic acids. This improves detection accuracy and reduces the likelihood of false positive or negative results. Fourth, because it uses the Argonaute protein, it does not require a PAM sequence for target recognition. This expands the range of detectable nucleic acid targets and enhances its utility in diagnostic applications. Fifth, its high stability allows for direct detection of pathogens (including strains and viruses) even in harsh environments. Sixth, it can be easily integrated into portable diagnostic devices, enabling rapid on-site diagnosis of pathogen (including strains and viruses) infections. This is especially useful in environments with limited resources or where immediate diagnosis is required.

[0057] The system according to the present invention can detect target pathogens added to human samples with 100% clinical sensitivity and specificity without nucleic acid amplification or separate extraction steps, and when used in conjunction with a three-dimensional nanopillar array structure, target pathogens collected from the surface of an object can be directly identified. In addition, since the trigger sequence cleaved by the subprotein is designed to be used as a guide, it is simpler to manufacture than existing nucleic acid detection circuits because it uses a single protein, and it has the advantage of reducing costs because it can detect target pathogens with high sensitivity even with a small amount of target nucleic acid sequence as long as the subprotein, reporter, and reporter complement according to the system of the present invention are not depleted. In addition, the system according to the present invention has the advantage of being easy to apply to the detection of various target nucleic acid sequences by proceeding in a single step and appropriately changing the nucleic acid recognition site to match the target pathogen.

[0058] Figure 1 is a schematic diagram showing the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention.

[0059] Figure 2 shows the results of the T0-T values ​​of the Argonaute protein and the artificial nucleic acid circuit (ANCA) method according to the form of the reporter (R) and reporter complement (R*) (T0: critical time for negative samples, T: critical time for positive samples). Here, the critical time is the reaction time at which the fluorescence intensity reaches 10,000.

[0060] Figure 3 shows the results of optimizing the reaction conditions of the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): the result of confirming the optimal reaction temperature, (b): the result of confirming the optimal concentration of the reporter (R), (c): the result of confirming the optimal concentration of the reporter complement (R*), (d): the result of confirming the optimal concentration of the agonist protein, (e): the result of confirming the optimal concentration of MgCl2).

[0061] Figure 4 shows the results of evaluating the sensitivity of the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention to Klebsiella pneumoniaecarbapenemase (KPC) and Klebsiella pneumoniaeImipenemase (IMP) ((a): Results of confirming the change in fluorescence intensity over time for various concentrations of KPC, (b) Results of confirming the critical time according to the concentration of KPC, (c): Results of confirming the change in fluorescence intensity over time for various concentrations of IMP, (d): Results of confirming the critical time according to the concentration of IMP). Here, the critical time is the reaction time at which the fluorescence intensity reaches 10,000.

[0062] FIG. 5 shows the results of evaluating the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): schematic diagram of ANCA according to the present invention, (b): schematic diagram of ANCA without reporter complement (R*), (c): target DNA detection results using ANCA (left panel: change in fluorescence intensity according to reaction time, right panel: PAGE analysis results), (d): target DNA detection results using ANCA without reporter complement (R*) (left panel: change in fluorescence intensity according to reaction time, right panel: PAGE analysis results)).

[0063] Figure 6 shows the results of RT-PCR detecting KPC and IMP positive (producing) strains using specific primers ((a): KPC detection, (b): IMP detection).

[0064] FIG. 7 shows the results of directly detecting antibiotic-resistant strains using the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): schematic diagram of ANCA reaction, (b): detection of KPC through ANCA method, (c): detection of IMP through ANCA method, (d): detection of KPC through RT-PCR reaction, (e): detection of IMP through RT-PCR reaction).

[0065] FIG. 8 shows the results of directly detecting antibiotic-resistant strains in urine samples using the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): schematic diagram of ANCA reaction, (b): detection of KPC using ANCA method, (c): detection of IMP using ANCA method).

[0066] FIG. 9 shows the results of directly detecting antibiotic-resistant strains in blood samples using the agonist protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): schematic diagram of ANCA reaction, (b): detection of KPC using ANCA method, (c): detection of IMP using ANCA method).

[0067] FIG. 10 shows the results of diagnosing antibiotic-resistant strains using the Argonaute protein and the artificial nucleic acid circuit (ANCA) method according to the present invention ((a): Schematic diagram of diagnosis of antibiotic-resistant strains in transport medium soaked with rectal swabs using the ANCA method, (b): Results showing T0-T values ​​according to clinical samples (transport medium soaked with rectal swabs), (c): Results showing T0-T values ​​(red) and Ct0-Ct values ​​(blue) for KPC-positive clinical samples). T0 is the critical time of a solution containing only the transport medium, and T is the critical time of a clinical sample. Ct0 is set to 50, and Ct represents the PCR critical cycle value of a clinical sample.

[0068] FIG. 11 is a ROC curve obtained from the diagnostic results of clinical samples and antibiotic-resistant strain-added samples using the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): Evaluation for a total of 200 clinical (143) and KPC-added samples (57 transport media soaked with rectal swabs), (b): Evaluation for a total of 160 clinical (80) and IMP-added samples (80 transport media soaked with rectal swabs)).

