KR20230071518A - Gene detection method using personal glucose meter - Google Patents

Abstract

The present invention relates to a method for detecting genes and/or nucleic acids using a personal glucose meter (PGM).
In the method of the present invention, when a nucleic acid sequence of a target to be detected (eg, SARS, COVID 19, cancer-causing factor, etc.) is present in a sample, the nucleic acid sequence can be decomposed and quickly detected through PGM. , By changing the nucleic acid sequence according to the user's purpose, the disease to be diagnosed can be changed, so it can be used universally for diagnosis of various diseases.

Description

Gene detection method using personal glucose meter}

The present invention relates to a method for detecting genes and/or nucleic acids using a personal glucose meter (PGM).

Most of the existing point of care (POCT) devices are difficult to use or operate, require specialized knowledge, and have a limited number of substances that can be diagnosed on the spot, so they are not suitable for use by the general public. There was a problem that was difficult to apply in practice.

For example, in the case of a self-in-situ diagnostic device using CRISPR-Cas protein, the presence of a target nucleic acid was confirmed using fluorescence or LFA method, but in the case of fluorescence method, additional equipment that can confirm fluorescence is required, The method had a disadvantage that it was impossible to use continuously because it was disposable.

Accordingly, there has been a demand for the development of a self-in-situ diagnosis device that can derive diagnosis results within a short time with easy operation even by ordinary people without specialized knowledge and equipment.

On the other hand, a personal glucose meter (PGM) is small in equipment, easy to carry, inexpensive, and simple to use, so it is widely distributed and used by the general public. In addition, since PGM is convenient to use and provides quick results, it has recently been applied to a highly versatile POCT method by relating other substances to glucose as well as measuring blood glucose (US 8,945,943).

Under this background, the present inventors have made diligent efforts to develop an easy and fast universal diagnostic method, and as a result, when a nucleic acid sequence of a detection target is present in a sample using PGM, the present inventors can decompose and detect the nucleic acid sequence on site. A diagnostic method was developed to complete the present invention.

One object of the present invention is to: (a) produce AMP by cleaving a nucleic acid to be detected in a sample with a Cas13 protein in the presence of a guide RNA and a target RNA; (b) phosphorylating the AMP of step (a) using myokinase to produce ADP; (c) converting glucose into glucose-6-phosphate by adding ADP from step (b) to a composition for detecting ATP containing glucose, hexokinase and pyruvate kinase; and (d) detecting or quantifying ATP by measuring the glucose concentration of the composition for detecting ATP in step (c) with a blood glucose meter.

Another object of the present invention is a first container comprising a guide RNA, target RNA and Cas13 protein; a second container containing myokainase; a third container containing a composition for detecting ATP including glucose, hexokinase and pyruvate kinase; And to provide a kit for detecting or quantifying nucleic acid in a sample using a blood glucose meter, including a blood glucose meter.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is (a) producing AMP by cleaving a nucleic acid to be detected in a sample with a Cas13 protein in the presence of a guide RNA (gRNA) and a target RNA; (b) phosphorylating the AMP of step (a) using myokinase to produce ADP; (c) converting glucose into glucose-6-phosphate by adding ADP from step (b) to a composition for detecting ATP containing glucose, hexokinase and pyruvate kinase; and (d) detecting or quantifying ATP by measuring the glucose concentration of the composition for detecting ATP in step (c) with a blood glucose meter.

In the present invention, there is no time interval between steps such as "(a), (b), (c), (d) ...", performed simultaneously, or at an arbitrary interval such as several seconds, several minutes, several hours, etc. can be performed

In the method of the present invention, step (a) may include producing AMP by cleaving the nucleic acid to be detected in the sample by Cas13 protein in the presence of guide RNA and target RNA.

Samples of the present invention include samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine isolated from an individual, and are not particularly limited as long as they contain a nucleic acid to be detected.

The nucleic acid to be detected in the present invention may be any one or more selected from the group consisting of viruses, bacteria, and oncogenes.

