WO2020116812A1 - Method for detecting target nucleic acid using blood glucose self-monitoring device - Google Patents

Abstract

The present invention relates to a method for conveniently detecting a target nucleic acid using a blood glucose self-monitoring device, and according to the present invention, by using a blood glucose self-monitoring device that is cheap and can be easily and conveniently used by anyone, the target nucleic acid of the nucleic acid amplification product can be rapidly and conveniently detected and quantified.

Description

Target nucleic acid detection method using an auto-glycemic meter

The present invention relates to a method for easily detecting a target nucleic acid using an auto-glycemic meter, and more specifically, by adding a target nucleic acid detection reaction solution containing an enzyme involved in glucose and dNTP regeneration to amplification reaction solution of the target nucleic acid, After converting glucose into glucose-6-phosphate by dNTP remaining in the amplification reaction solution, the glucose concentration is measured using a blood glucose meter, and the target nucleic acid is used using a blood glucose meter, characterized in that it detects or quantifies the target nucleic acid. It relates to a detection or quantitative method.

Target nucleic acid detection technology is gaining importance in various technical fields such as genetics, clinical chemistry, forensic and diagnostics. The most representative target nucleic acid detection method used in this technical field is to amplify a specific sequence of target nucleic acid using PCR, and then to confirm the amplification product generated through electrophoresis. However, in the case of such an electrophoresis-based PCR analysis technique, not only a lot of time is required for detection, but also the disadvantage that the entire process is labor intensive. As a technique for solving these shortcomings, a real-time PCR (hereinafter RT-PCR) technique for detecting a PCR reaction in real time has been developed. RT-PCR technology is widely used as a representative technology in the molecular diagnostics market by enabling real-time detection of target nucleic acid amplification reactions as well as quantitative detection of target nucleic acids. However, despite these advantages, expensive reagents and detection equipment are required, and since the detection equipment is not portable, it has a limitation that it can be used only in an environment such as a large hospital or a specialized laboratory.

On the other hand, a personal glucose meter (PGM) is the most widely used field-based in vitro diagnostic device worldwide, and a technique for detecting target substances other than glucose using an autoglucometer has recently been reported ( U.S. Patent No. 8,945,943). In the case of the above technique, several target substances (cocaine, adenosine, interferon) are produced by using a strand displacement reaction by binding of a target material and an aptamer and a glucose production reaction using an invertase activity. -γ, and UO22+, etc.) were easily detected using an auto-glucometer. However, the implementation of the above technique requires not only the modification of the aptamer probe and the enzyme, but also requires a separation process of a sample using a magnetic particle (magnetic bead).

In order to solve the disadvantages of the above-mentioned existing technology, the present inventors have developed adenosine 5'-triphosphate (hereinafter ATP) detection technology utilizing an auto-glucose meter (Korean Patent Publication: 10-2018-0107875). Specifically, in the case of the above technique, it relates to a technique for detecting ATP by measuring a change in glucose concentration induced by a phosphatase chain reaction that occurs when ATP is present in a solution with a commercially available autoglucometer.

Thus, the present inventors tried to develop a method for more easily detecting or quantifying target nucleic acid in a nucleic acid amplification product, and as a result, when the target nucleic acid is present in the samples of the present inventors, a PCR reaction occurs, resulting in a lower dNTP concentration of the amplification product. With this in mind, glucose, hexokinase and pyruvate kinase are added to the PCR reaction product to react, and then, when measuring glucose concentration using an auto-glucometer, the target nucleic acid of the sample is detected. Or it was confirmed that it can be quantified, and the present invention has been completed.

Summary of the invention

An object of the present invention is to provide a method for more easily detecting or quantifying a target nucleic acid in a nucleic acid amplification product.

In order to achieve the above object, the present invention (a) a nucleic acid for detection; dNTP; A target nucleic acid amplification primer; And amplifying the target nucleic acid using a sample containing the nucleic acid polymerase to obtain a target nucleic acid amplification reaction solution; (b) glucose in the target nucleic acid amplification reaction solution; Phosphoenolpyruvate (PEP); Hexokinase; And adding a target nucleic acid detection reaction solution containing pyruvate kinase, and converting the glucose into glucose-6-phosphate; And (c) detecting or quantifying target nucleic acid in the sample by measuring the glucose concentration of the target nucleic acid detection reaction solution of step (b).

1 is a diagram showing a method for detecting a target nucleic acid using an auto-glycemic meter according to the present invention.