[0069] Figure 12 shows the results of diagnosing clinical samples and samples with antibiotic-resistant strains added using the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention (a): Calculated results of negative percent agreement (NPA) and positive percent agreement (PPA), (b): Results showing T0-T values ​​for clinical samples and KPC-added samples (medium in which rectal swabs were soaked), (c): Results showing T0-T values ​​for clinical samples and IMP-added samples (medium in which rectal swabs were soaked). T0 is the critical time for the transport medium without any addition, and T is the critical time for clinical samples and mock clinical samples.

[0070] Figure 13 shows the results of directly diagnosing the presence of antibiotic-resistant strains in clinical samples (rectal swabs) using the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): schematic diagram of the diagnostic method, (b): results showing T0-T values). T0 is the critical time for a clean rectal swab, and T is the critical time for a clinical sample.

[0071] Figure 14 is a comparative graph of T0-T values ​​obtained from rectal specimens (red) and transport media (black) of KPC-positive clinical samples using the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention. T0 is the critical time for the transport media without any additives, and T is the critical time for the clinical sample.

[0072] Figure 15 shows the results of a scatter plot of T0-T values ​​obtained from rectal swabs (red) versus T0-T values ​​obtained from transport media (black) of KPC-positive clinical samples and mock clinical samples using the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): clinical samples, (b): mock clinical samples).

[0073] FIG. 16 shows the results of detecting the capture of antibiotic-resistant bacteria using a 3D nanofiller array swab and the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention ((a): Photograph and schematic diagram of capturing antibiotic-resistant strains from micropig skin, desk, scissors, and gloves using a 3D nanofiller array swab, (b): Scanning electron microscope (SEM) images before (top) and after (bottom) capturing antibiotic-resistant strains using a nanofiller array swab, (c): Results showing the T0-T values ​​for KPC on a contaminated surface, (d): Results showing the T0-T values ​​for IMP on a contaminated surface). T0 is the critical time for a negative control sample, and T is the critical time for a test sample.

[0074] FIG. 17 shows the results of diagnosing whether a clinical specimen (transport medium soaked with a rectal swab) is infected with an antibiotic-resistant strain by applying the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention to a portable device ((a): schematic diagram, (b): diagnosis result for whether or not an antibiotic-resistant strain is infected).

[0075] FIG. 18 shows the results of evaluating the sensitivity of the Argonaute protein and artificial nucleic acid circuit (ANCA) method according to the present invention to Verona integron-encoded metallo-beta-lactamase (VIM), New Delhi metallo-beta-lactamase (NDM), and oxacillinase-48 (OXA-48) ((a): Results of confirming the change in fluorescence intensity over time for various concentrations of VIM, (b) Results of confirming the critical time according to VIM concentration, (c): Results of confirming the change in fluorescence intensity over time for various concentrations of NDM, (d): Results of confirming the critical time according to NDM concentration, (e): Results of confirming the change in fluorescence intensity over time for various concentrations of OXA-48, (f): Results of confirming the critical time according to OXA-48 concentration). Here, the critical time is the reaction time at which the fluorescence intensity reaches 10,000.

[0076] Figure 19 shows the results of an attempt to detect carbapenemase-producing Klebsiella pneumoniae (CPKP) using the VIM, NDM, and OXA-48 circuits, confirming that each circuit can well distinguish between WT and CPKP.

[0077] Figures 20 to 24 illustrate schematic diagrams of individual systems. Figure 20 is a sequence-based schematic diagram for a KPC circuit, Figure 21 is a sequence-based schematic diagram for an IMP circuit, Figure 22 is a sequence-based schematic diagram for a VIM circuit, Figure 23 is a sequence-based schematic diagram for an NDM circuit, and Figure 24 is a sequence-based schematic diagram for an OXA-48 circuit.

[0078] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0079] Experimental Materials and Methods

[0080] 1. Experimental materials

[0081] All oligonucleotides used in this invention were purchased from Integrated DNA Technology (Coralville, IA, USA). TtAgo (Thermus thermophilus argonaute) was purchased from NEB (Beverly, MA, USA). Gel-Red Nucleic Acid Stain (41003) was purchased from Biotium (Fremont, CA, USA), and 6X DNA loading buffer and SYBR Green I Nucleic Acid Gel Stain were purchased from Takara Korea Biomedical Inc. (Seoul, Korea). The TOPsimple preMIX (aliquot)-nTaq kit was purchased from Enzynomics (Daejeon, Korea), and human urethra and blood samples were purchased from Innovative Research (Novi, MI, USA). Finally, transport medium was purchased from Asanpharm (Seoul, Korea).