The virus is, for example, severe acute respiratory syndrome (SARS), COVID 19, Epstein-Barr Virus (Epstein-Barr Virus, EBV), Human Immunodeficiency Virus (HIV), Influenza virus, Papilloma virus, Hepatitis virus, Adenovirus, herpes simplex type 1, herpes simplex type 2, Varicella-zoster virus, Epstein-Barr virus, human cytomegalo virus, Kaposi's sarcoma-associated herpes virus (KSHV), BK virus, JC virus (John Cunningham virus), Smallpox Virus, Parvovirus B19, Human Astrovirus, Norovirus, Coxsackie Virus, Hepatitis A Virus, Polio Virus, Rhino Virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Yellow Fever Virus, Dengue Virus, West Nile Virus, TBE Virus, Rubella Virus, Orthomyxoviridae, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Hantaan Virus, Ebola Virus, Marburg Virus, Measles Virus, Mumps Virus, Parainfluenza Virus , respiratory syncytial virus (RS virus), Rabies virus, rotavirus, Orbi virus, Colti virus and Banna virus, but not limited thereto.

The bacteria are, for example, Escherichia coli ( E. coli ), Brucella ( Brucella ), Campylobacter ( Campylobacter ), Vibrio cholera ( Vibrio cholera ), Gonorrhea ( Gonorrhea ), Legionella ( Legionella ), Meningococcus ( Meningococcus ) , Pertussis , Yersinia pestis , Pseudomonas , Salmonella, Shigella , Francisella tularensis , Francisella tularensis, Anthrax , diphtheria ( Diphtheria ), Enterococci ( Enterococci ), Swine monobacteria ( Erysipelothrix rhusiopathiae ), Listeria monocytogenes ( Listeria monocytogenes ), Nocardia ( Nocardia ), Pneumococcus ( Pneumococcus ), Streptococcus ( Streptococcus ), Treponema ( Treponema ), Leptospira ( Leptospira ), Borrelia ( Borrelia ), Moniliformis chain rod bacteria ( Moniliformis streptococcus ), Actinomyces israeli ( Actinomyces israeli ), Bacteroides ( Bacteroides ), Clostridium botulinum ( Clostridium botulinum ) and tetanus bacteria ( Clostridium tetani ), etc., but are not limited thereto.

The oncogene is, for example, c-myc, N-myc, L-myc, c-jun, jun-B, jun-D, c-fos, fos-B, ets-1, ets-2, myb, erbA , rel, ski, spi-1, evi-1, Ha-ras, Ki-ras, N-ras, erbB-1, erbB-2, erbB-2/neu, src, fms, mos, abl, ros, raf , yes, fps, trk, sis, Adeno E1A, Polyoma large T, Papilloma E7, SV40 large T, and Polyoma middle T, but not limited thereto.

As used herein, the term "Cas13 (Cas13a) protein" refers to a protein that recognizes a target RNA (e.g., single-stranded RNA (ssRNA)) when it is bound to gRNA and cleave the target RNA and surrounding ssRNA. At this time, the presence or absence of gRNA serves as a switch such as on or off of Cas13. When Cas13 and gRNA are bound, the presence or absence of the target RNA acts as a switch such as turning Cas13 on or off. The nucleic acid cleaved by the Cas13 reaction is Adenosine 2', 3'-cyclic Phosphate, which is structurally similar to AMP.

In one embodiment of the present invention, as a result of confirming whether one base is cleaved by Cas 13a protein using a composition containing target RNA and a fluorescently labeled (Cy5) probe (FIG. 2), fluorescence depends on the presence or absence of target RNA. It was confirmed that there was a difference in the value (Fig. 3), and the fluorescence signal was amplified when the target RNA was present, which is a Cas reaction from the predicted cleavage site (Fig. 2) to Adenosine 2', 3'-, a nucleic acid similar in structure to AMP. It can be seen that this is because cyclic Phosphate is cleaved and one rA base is removed and fluorescence is expressed.

In the present invention, step (a) may further include adding T4 polynucleotide kinase (PNK) to produce AMP.

In the method of the present invention, step (b) may include phosphorylating the AMP of step (a) using myokinase to produce ADP.

In the present invention, the term "Myokinase" is an enzyme that catalyzes the reaction of ATP　+　AMP　⇔ 2　ADP, and uses AMP and triphosphate pentabasic to generate ADP. The myokinase is an enzyme that balances ATP, AMP, and ADP by synthesizing AMP when an excess of ATP is present and a small amount of AMP, and synthesizing ATP when a small amount of ATP is present and an excess of AMP is present.