Figure 2 shows the results of measuring the glucose concentration in a solution that varies depending on the combination of the presence or absence of dNTP and the addition of hexokinase and pyruvate kinase enzymes with an autoglycoscopy (a: solution without enzymes added; b: addition of hexokinase) Solution; c: solution of addition of hexokinase and pyruvate kinase; d: solution of addition of hexokinase and pyruvate kinase but no addition of dNTP).

Figure 3 shows the results of measuring the glucose concentration in a solution that varies depending on whether hexokinase and pyruvate kinase enzymes are added when different types of dNTP are added, and (a), (b), and (c) ), and (d) are solutions to which dATP, dGTP, dCTP, and dTTP were added, respectively.

Figure 4 shows the results of measuring the glucose concentration in a solution that is changed according to the combination of the presence or absence of target nucleic acid (gray bar: target nucleic acid x, pink rod target nucleic acid o) and the addition or absence of hexokinase and pyruvate kinase enzymes with a glucose meter. (a: a solution without an enzyme added; b: a solution with only hexokinase added; c: a solution with both hexokinase and pyruvate kinase added).

Figure 5 shows the results of measuring the glucose concentration change in the solution according to the concentration of the amplification product generated by PCR with an autoglycometer.

Figure 6 shows the results of measuring the glucose concentration change in the solution according to the type of nucleic acid with an auto-glycemic meter (a: solution without nucleic acid added; b: target nucleic acid (Hepatitis B virus (hereinafter HBV) gDNA is added) Solution, c: Escherichia coli gDNA added solution; d: Enterococcus faecium gDNA added solution).

Detailed description of the invention and preferred embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and is commonly used.

In the present invention, to develop a method for more easily detecting or quantifying a target nucleic acid in a nucleic acid amplification product using an autologous glucose meter, which is the world's most widely used on-site in vitro diagnostic device, and when dNTP is present, a phosphatase chain A method for detecting target nucleic acids was developed using the principle of reducing the glucose concentration in a solution by reaction.

Specifically, in the present invention, when a target nucleic acid is present in a nucleic acid amplification reaction solution containing dNTP and a target nucleic acid amplification primer and a nucleic acid polymerase, the concentration of dNTP in the solution is reduced by a target nucleic acid amplification reaction. It is a method of detecting target nucleic acid based on the principle that the degree of reduction in glucose concentration is finally reduced by reducing the driving efficiency of the enzymatic chain reaction.

Therefore, the present invention (a) a nucleic acid for detection; dNTP; A target nucleic acid amplification primer; And amplifying the target nucleic acid using a sample containing the nucleic acid polymerase to obtain a target nucleic acid amplification reaction solution; (b) glucose in the target nucleic acid amplification solution; Phosphoenolpyruvate (PEP); Hexokinase; And adding a target nucleic acid detection reaction solution containing pyruvate kinase, and converting the glucose into glucose-6-phosphate; And (c) detecting or quantifying target nucleic acid in the sample by measuring the glucose concentration of the target nucleic acid detection reaction solution of step (b).

In the target nucleic acid detection reaction solution of the method of the present invention (i) conversion of dNTP to dNDP by hexokinase activity; (ii) converting glucose to glucose-6-phosphate (G6P) by the hexokinase activity of (i); (iii) converting dNDP produced in (i) to dNTP by pyruvate kinase activity; (iv) converting PEP to pyruvate by pyruvate kinase activity of (ii); And (v) a phosphatase chain reaction consisting of the step of continuously decreasing the glucose concentration in the solution through the phosphatase chain reaction.

In the present invention, when the target nucleic acid is present in the sample, PCR is induced to exhaust the dNTP in the solution, the dNTP concentration decreases the driving efficiency of the phosphatase chain reaction, and the phosphorylase chain reaction decreases the driving efficiency to decrease the glucose concentration. The degree is reduced, and the reduced glucose concentration is measured by an auto-glucometer, whereby dNTP concentration decreases when the target nucleic acid is present, and the glucose concentration decreases less, and when the target nucleic acid does not exist, the dNTP decrease in solution. No, the phosphatase chain reaction increases and the glucose concentration decreases.

In the present invention, the amplification of the target nucleic acid in step (a) can be used without limitation as long as it is a nucleic acid amplification method, preferably PCR reaction, RT-PCR, Gap-LCR ligase chain reaction, Gap-LCR, transcription -It can be performed by a method selected from the group consisting of mediation amplification, self-retaining nucleotide sequence replication, consensus sequence priming polymerase chain reaction, and nucleic acid base sequence-based amplification.

In the present invention, the measurement of step (c) may be characterized by using a blood glucose meter, and when the concentration of glucose is higher than the result using a control group without target nucleic acid, the target nucleic acid is present in the sample. It can be characterized in that it is determined to be, preferably, when the concentration of glucose is higher than the result of using the control group without the target nucleic acid 105 ~ 200%, it can be determined that the target nucleic acid is present in the sample.