[0082] 2. Evaluation of the Argonaute Protein and Artificial Nucleic Acid Circuit (ANCA) Method

[0083] Target DNA (2 μl) for Klebsiella pneumoniaecarbapenemase (KPC) was mixed with reaction solution (18 μl). The reaction solution was composed of a reaction buffer containing Tris-HCl (20 mM), (NH4)2SO4 (10 mM), KCl (10 mM), MgCl2 (10 mM), and Triton X-100 (0.1%), and containing agonist protein (TtAgo, 200 nM), first guide nucleic acid (G1, 25 nM), second guide nucleic acid (G2, 25 nM), reporter sequence (R, 500 nM), and reporter complement (R*, 500 nM). The mixture was then reacted at 75 °C, and the fluorescence signal was measured at 1-min intervals using a CFX Opus 96 RT-PCR system (Bio-Rad, Hercules, CA, USA). ANCA reactions without reporter complement (R*) were performed under the same reaction conditions except for the reporter complement (R*).

[0084] Additionally, for PAGE analysis, 1 nM of target DNA was combined with the ANCA reaction solution and reacted at 75°C for 70 minutes. The reaction product was then combined with 6X DNA loading buffer and loaded onto a 15% polyacrylamide gel. After electrophoresis, the gel was stained with Gel-Red Nucleic Acid dye and images were extracted and visualized using the Geldoc Go Imaging system (Bio-Rad).

[0085] 3. Direct detection of antibiotic-resistant bacteria using the Argonaute protein and artificial nucleic acid circuit (ANCA) method.

[0086] All strains and clinical isolates were provided by Gyeongsang National University College of Medicine. The strains were cultured on LB (Luria-Bertani) agar medium at 37°C for 16 h, then harvested and resuspended in sterile PBS. The optical density (OD) of the obtained suspension 600) was measured using Nanophotometer P330 (Implen, Germany), and the number of strains was estimated from this value.

[0087] To confirm the presence of CPE-inducing genes in the strains, gene-specific primers for each KPC (Klebsiella pneumoniaecarbapenemase) and IMP (Klebsiella pneumoniaeImipenemase) gene were designed using the Primer 3 tool (http: / primer3.wi.mit.edu) and NCBI Reference sequence Database (https: / www.ncbi.nlm.nih.gov / nuccoreGCF_000240185.1 (WT), NG_049244.1 (KPC), MH909334 (IMP), AAY33963.1 (VIM), AFN84620.1 (NDM), JN626286.1 (OXA-48)).

[0088] Then, qRT-PCR analysis was performed using these primers. Specifically, qRT-PCR was performed by combining antibiotic-resistant strains with a PCR reaction solution containing gene-specific primer sets (each 0.5 μM), 1X TOPsimple preMIX (aliquot)-nTaq, and 1X SYBR Green I dye. The amplification reaction proceeded through the following steps: denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, repeated 50 times in total. The fluorescence signal of the PCR was measured at each extension step using a CFX Opus 96 RT-PCR system.

[0089] For direct detection of antibiotic-resistant strains, 10 5The strains were combined with the ANCA reaction components at cell / ml and reacted according to the procedure described above. Each strain sample was individually mixed with the KPC, IMP, VIM, NDM, and OXA-48 cycle mixture.

[0090] To verify the applicability of the ANCA method for strain detection in clinical samples, each strain was tested against human urine and blood samples (10 5 cells / mL) were added. The urine and blood samples with added strains were mixed with KPC (Klebsiella pneumoniaecarbapenemase) and IMP (Klebsiella pneumoniaeImipenemase) cycle mixtures and processed according to the same procedure described above.

[0091] 4. Diagnosis of antibiotic-resistant bacteria using the Argonaute protein and artificial nucleic acid circuit (ANCA) method

[0092] One hundred and forty-three clinical specimens (63 KPC-positive, 80 KPC-negative) were obtained from Gyeongsang National University College of Medicine. Rectal specimens were collected from patients using cotton swabs, placed in transport media (5 ml), and stored at -80°C until further use. This study was rigorously reviewed and approved by the Ethics Committee of Gyeongsang National University Changwon Hospital (IRB approval number 2022-10-012).

[0093] For the diagnosis of antibiotic-resistant strains, the transport medium (2 μl) in which the rectal swab was soaked was mixed with the ANCA reaction components, and the reaction was performed according to the procedure mentioned above. For the comparison of the ANCA method and the qRT-PCR results, the transport medium in which the rectal swab was soaked was mixed with the PCR reaction mixture containing the Klebsiella pneumoniaecarbapenemase (KPC) gene-specific primer set (0.5 μM each), 1X TOPsimple preMIX (aliquot)-nTaq, and 1X SYBR Green I dye. The fluorescence signal at each extension step was measured using a CFX Opus real-time system (Bio-Rad), and C t Related software (CFX Maestro) was used to obtain the values.

[0094] To prepare mock clinical samples for Klebsiella pneumoniaecarbapenemase (KPC), rectal swabs stored in transport medium that had tested negative at a hospital were randomly selected. These swabs were then randomly sprayed with various concentrations of KPC (Klebsiella pneumoniaecarbapenemase) and immediately immersed in clean transport medium. Finally, the prepared mock clinical samples (2 μL) were mixed with the ANCA reaction components, and the reaction was performed according to the above-mentioned procedure.

[0095] To prepare mock clinical samples for IMP (Klebsiella pneumoniae imipenemase), transport media (500 μl each) containing rectal swabs that tested negative in hospitals were randomly collected. Various concentrations of IMP (Klebsiella pneumoniae imipenemase) were then randomly mixed with the transport media. Finally, the prepared mock clinical samples (2 μl) were mixed with the ANCA reaction components, and the reaction was performed according to the above-mentioned procedure.