In one embodiment of the present invention, as a result of confirming whether ATP is produced by myokinase, an ATP signal was measured when ATP was present, and an ATP signal was not measured when AMP was present, but myokinase was added in the presence of AMP When the ATP signal was generated, it was confirmed that AMP was converted to ATP through the myokinase reaction (FIG. 4).

In the method of the present invention, step (c) includes adding ADP from step (b) to a composition for detecting ATP containing glucose, hexokinase and pyruvate kinase to convert glucose into glucose-6-phosphate can do.

In the present invention, step (c) may be CER (Cascade enzyme reactions) by myokinase, hexokinase, and pyruvate kinase.

In the present invention, the term "Cascade enzyme reactions (CER)" means that several enzymes share a product and a reactant to cause a positive feedback action, and even if the amount of the nucleic acid to be detected in the sample is extremely small, the CER Through this, a positive feedback action is induced, and the reaction is amplified through each reaction step, so that nucleic acid detection can be facilitated. For the purposes of the present invention, the CER may be one in which hexokinase and pyruvatekinase cause a positive feedback action.

As used herein, the term "hexokinase" refers to an enzyme that converts ATP into ADP.

As used herein, the term "pyruvate kinase" refers to an enzyme that converts ADP into ATP and converts phosphoenolpyruvic acid (PEP) into pyruvate.

Specifically, ATP is converted to ADP and glucose is converted to glucose-6-phosphate by the hexokinase, and at the same time as the above reaction, ADP produced by the hexokinase enzyme reaction is converted to ATP again by pyruvate kinase, CER may be induced by converting forenolpyruvic acid to pyruvate and causing a positive feedback action.

In the method of the present invention, step (d) may include detecting or quantifying ATP by measuring the glucose concentration of the composition for detecting ATP of step (c) with a blood glucose meter.

In the present invention, the term "Personal glucose meter (PGM)" refers to the conversion of hydrogen peroxide, which is produced by oxidation of glucose in the blood by glycolytic enzyme, into oxygen, and converts the electrons into electric current using electrodes and quantifies the blood It means a device that analyzes the blood sugar concentration in the blood. The need to evaluate the accuracy and precision of blood glucose meters with standardization methods different from equipment used in laboratories has emerged, and the International Organization for Standardization (ISO) stipulates the accuracy of self-monitoring blood glucose meters as ISO15197. The blood glucose meter may be used in combination with a self-monitoring blood glucose level.

In one embodiment of the present invention, as a result of confirming whether AMP is converted to ATP by CER by myokinase, hexokinase, and pyruvate kinase to induce a change in glucose concentration, glucose measurement according to AMP concentration The value decreased linearly, confirming that the glucose concentration was changed by myokainase and CER (FIG. 5).

In the present invention, nucleic acid detection in a sample may be determined based on the following criteria.

For example, when the glucose concentration decreases as a result of blood glucose measurement in step (d), it may be determined that the nucleic acid to be detected is present in the sample.

As another example, when the glucose concentration is maintained or increased as a result of blood glucose measurement in step (d), it may be determined that the nucleic acid to be detected is not present in the sample.

In addition, in the present invention, nucleic acid quantification in a sample may be determined based on the following criteria.

For example, when the blood glucose measurement result in step (d) shows that the level of reduction in glucose concentration is 30 mg/dL or more, specifically about 43 mg/dL or more, the AMP generated by the Cas13 reaction in the sample is 1 uM or more, specifically about 43 mg/dL or more. It may be determined to be present at a concentration of 2 uM or more, but is not limited thereto. For example, when the level of reduction in glucose concentration is 30 mg/dL or more as a result of blood glucose measurement, the AMP generated by the Cas13 reaction in the sample is 1 uM or more, 2 uM or more, 3 uM or more, 4 uM or more, 5 uM or more, 6 It can be determined that it is present at a concentration of uM or more, 7 uM or more, 8 uM or more, 9 uM or more, or 10 uM or more.

As another example, when the glucose concentration reduction level is less than 2 mg/dL, specifically less than about 1 mg/dL as a result of the blood glucose measurement in step (d), the AMP generated by the Cas13 reaction in the sample is less than 150 nM, specifically It may be determined to be present at a concentration of less than about 125 nM, but is not limited thereto. For example, when the level of reduction in glucose concentration is less than 2 mg / dL as a result of blood glucose measurement, the AMP generated by the Cas13 reaction in the sample is less than 150 nM, less than 140 nM, less than 130 nM, less than 125 nM, less than 120 nM, 115 It can be judged to be present at a concentration of less than nM, less than 110 nM, less than 105 nM or less than 100 nM.