In the autologous glucose-based target nucleic acid detection technology proposed in the present invention, dNTP is converted to dNDP by hexokinase activity, and glucose is converted to glucose-6-phosphate (G6P). Then, by the activity of pyruvate kinase, the generated dNDP is converted to dNTP, while the PEP added to the solution is converted to pyruvate, and the dNTP generated in the reaction is used again as a substrate of the hexokinase reaction. The phosphatase chain reaction is constituted by the above-mentioned series of reactions, and the target nucleic acid detection technology using an auto-glycoscopy device may be implemented through the phosphatase chain reaction.

More specifically, when the target nucleic acid is not present in the solution, the PCR reaction does not occur, and the amount of dNTP initially added remains in the solution. Therefore, the initial high concentration of dNTP, the phosphorylase chain reaction is actively induced, and finally, a large amount of glucose is converted to G6P, the concentration of glucose in the solution is greatly reduced. On the other hand, when the target nucleic acid is present in the solution, the PCR reaction is active, and a certain amount of dNTP initially put in the solution is consumed. DNTP is consumed by PCR driving, the driving efficiency of the phosphorylase chain reaction is lowered, and as a result, the degree of decrease in the glucose concentration in the solution is reduced. As described above, the target nucleic acid in the solution can be detected by measuring the glucose concentration determined according to the presence or absence of the target nucleic acid using an auto-glycemic meter (FIG. 1).

In addition, in one embodiment of the present invention, the correlation between the concentration of the amplification product generated by PCR and the glucose concentration determined by the phosphatase chain reaction was confirmed, and as the amplification product concentration (0 to 75 nM) changed, It was confirmed that the glucose concentration in the solution changed linearly (see FIG. 5 ), and it was confirmed that quantitative detection of the PCR amplification product was possible using the autologous glucose-based target nucleic acid detection technology of the present invention.

The primer used in the present invention is hybridized or annealed to one site of the target nucleic acid to form a double chain structure. Conditions for nucleic acid hybridization suitable for forming such a double chain structure include Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used for amplification of the present invention, and include “Klenow” fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtained from various bacterial species, which includes Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu). Includes. When carrying out the polymerization reaction, it is preferable to provide the reaction vessel in excess of the components necessary for the reaction. The excess amount of components required for the amplification reaction means an amount such that the amplification reaction is not substantially limited to the concentration of the components. It is desired to provide a promoter such as Mg2+, dATP, dCTP, dGTP and dTTP to the reaction mixture to the extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers ensure that all enzymes are close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reactant without changing conditions such as the addition of reactants.

In the present invention, annealing or hybridization is carried out under stringent conditions that allow specific binding between the target nucleic acid sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary depending on ambient environmental variables.

The term "amplification reaction" described herein refers to a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which are polymerase chain reactions (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reactions (hereinafter referred to as RT-PCR) (Sambrook et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction ( ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) ( WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276) , Consensus sequence primed polymerase chain reaction (CP-PCR) (US Pat. No. 4,437,975), arbitrary priming polymerase chain reaction (AP-PCR) (US Pat. No. 5,413,909 And 5,861,245), nucleic acid sequence based amplification (NASBA) (US Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displace amplification ment amplification and loop-mediated isothermal amplification; LAMP).

Other amplification methods that can be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09/854,317. In the most preferred embodiment of the present invention, the amplification process is carried out according to the polymerase chain reaction (PCR) disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159.

PCR is the most well-known nucleic acid amplification method, and many modifications and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction Chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR) and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000).

The term "primer" herein is a single-strand capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. Means oligonucleotide. The suitable length of the primer varies depending on various factors, such as temperature and the use of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.

The sequence of the primer need not have a completely complementary sequence with some sequences of the template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with the template and working with the primer. Therefore, the primer pair in the present invention need not have a sequence perfectly complementary to the target nucleic acid sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing with this sequence and acting as a primer.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Establishment of target nucleic acid amplification reaction conditions using PCR

In order to detect the target nucleic acid using an autoglycoscopy, a PCR reaction must be preceded. PCR reaction conditions for the target nucleic acid amplification used in the following examples are as follows.