[0096] To diagnose antibiotic-resistant strains, rectal swabs collected from patients and a mock rectal swab containing Klebsiella pneumoniae imipenemase (IMP) were soaked in distilled water (500 μl). The distilled water containing the extrarectal swab (2 μl) was then mixed with the ANCA reaction components, and the reaction was performed according to the procedure described above.

[0097] A portable isothermal nucleic acid amplification device was purchased from Revosketch (Daejeon, Korea). Transport medium (2 μl each) containing 20 rectal swabs was mixed with the ANCA reaction components, and the reaction was performed according to the procedure described above.

[0098] 5. Capture and Detection of Antibiotic-Resistant Bacteria Using Argonaute Protein and Artificial Nucleic Acid Circuit (ANCA) Method and 3D Nanofiller Swabs

[0099] Each strain (10 5 (cell / mL) was sprayed onto micropig Franz cell membranes (APURES, Seoul, Korea), desks, scissors, and gloves. Then, the strains present on the contaminated surface were gently rubbed using a 3D nanopillar array swab to capture them. SEM images were taken using a Nova 230 system (FEI, Hillsboro, OR, USA) at an accelerating voltage of 15 keV. The 3D nanopillar array with the strains captured was cut into 0.25 cm X 0.25 cm pieces that could fit into a PCR tube and placed in the ANCA reaction solution. The reaction was then performed according to the protocol mentioned above.

[0100] Example 1. Design of the Argonaute Protein and Artificial Nucleic Acid Circuit (ANCA) Method

[0101] The Argonaute protein and artificial nucleic acid circuit (ANCA) method was designed to construct a circular circuit by fully utilizing the two cleavage activities of the Argonaute protein: 1) the cleavage sites of the Argonaute protein are designed to be at the 10th and 11th positions from the 5' end of the guide DNA, and 2) the strand cleaved by the Argonaute protein has a phosphate group attached to the 5' end of the strand, so that it can be utilized as a guide DNA. The overall procedure of the ANCA method is shown in Fig. 1.

[0102] Specifically, to implement the ANCA method, four signal processors, including a first guide nucleic acid (G1), a second guide nucleic acid (G2), a reporter (R), and a reporter complement (R*), were designed to work cooperatively. The first guide nucleic acid (G1) and the second guide nucleic acid (G2) were designed according to the guide DNA design guidelines on the New England Biolabs Inc. (NEB) website, and the reporter (R) was designed to have both a quencher and a fluorophore. In addition, the sequences of all signal processors were carefully adjusted so that trigger 1 (T1) and trigger 2 (T2), which are generated during the reaction, can be used as guide DNAs for the agonist protein. The sequences of the oligonucleotides used in this experiment are shown in Table 1 below. In one embodiment, FAM was used as the fluorescent probe and BHQ2 was used as the quencher. In addition, details regarding each reaction are individually shown in FIGS. 20 to 24 . FIG. 20 is a sequence-based schematic diagram for a KPC circuit, FIG. 21 is a sequence-based schematic diagram for an IMP circuit, FIG. 22 is a sequence-based schematic diagram for a VIM circuit, FIG. 23 is a sequence-based schematic diagram for an NDM circuit, and FIG. 24 is a sequence-based schematic diagram for an OXA-48 circuit.

[0103] [Table 1]

[0104]

[0105]

[0106]

[0107] Example 2. Optimization and Sensitivity Evaluation of the Argonaute Protein and Artificial Nucleic Acid Circuit (ANCA) Method

[0108] To determine the optimal conditions for the ANCA method, we first observed changes in fluorescence intensity depending on the structures of the reporter (R) and reporter complement (R*). As shown in Figure 2, when the fluorophore and quencher were attached only to the reporter (R), the ANCA method exhibited excellent target DNA detection performance. In contrast, when the fluorophore and quencher were attached to both the reporter (R) and reporter complement (R*), the reaction efficiency was significantly reduced. This is presumably due to steric hindrance that interferes with the cleavage activity of the Ago / guide DNA complex.

[0109] Next, the reaction temperature and the reporter (R), reporter complement (R*), argon protein and Mg 2+ The concentration of was optimized. As shown in Fig. 3, the conditions for obtaining optimal detection performance were confirmed to be a reaction temperature of 75°C, 500 nM of each reporter (R) and reporter complement (R*), 200 nM of agonist protein, and 10 mM of MgCl2. Therefore, the conditions were used in subsequent experiments.

[0110] Meanwhile, the sensitivity of the ANCA method was evaluated for the target base sequences KPC (Klebsiella pneumoniaecarbapenemase) and IMP (Klebsiella pneumoniaeImipenemase) under the optimized conditions as described above.

[0111] As shown in Fig. 4, a linear relationship with an R² value of 0.99 was observed in the range of 100 fM to 10 nM for KPC (Klebsiella pneumoniaecarbapenemase), and a linear relationship with an R² value of 0.99 was observed in the range of 10 fM to 10 nM for IMP (Klebsiella pneumoniaeImipenemase).