In the present invention, the term “about” may be preceded by a specific numerical value. As used in this application, the term "about" includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented. As an example, the term “about” can refer to a range of -10% to +10% of a numerical value. As another example, the term "about" can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.

In one embodiment of the present invention, as a result of confirming whether AMP generated by the Cas13 reaction can be measured as the glucose signal of the PGM using the target gene (25nM), the level of reduction in the glucose concentration measured through the PGM is 43 mg / dL or more, the AMP produced by the Cas13 reaction in the sample is present at a concentration of 2 uM or more, and when the level of reduction in glucose concentration is less than 1 mg / dL, the AMP produced by the Cas13 reaction in the sample is less than 125 nM It was confirmed that the concentration was present (FIG. 8). From this, it can be seen that Cas13 is activated when the target nucleic acid is present, and when the glucose concentration decreases through a series of processes (CER), the change in signal can be confirmed by PGM.

Most of the existing point of care (POCT) devices are difficult to use or operate, require specialized knowledge, and have a limited number of substances that can be diagnosed on the spot, so they are not suitable for use by the general public. There was a problem that was difficult to apply in practice.

For example, in the case of a self-in-situ diagnostic device using CRISPR-Cas protein, the presence of a target nucleic acid was confirmed using fluorescence or LFA method, but in the case of fluorescence method, additional equipment that can confirm fluorescence is required, The method had a disadvantage that it was impossible to use continuously because it was disposable.

Therefore, the present invention is meaningful in that it has improved the difficulty of using conventional diagnostic equipment by being able to derive diagnostic results in a short time with easy operation even by ordinary people without professional knowledge without the need for other equipment like the conventional fluorescence detection method. .

The present invention can quantitatively confirm the results within 5 seconds after sample processing, and can detect many types of nucleic acids by replacing only the guide RNA of CRISPR according to the type of nucleic acid to be detected, enabling universal use. In addition, there is an advantage in that accurate detection is possible even if the amount of the nucleic acid to be detected in the sample is extremely small through reaction amplification using CER.

In one embodiment of the present invention, the presence or absence of a target RNA has been detected through a PGM signal using a part of the SARS-CoV-2 gene sequence as a target RNA (Example 4-5).

Another aspect of the present invention is a first container comprising a guide RNA, a target RNA and a Cas13 protein; a second container containing myokainase; a third container containing a composition for detecting ATP including glucose, hexokinase and pyruvate kinase; and a kit for detecting or quantifying nucleic acids in a sample using a blood glucose meter, including a blood glucose meter.

Terms used herein are as described above.

The kit of the present invention comprises a first container containing the guide RNA, target RNA and Cas13 protein, a second container containing myokinase, and a third container containing a composition for detecting ATP including glucose, hexokinase and pyruvate kinase. Means a tool that can be used to detect and quantify nucleic acids in a sample, including containers and glucometers. The type of the kit is not particularly limited, and kits of a type commonly used in the art may be used.

The kit of the present invention includes i) guide RNA, target RNA, and Cas13 protein, or ii) myokinase, or iii) glucose, hexokinase, and pyruvate kinase, each contained in an individual container, or one divided into one or more compartments. It may be packaged in a form contained in a container, wherein i) guide RNA, target RNA and Cas13 protein or ii) myokinase or iii) glucose, hexokinase and pyruvate kinase are packaged in a unit dose form of a single dose, respectively may have been

The i) guide RNA, target RNA and Cas13 protein or ii) myokinase or iii) glucose, hexokinase and pyruvatekinase in the kit can be sequentially administered at an appropriate time according to an experimental plan of a person skilled in the art.

The kit of the present invention includes i) guide RNA, target RNA, and Cas13 protein, or ii) myokinase, or iii) glucose, hexokinase, and pyruvate kinase. Alternatively, a user manual describing how to use the blood glucose meter may be further included.

In the method of the present invention, when a nucleic acid sequence of a target to be detected (eg, SARS, COVID 19, cancer-inducing factor, etc.) is present in a sample, the nucleic acid sequence is decomposed and used for a personal glucose meter (PGM). It can be quickly detected through the nucleic acid sequence, and the disease to be diagnosed can be changed by changing the nucleic acid sequence according to the user's purpose, so it can be used universally for diagnosis of various diseases.