[0112] Based on the above results, the limit of detection (LOD) of the ANCA method according to the present invention was calculated using the following formula, and the limit of detection (LOD) of the ANCA method according to the present invention was found to be 2.5 fM: (LOB = mean of negative samples + 1.645 × standard deviation of negative samples, LOD = LOB of low concentration samples + 1.645 × standard deviation).

[0113] The LOD was calculated to be 2.5 fM, demonstrating a significant improvement over previous results using Argonaute protein without prior amplification. Furthermore, the probe used in the ANCA method according to the present invention successfully detected KPC and IMP sequences with only minor sequence modifications. These results demonstrate the flexibility and broad target applicability of the ANCA method.

[0114] Example 3. Evaluation of the Argonaute Protein and Artificial Nucleic Acid Circuit (ANCA) Method

[0115] Using the optimal reaction conditions confirmed in Example 2 above, the ANCA method according to the present invention was evaluated.

[0116] To this end, as shown in Figure 5a, the ANCA method was developed to achieve exponential fluorescence signal amplification through a positive feedback circuit. We first targeted Klebsiella pneumoniaecarbapenemase (KPC) as a target substance and evaluated how the results of the ANCA reaction varied depending on the component.

[0117] As shown in the red spectrum of Figure 5c, a strong fluorescence signal was observed during the ANCA reaction when all reaction components of ANCA, including the target DNA, were present. In contrast, as shown in the black and blue spectra of Figure 5c, a negligible level of fluorescence signal was observed when the target DNA and / or Ago protein were absent.

[0118] To more closely validate the ANCA method, gel electrophoresis (polyacrylamide gel electrophoresis, PAGE) analysis was additionally performed. As shown in the right panel of Fig. 5 c, a band representing the intact reporter (R)-reporter complement (R*) structure was observed before ANCA reaction in the absence of target DNA or agonist protein (lanes 1 and 2 in Fig. 5 c). However, after intact ANCA reaction in the presence of both target DNA and ANCA response elements, the band representing the reporter (R)-reporter complement (R*) structure was completely resolved (lane 3 in Fig. 5 c).

[0119] Next, as shown in Figure 5b, a comparative experiment was performed to evaluate the amplification efficiency of the ANCA method. Specifically, this is as follows. In an ANCA reaction without a reporter complement (R*), the Ago / G1 and Ago / G2 complexes cleave the target DNA to form the Ago / Trigger 1 (T1) complex. Subsequently, the Ago / T1 complex cleaves the reporter (R) to generate a reactant (Output) and Trigger 2 (T2).

[0120] However, as shown in the red spectrum of Fig. 5d, the absence of the reporter complement (R*) resulted in no further reaction, and thus the fluorescence signal did not increase further over the 70-minute reaction even in the presence of the target nucleic acid sequence. Furthermore, as shown in the black and blue spectra of Fig. 5d, the fluorescence signal did not increase in the absence of the target DNA and / or the agonist protein. These results are also confirmed in the PAGE results on the right panel of Fig. 5d, where bands representing the intact, uncut reporter (R) structure were confirmed under all conditions.

[0121] Therefore, these results imply that the ANCA method consists of a positive feedback circuit, provides high amplification efficiency, and facilitates sensitive detection of target DNA.

[0122] Example 4. Direct detection of antibiotic-resistant strains using Argonaute protein and artificial nucleic acid circuit (ANCA) method.

[0123] 1. Direct detection of antibiotic-resistant strains

[0124] In this experiment, we attempted to detect antibiotic-resistant strains (CPE) without a separate DNA purification process using the ANCA method. To prevent cross-contamination, K. pneumoniae producing KPC (hereinafter referred to as KPC-positive (producing) strain) and K. pneumoniae producing IMP (hereinafter referred to as IMP-positive (producing) strain) were cultured separately and used in the experiment. As shown in Figure 6, PCR was performed using primers specific to the KPC and IMP gene sequences, confirming that the strains were cultured well.

[0125] Next, to verify the ANCA method according to the present invention, as shown in Fig. 7 a, antibiotic-resistant strains were directly added to the tube containing the ANCA reaction mixture and reacted at 75˚. As shown in Fig. 7 b, the largest T0-T value was observed when KPC (Klebsiella pneumoniaecarbapenemase) was present in the sample (blue bars), which proves that the target antibiotic-resistant strain can be directly detected using the ANCA reaction for KPC. In addition, as shown in Fig. 7 c, it was confirmed that IMP (Klebsiella pneumoniaeImipenemase) could also be directly detected like KPC. This result can also be confirmed through Figs. 7 d and e, which confirmed real-time fluorescence from RT-PCR using primers specific for the KPC and IMP gene sequences.

[0126] The above results imply that the ANCA method can detect antibiotic-resistant strains in a very simple manner, without prior concentration, lysis, or DNA purification steps. This is believed to be due to the ANCA method's high activity at a high reaction temperature of 75°C.

[0127] 2. Detection of antibiotic-resistant strains from clinical samples

[0128] To determine whether the ANCA method can detect antibiotic-resistant strains in clinical samples, human urine and blood samples containing antibiotic-resistant strains were prepared.

[0129] As shown in Figure 8a and Figure 9a, urine and blood samples with added strains were directly added to test tubes containing the ANCA reaction mixture and reacted.

[0130] As shown in b and c of Fig. 8, KPC-producing strains (Klebsiella pneumoniaecarbapenemase) and IMP-producing strains (Klebsiella pneumoniaeImipenemase) contained in urine (99%) were specifically detected.

[0131] In addition, as shown in b and c of Fig. 9, KPC producing strains (Klebsiella pneumoniaecarbapenemase) and IMP producing strains (Klebsiella pneumoniaeImipenemase) contained in blood (99%) were specifically detected.

[0132] These results suggest that the ANCA method can be used to detect antibiotic-resistant strains under challenging conditions. Given that monitoring antibiotic-resistant strains in urine and blood samples can provide valuable epidemiological data on the prevalence and distribution of antibiotic-resistant bacteria, these results have important implications in the fields of diagnostics and biology.

[0133] Example 5. Diagnosis of antibiotic-resistant strains using Argonaute protein and artificial nucleic acid circuit (ANCA) method

[0134] 1. Detection of antibiotic-resistant strains in clinical samples

[0135] The standard diagnostic procedure for antibiotic-resistant strains involves several steps, including sample collection, strain culture, strain identification and isolation, antibiotic susceptibility testing, and carbapenem resistance determination. Because the entire process takes several days, an efficient method for rapid identification of antibiotic-resistant strains is needed. Diagnosis of antibiotic-resistant strains is typically performed using rectal swabs collected from patients. To preserve the integrity of the collected rectal swab sample, it is immediately immersed in transport medium. Transport medium is specially formulated to maintain the integrity of the strain sample during transport and storage, preserving its characteristics and preventing microbial overgrowth. We aimed to diagnose antibiotic-resistant strains in clinical specimens stored in transport medium using the ANCA method.

[0136] For this purpose, a total of 143 rectal swab samples (63 KPC-positive and 80 negative) were collected from each patient using swabs, and each swab was soaked in a transport medium and stored. As shown in Fig. 10 a, the transport medium containing the rectal swabs was mixed with the KPC-specific ANCA reactant to carry out the reaction, and the change in fluorescence signal was observed. The cutoff T0- T value was calculated as 10.11 using the following formula (cutoff T0- T value = average T0- T value of 80 negative samples + 5 × average standard deviation of 80 negative samples). The ANCA-based diagnostic test was performed in a blind manner, and the positive samples were marked sequentially from #81 to #143 according to their T0- T values ​​after the experiment.

[0137] As shown in Fig. 10b, the ANCA method was able to accurately distinguish 63 KPC positive samples and 79 negative samples based on the cutoff line.

[0138] Furthermore, as a result of comparative analysis of the diagnostic results of the ANCA method and PCR, a strong correlation was observed between the T0-T values ​​and the Ct0-Ct values ​​for KPC-positive clinical samples, as shown in Fig. 10c. These results demonstrate that the ANCA method according to the present invention can be used for simple and accurate diagnosis of antibiotic-resistant strains.

[0139] 2. Detection of antibiotic-resistant strains in clinical and simulated clinical samples

[0140] Meanwhile, to evaluate the clinical sensitivity and specificity of the ANCA method, rectal swabs spiked with 57 strains were used in addition to clinical samples. Given that the clinical sensitivity and specificity of molecular diagnostics approved by the Korea Food and Drug Safety (KFDA) are both 95%, a minimum of 80 positive samples needed to be tested using the ANCA method. Therefore, additional mock clinical samples were prepared by spiked with the KPC-producing strain (Klebsiella pneumoniaecarbapenemase) to rectal specimens diagnosed as negative.

[0141] The amount of added KPC-producing strain (Klebsiella pneumoniaecarbapenemase) was determined randomly, and the experiment was conducted in a blind manner.

[0142] Figure 11a shows the diagnostic results of a total of 200 clinical (143) and Klebsiella pneumoniaecarbapenemase (KPC)-producing strain-spiked samples (57), demonstrating 100% sensitivity and 98.7% specificity. In particular, the area under the curve (AUC) value was 0.9997, indicating that the ANCA method exhibits comparable accuracy even when compared to the PCR technique.

[0143] A graph of the T0-T values ​​of the above samples is shown in Fig. 12b, and the simulated clinical samples are marked sequentially from #144 to #200 based on the T0-T values ​​after the experiment. These results are noteworthy, considering that the ANCA method directly uses clinical samples without DNA purification and amplification steps.

[0144] Furthermore, the diagnostic ability of the IMP-specific ANCA reaction was verified using a total of 160 clinical and simulated clinical samples (80 IMP-positive and 80 IMP-negative samples). Patients were monitored in the hospital for several months to collect IMP-positive rectal swab samples, but no infected patients were found. Therefore, IMP-positive samples were prepared by randomly adding an IMP-producing strain (Klebsiella pneumoniae imipenemase) to the transport medium in which the negative rectal swabs were soaked.