1 is a schematic diagram of a method for detecting or quantifying nucleic acids in a sample using a personal glucose meter (PGM) of the present invention.
2 is a diagram showing the structure of a fluorescently labeled probe used in the Cas 13 reaction. The probe is a single-stranded RNA (ssRNA), and is a fluorescently labeled probe for evaluating the cleavage activity of nearby target RNA (ssRNA) when Cas13a is activated in the presence of gRNA and target RNA. In the probe, rA is RNA adenine, A is DNA adenine, a quencher is immobilized at the 5' end, and Cy5 as a fluorescent material is immobilized at the second A.
3 is a diagram showing fluorescence values according to the presence or absence of target RNA in Cas 13 reaction.
4 is a diagram showing ATP signals according to whether myokinase is added in the presence of ATP or AMP. One; A signal occurs when ATP is present. 2; In the case of AMP, no signal is generated. 3; When myokinase is added to AMP, the AMP is converted to ATP and a signal is generated.
5 is a result of measuring the change in glucose concentration according to the AMP concentration by PGM.
6 is a schematic diagram showing target nucleic acid detection using Cas 13 reaction and CER (Cascade enzyme reactions) reaction.
7 is a cleavage reaction by Cas 13 and T4 polynucleotide kinase (T4 polynucleotide kinase, PNK) reaction indirect verification results.
8 is a result of measuring the glucose signal value according to the presence or absence of a target gene (25 nM) by PGM.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Cas 13 reaction verification

Detection or quantification of nucleic acids in the sample was performed using a personal glucose meter (PGM) as shown in FIG. 1 .

In order to confirm whether one base is cleaved by the Cas 13 protein of FIG. 1, Cut smart buffer 3 μL, 0.5 uM Cas13a 3 μL, 0.5 uM gRNA (guide RNA) (SEQ ID NO: 1) 1.5 μL and distilled water (Nuclase free D.W.) 19.5 μL was placed in an incubator at 25° C. for 30 minutes to allow the Cas13a protein and gRNA to bind first before encountering the target RNA. To this, 0.5 uM target RNA or 1.5 μL of distilled water and 1.5 μL of 100 uM fluorescently labeled (Cy5) probe (Fig. 2) were added to make the total volume 30 μL, followed by reaction at 37°C for 1 hour and reaction at 95°C for 10 minutes. to inactivate the activity of the enzyme. At this time, it was observed that the previously bound Cas13a protein and gRNA bound to the target RNA, and the activated Cas13a cleaved the target RNA and the fluorescently labeled probe, while fluorescence was expressed from the probe.

As a result, it was confirmed that there was a difference in fluorescence value depending on the presence or absence of the target RNA (FIG. 3), and the fluorescence signal was amplified when the target RNA was present, indicating that AMP and structure were formed from the expected cleavage site (FIG. 2) by Cas reaction. It can be seen that this is because Adenosine 2', 3'-cyclic Phosphate, a similar nucleic acid, was cleaved and one rA base was removed and fluorescence was expressed.

Example 2. Myokinase Reaction Verification

In order to confirm whether ATP is produced by myokinase of FIG. 1, the samples shown in Table 1 below were mixed and reacted at 30 ° C. for 16 hours in a thermostat and reacted at 95 ° C. for 10 minutes to inactivate the enzyme activity. . In order to verify the generated ATP, the degree of luminescence was measured in the sample after the reaction using an ATP assay kit. The plate used for the measurement of luminescence is a white well, and 40 μL of the sample, 60 μL of 1X reaction buffer, and 100 μL of ATP assay mixture were put thereinto, and the degree of luminescence was measured at 560 nm. In Table 1 below, triphosphate pentabasic and phosphoenolpyruvic acid (PEP) serve as fuels in the ADP and ATP cycles to keep the signal reduction high even when the cycle returns.

As a result, the ATP signal was measured when ATP was present, and the ATP signal was not measured when AMP was present. However, when myokinase was added in the presence of AMP, an ATP signal was generated and AMP was converted to ATP through the myokinase reaction. It was confirmed that it was done (FIG. 4).

reaction I volume 10X reaction buffer 5 μL 100 mM phosphoenolpyruvic acid (PEP) 1 μL 1 uM AMP (or ATP) 5 μL 10 uM triphosphate pentabasic 5 μL D.W. 24 µL 1 kU/mL myokinase 5 μL 1 kU/mL pyruvate kinase 5 μL total reaction volume 50 µL

Example 3. Myokinase and CER (Cascade enzyme reactions) reaction verification

In order to confirm whether AMP is converted to ATP by CER by myokainase, hexokinase, and pyruvate kinase of FIG. 1 to induce a change in glucose concentration, the samples shown in Tables 2 and 3 below mixed with each other. After adding 0.5, 1, 1.5, and 2 uM of AMP to the sample mixture in Table 2 and reacting at 30° C. for 30 minutes, the amount of glucose remaining was measured with PGM.

As a result, the measured glucose value decreased linearly according to the AMP concentration, confirming that the glucose concentration was changed by myokinase and CER (FIG. 5).

Example 4. Cleavage reaction by Cas 13 and T4 polynucleotide kinase (T4 polynucleotide kinase, PNK) reaction verification

Cas 13 activity according to the presence or absence of target nucleic acid and PNK activity according to the presence or absence of PNK were confirmed to verify whether AMP was produced by Cas 13 cleavage reaction and PNK action.

First, in order to confirm PNK activity according to the presence or absence of PNK, 2 μL of Cut smart buffer, 2 μL of 0.5 uM Cas13a, 1 μL of 0.5 uM gRNA (SEQ ID NO: 1), and 1 μL of Nuclase free D.W were mixed and incubated at 25 ° C for 30 minutes. and reacted. The samples shown in Table 2 were added to the reactants and reacted at 37 ° C for 2 hours, and the samples shown in Table 3 were added to the reactants and reacted at 30 ° C for 1 hour and 30 minutes to induce myokinase and CER reactions. After that, glucose concentration was measured by PGM.

0.5 uM SARS-CoV-2 gene sequence (target RNA) (SEQ ID NO: 3) 1 μL 100uM AMP probe (rArUTCCCA) (SEQ ID NO: 2) 8 μL 10kU/ml PNK or free D.W. 5 μL total reaction volume 20 μl

Reaction II Volume Reaction I 20 µL 1 kU/mL Myokinase Rabbit 5 μL 50 mM glucose 5 μL 10 μM triphosphate pentabasic 5 μL 1 kU/mL pyruvate kinase 5 μL 1 kU/mL hexokinase 5 μL Cut smart buffer 3 μL 100 mM PEPs 1 μL Nuclase free D.W. 1 μL total reaction volume 50 μl

To confirm Cas 13 activity according to the presence or absence of target nucleic acids, cut smart buffer 2 μL, 0.5 uM Cas13a 2 μL, 0.5 uM gRNA (SEQ ID NO: 1) 1 μL and Nuclase free D.W 1 μL were mixed and incubated at 25 ° C for 30 minutes. and reacted. The samples shown in Table 4 were added to the reaction mixture, reacted at 37° C. for 2 hours, and myokinase and CER reactions were induced in the same manner as above, and then the glucose concentration was measured by PGM.

0.5 uM target RNA (SEQ ID NO: 3) or free D.W. 1 μL 100uM AMP probe (rArUTCCCA) (SEQ ID NO: 2) 8 μL 10 kU/ml PNK 5 μL total reaction volume 20 μl

Through the above process, when the bound gRNA and Cas13a first meet the target RNA, rA is concomitantly cleaved by an AMP probe (rArUTCCCA) (SEQ ID NO: 2) to produce cAMP (Adenosine-2',3'-cyclic monophosphat) . cAMP interacts with PNK to convert to AMP, which is a starting material for myokinase and CER reactions (first step). The converted AMP reacts with myokainase to produce ADP, ADP reacts with pyruvatekinase to produce ATP, and hexokinase consumes ATP and glucose to produce ADP (step 2). Confirmation was possible (FIG. 6).

As a result, in the PNK activity test results, even if the target RNA was present, the glucose concentration decreased only when PNK was present, which indirectly confirmed the conversion of PNK from cAMP to AMP (FIG. 7).

As a result of the PNK activity experiment, the glucose concentration decreased in the presence of the target RNA (FIG. 7). Specifically, in the presence of the target RNA, Cas13 was activated to generate cAMP through concomitant cleavage, and cAMP interacted with PNK to convert to AMP. AMP is circulated in the form of ATP and ADP through CER, and as glucose is consumed in this process, the glucose concentration is lowered, which can be confirmed as a PGM signal.