[0145] As shown in Fig. 11 b, the clinical sensitivity and specificity for the IMP-producing strain (Klebsiella pneumoniae Imipenemase) were both 100% and 100%, respectively, and the AUC was confirmed to be 1.0. A diagram of the T0-T values ​​of the above samples is shown in Fig. 12 c.

[0146] 3. Detection of antibiotic-resistant strains directly from rectal swabs

[0147] Based on the previous results, the ANCA method demonstrated excellent stability, enabling the detection of antibiotic-resistant strains even under challenging conditions. Accordingly, we conducted additional experiments to determine whether the method could directly detect KPC-producing strains (Klebsiella pneumoniaecarbapenemase) using patient swabs.

[0148] As shown in Fig. 13 a, a cotton swab was dipped into a test tube containing a KPC-specific ANCA reaction product, and the reaction was performed at 75˚.

[0149] As shown in Figure 13b, the ANCA method successfully identified the KPC-producing strain (Klebsiella pneumoniaecarbapenemase) even when rectal swabs were used directly. The diagnostic results obtained from rectal swabs (sensitivity = 100%, specificity = 100%) demonstrated superior accuracy compared to those obtained using transport media in which rectal swabs were soaked (sensitivity = 98.7%, specificity = 100%).

[0150] Moreover, as shown in Figure 14, a strong correlation was observed between the T0-T values ​​of KPC-positive rectal swabs and transport media. This suggests that the collected antibiotic-resistant strains were present simultaneously in the rectal swabs and transport media.

[0151] Interestingly, as shown in Figure 15a, the mean T0-T value (54.89) of KPC-positive rectal swabs was higher than that of the transport medium (47.02). This suggests that rectal swabs harbor a higher abundance of KPC-producing strains (Klebsiella pneumoniaecarbapenemase) than the transport medium immersed in the rectal swabs.

[0152] To quantitatively compare this difference, we calculated the difference in the amount of target DNA using the calibration curve of the KPC circuit shown in Figure 4b. For the convenience of calculation, we assumed that the T0-T value would show a similar trend to the y value of the calibration curve, and based on this, the following calculation formula was derived: y t - y s = -5.7638(x t - x s ), where y t Wow y srepresents the T0- T values ​​of the transport medium and the rectal swab, respectively, and x t Wow x s represents the log value of KPC concentration in transport medium and rectal swab, respectively. In this calculation, y t = 47.02 and y s = Substituting 54.89 into the equation, x t - x s = 1.365, where x represents the value derived from the logarithm of the target nucleic acid concentration, so the actual concentration ratio between the two samples is 23.196. This means that when rectal swabs are used directly, KPC-producing strains can be detected more than 20 times more sensitively.

[0153] As shown in Figure 15b, a similar trend was observed in the experimental results derived from simulated clinical samples. In this comparative experiment, rectal swabs were first soaked in transport medium before use in the ANCA method. Given that the transport medium volume (5 ml) was sufficiently large to dilute the strain concentration, these results are consistent with logical expectations. However, it is important to note that a significant number of strains remained in the rectal swabs even after soaking in the transport medium.

[0154] These experimental results suggest that the ANCA method has significant potential for directly detecting antibiotic-resistant strains in rectal swabs. Furthermore, it suggests that the ANCA method can significantly contribute to accurate diagnostic results and appropriate treatment strategies in healthcare settings.

[0155] Example 6. Capture and detection of antibiotic-resistant strains based on the Argonaute protein and artificial nucleic acid circuit (ANCA) method using a 3D nano-filler swab.

[0156] Hospital-acquired infections can occur through various routes, including direct or indirect contact with infected individuals, respiratory droplets or airborne transmission, and other vector-borne transmission. In particular, infections with antibiotic-resistant strains frequently occur through contaminated skin and medical equipment. Therefore, regular monitoring of patients and contaminated surfaces can aid in the early detection and control of potential antibiotic-resistant strains. Given our previous success in detecting KPC and IMP-producing strains in clinical specimens using the ANCA method, we set an additional goal of using the ANCA method to capture and detect antibiotic-resistant strains from contaminated surfaces.

[0157] As shown in Figure 16a, a 3D nanofiller array swab was used in this experiment to efficiently capture strains present on surfaces. For the experiment, KPC and IMP-producing strains were intentionally sprayed onto micropig skin, a desk, gloves, and scissors. The swab was then gently placed in contact with the contaminated surface and rubbed to capture the strains. The strain-captured swab was then directly immersed in a test tube containing the ANCA reagent and the ANCA reaction was performed. The complex 3D nanofiller array structure promotes strain attachment through nanotopographic interactions, irreversibly capturing the strains and preventing secondary infections.

[0158] Figure 16b is a scanning electron microscope (SEM) image of the nanofiller array structure according to the capture of antibiotic-resistant strains, showing that the strains were successfully captured by the swabs. As shown in Figure 16c, using the KPC-specific ANCA reactant, the fluorescence signal was rapidly amplified only when the KPC-producing strain (Klebsiella pneumoniaecarbapenemase) was captured by the 3D nanofiller swab.