Example 5. One-pot reaction verification

The cleavage reaction by Cas 13 and the CER reaction of the first and second steps, in which AMP is produced by PNK action, verified in Example 4, were performed as a one-pot reaction, and Cas13 reaction It was confirmed whether the generated AMP could be measured as the glucose signal of the PGM.

Specifically, 2 μL of Cut smart buffer, 2 μL of 0.5 uM Cas13a, and 1 μL of 0.5 uM gRNA (SEQ ID NO: 1) were mixed and incubated at 25° C. for 30 minutes to react. The samples shown in Table 5 were added to the reactants and reacted at 37° C. for 0 to 2 hours to induce myokinase and CER reactions.

As a result, a difference occurs in the glucose signal value depending on the presence or absence of the target gene (25nM). Specifically, when the level of reduction in glucose concentration is 43 mg/dL or more as a result of blood glucose measurement, the AMP generated by the Cas13 reaction in the sample is 2 uM It was confirmed that AMP generated by the Cas13 reaction in the sample was present at a concentration of less than 125 nM when present at a concentration equal to or greater than 1 mg/dL and the decrease in glucose concentration was less than 1 mg/dL (FIG. 8).

Through this, it was confirmed that when the target nucleic acid is present, Cas13 is activated, and when the glucose concentration decreases through CER, the change in signal can be confirmed by PGM.

0.5 uM target RNA (SEQ ID NO: 3) or DEPC D.W. 1 μL 100uM AMP probe (rArUTCCCA) (SEQ ID NO: 2) 9 μL 10 kU/ml T4 polynucleotide kinase (PNK) 5 μL 1 kU/mL Myokinase Rabbit 5 μL 50 mM glucose 5 μL 10 μM triphosphate pentabasic 5 μL 1 kU/mL pyruvate kinase 5 μL 1 kU/mL hexokinase 5 μL Cut smart buffer 3 μL 100 mM PEPs 1 μL DEPC D.W. 1 μL total reaction volume 50 μl

It can be seen that according to the method of the present invention, when a nucleic acid sequence of a detection target is present in a sample using PGM, the corresponding nucleic acid sequence can be decomposed and detected. This method suggests that the detection speed is as fast as 5 seconds or less, and the disease to be diagnosed can be changed by changing the nucleic acid sequence according to the user's purpose, so that it can be used universally for diagnosis of various diseases.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

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Claims (8)

(a) producing AMP by cleavage of a nucleic acid to be detected in a sample by Cas13 protein in the presence of guide RNA and target RNA;
(b) phosphorylating the AMP of step (a) using myokinase to produce ADP;
(c) converting glucose into glucose-6-phosphate by adding ADP from step (b) to a composition for detecting ATP containing glucose, hexokinase and pyruvate kinase; and
(d) a method for detecting or quantifying nucleic acid in a sample using a blood glucose meter, comprising the step of detecting or quantifying ATP by measuring the glucose concentration of the ATP detection composition of step (c) with a blood glucose meter.
The method of claim 1, wherein the sample is any one or more selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine isolated from the subject.
The method of claim 1, wherein the nucleic acid to be detected is at least one selected from the group consisting of viruses, bacteria, and oncogenes.
The method according to claim 1, wherein the method determines that the nucleic acid to be detected is present in the sample when the glucose concentration decreases as a result of blood glucose measurement in step (d).
The method according to claim 1, wherein the method determines that the nucleic acid to be detected is not present in the sample when the glucose concentration is maintained or increased as a result of the blood glucose measurement in step (d).
The method of claim 1, wherein the method determines that AMP generated by the Cas13 reaction in the sample is present at a concentration of 1 uM or more when the level of decrease in glucose concentration is 30 mg/dL or more as a result of blood glucose measurement in step (d). which way.
The method according to claim 1, wherein, when the blood glucose measurement result in step (d) shows that the level of decrease in glucose concentration is less than 2 mg/dL, it is determined that AMP produced by the Cas13 reaction in the sample is present at a concentration of less than 150 nM. How to do it.
A first container containing guide RNA, target RNA and Cas13 protein; a second container containing myokainase; a third container containing a composition for detecting ATP including glucose, hexokinase and pyruvate kinase; And a kit for detecting or quantifying nucleic acid in a sample using a blood glucose meter, comprising a blood glucose meter.

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