[0159] Similarly, as shown in Figure 16d, for the IMP circuit, the signal increased rapidly only when the IMP-producing strain (Klebsiella pneumoniae imipenemase) was captured. These results suggest that the ANCA method, combined with a 3D composite nanofiller array swab, is capable of on-site capture and identification of antibiotic-resistant strains.

[0160] Example 7. Diagnosis of antibiotic-resistant strain infection based on the Argonaute protein and artificial nucleic acid circuit (ANCA) method using a portable isothermal nucleic acid amplification device.

[0161] As shown in Figure 17 a, the functionality of the ANCA method was further confirmed using a commercially available portable isothermal nucleic acid amplification device.

[0162] As shown in Figure 17b, a total of 20 clinical specimens (10 KPC-positive, 10 negative) were tested, and the ANCA method according to the present invention accurately diagnosed KPC-positive specimens. These results demonstrate the versatility of the ANCA method in various experimental environments and its potential for detecting antibiotic-resistant strains in the field.

[0163] Example 8. Detection of New Delhi metallo-betalactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase-48 (OXA-48) using Argonaute protein and artificial nucleic acid circuit (ANCA) method

[0164] As described in Example 2, the sensitivity of the ANCA method was evaluated for the target sequences New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase-48 (OXA-48) under optimized conditions. The system was designed and evaluated based on the sequences described in Example 1 above.

[0165] As shown in Fig. 18, a linear relationship (R² = 0.99) was observed in all cases in the range of 1 fM to 1 nM, and the LODs of VIM, NDM, and OXA-48 were calculated to be 529, 120, and 144 aM, respectively. This is a level that is much superior to previous results utilizing Ago proteins without pre-amplification. In addition, Fig. 18 demonstrates the flexibility and wide target applicability of the present method, and shows the result of successfully detecting the target DNA sequence.

[0166] Example 9. Direct detection of antibiotic-resistant bacteria

[0167] An attempt was made to detect CPKP by the ANCA method without DNA purification, as described in Example 4.

[0168] The results are shown in Figure 19.

[0169] As shown in a, b, and c of Fig. 19, when the VIM, NDM, and OXA-48 circuits were used to detect carbapenemase-producing Klebsiella pneumoniae (CPKP), it was confirmed that each circuit could well distinguish between WT and CPKP. In other words, it was confirmed that the ANCA method can detect CPKP in a simplified manner without the need for pre-concentration, lysis, and DNA purification steps, and that it has the potential for general use. In particular, it was confirmed that the method has high utility in that detection is possible without the complicated, difficult, and time-consuming steps associated with lysis and nucleic acid extraction.

Claims

1. A composition for detecting a target nucleic acid, comprising: an agonist protein; a first guide nucleic acid (G1); a second guide nucleic acid (G2); a reporter sequence to which a quencher and a fluorescent probe are bound; and a reporter complement; The above reporter complement is composed of the same sequence as the reporter sequence without the quencher and fluorescent probe conjugated thereto, A composition wherein the first guide nucleic acid, the second guide nucleic acid, or both have a phosphate attached to the 5' end.

2. A composition according to claim 1, wherein the first guide nucleic acid, the second guide nucleic acid, or both of them are designed such that trigger 1 (T1) and trigger 2 (T2), which are reaction products cleaved by an agout protein, can function as separate guide nucleic acids.

3. In the first paragraph, the composition, wherein the first guide nucleic acid, the second guide nucleic acid, or both have at least one condition selected from the following conditions: 1) Consisting of 16 to 18 bases, 2) Having a CG content of between 10 and 30%; 3) Thymine is located at the first base sequence of the nucleic acid, and 4) The 12th base sequence of the nucleic acid is not adenine.

4. A composition for detecting a target nucleic acid, wherein the quencher is any one selected from the group consisting of DABCYL, BHQ, ECLIPSE, and TAMRA.

5. A composition for detecting a target nucleic acid, wherein the fluorescent probe is any one selected from the group consisting of ALEX-350, FAM, VIC, TET, CAL Fluor®Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, and Quasar 705.

6. A kit for detecting a target nucleic acid comprising a composition according to any one of claims 1 to 5.

7. A kit according to claim 6, wherein the kit comprises 150 to 400 nM of argon protein; 250 to 1,000 nM of reporter sequence; and 250 to 1,000 nM of reporter complement.

8. A kit according to claim 6, wherein the kit further comprises a reaction buffer containing MgCl2.

9. A kit according to claim 8, wherein the MgCl2 has a concentration of 5 to 30 mM.

10. In the 6th paragraph, the kit further comprises at least one selected from the group consisting of an absorbent pad, a cotton swab, a dropper, a spoon, a swab, and a toothpick as a collection device for collecting a sample.

11. In the 10th paragraph, the collection device is a cotton swab.

12. A method for detecting a target nucleic acid, comprising the step of reacting a composition for detecting a target nucleic acid according to any one of claims 1 to 5 with a sample.

13. A method further comprising a step of visually confirming a change in fluorescence color development of a reactant in the 12th paragraph; or a step of measuring a change in fluorescence color development of a reactant.

14. A method according to claim 12, wherein the reaction is performed at 65 to 75°C.

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