The PCR solution used (final 50 μL) is 5 μL of dNTP (1 mM each), 4 μL of the forward/reverse primer (10 mM each) of the following sequence, and HBV (Hepatitis B virus) gDNA (Korea Research Institute of Bioscience and Biotechnology) as target nucleic acid 107 copies/μL) 1 μL, and 0.5 μL of Taq DNA polymerase (5 U/μL) were added to the PCR buffer solution, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 2 mM MgCl2 PCR buffer solution containing was used. After preheating the PCR solution at 95°C for 5 minutes, the temperature control process of 95°C 30 seconds, 55°C 30 seconds, and 72°C 1 minute was repeated 40 times, and then treated at 72°C for 5 minutes, Target nucleic acid was amplified.

Primer sequences used are as follows.

Forward primer: 5'-CTC GCC AAC TTA CAA GGC-3' (SEQ ID NO: 1)

Reverse primer: 5'-CAG AGG TGA AGC GAA GTG-3' (SEQ ID NO: 2)

Example 2: Establishment of target nucleic acid detection reaction conditions using an autoglucometer

The target nucleic acid detection reaction solution (final 50 μL) using an autoglucometer is 20 μm glucose 5 μL, 50 mM PEP (phosphoenolpyruvic acid) 2 μL, hexokinase (2.5 U/μL) 2 μL, pyruvate kinase ) (1U/μL) 5 μL, and 25 μL of the PCR solution subjected to the reaction in Example 1 was prepared by adding to the phosphatase chain reaction buffer solution (100 mM Tris-HCl (pH 7.4) and 10 mM MgCl ₂ ).

After reacting the target nucleic acid detection reaction solution at 30°C for 30 minutes, the glucose concentration in the solution was measured using an auto-glycemic meter.

Example 3: Verification of phosphatase chain reaction by dNTP

An experiment was conducted to verify the driving of the phosphorylase chain reaction by dNTP. In the experimental group, a solution in which no enzyme was added (a). Hexokinase (hexokinase) only solution (b), hexokinase and pyruvate kinase (pyruvate kinase) all added solution (c) and hexokinase and pyruvate kinase were added but dNTP was not added (d After dividing ), the reaction solution was reacted at 30° C. for 30 minutes, and then the glucose concentration in the solution was measured using an auto-glucometer.

As a result, as shown in FIG. 2, it was confirmed that the concentration of glucose in the solution of the solution (b) to which hexokinase (hexokinase) was added decreases, compared to the solution (a) to which no enzyme was added. In addition, in the case of the solution (c) in which both hexokinase and pyruvate kinase were added, it was confirmed that the glucose concentration was significantly reduced compared to the solution (b) in which only hexokinase was added. Through the above experimental results, it was confirmed that the phosphatase chain reaction used in the present invention is driven with high efficiency only when hexokinase and pyruvate kinase have activity simultaneously. In addition, both hexokinase and pyruvate kinase were added, but the glucose concentration was not decreased in the solution (d) without dNTP. Through this, it was confirmed that the phosphatase chain reaction used in the present invention is induced only when dNTP is present in the solution.

Example 4: Verification of the kinase chain reaction by dNTP of different types

Experiments were conducted to verify the driving of the phosphorylase chain reaction by different types of dNTPs. The experimental bacteria were divided into a solution containing only hexokinase and a solution containing hexokinase and pyruvate kinase, each containing dATP (a), dGTP (b), dCTP (c) and dTTP (d), respectively. .

As a result, as shown in FIG. 3, in the case of a solution in which dATP (a) and dGTP (b) were added, when hexokinase and pyruvate kinase were added, the concentration of glucose in the solution was significantly reduced by phosphatase chain reaction. Was confirmed. On the other hand, in the case of the solution to which dCTP (c) and dTTP (d) were added, it was confirmed that even if hexokinase and pyruvate kinase were added, the decrease in glucose concentration in the solution was not significant. Through the above experimental results, it was confirmed that the phosphorylase chain reaction used in the present invention is mainly induced by dATP and dGTP.

Example 5: Target nucleic acid detection reaction system verification using an auto-glucometer

Using the above-mentioned phosphatase chain reaction, an experiment was conducted to confirm the change in glucose concentration depending on the presence or absence of target nucleic acid.

The experiment was carried out in the same manner as in Example 2, and when no enzyme was added (a), when hexokinase was added (b), when both hexokinase and pyruvate kinase were added (c) Was performed.

As a result, when no enzyme was added (a), there was no change in glucose concentration depending on whether the target nucleic acid was added. However, when hexokinase was added (b), a difference in glucose concentration according to the presence or absence of target nucleic acid was confirmed, and when both hexokinase and pyruvate kinase were added to induce the phosphatase chain reaction (c), target nucleic acid It was confirmed that the difference in glucose concentration according to the presence or absence was further increased.

Example 6: Verification of glucose concentration change in solution according to amplification product concentration generated by PCR

An experiment was conducted to confirm the correlation between the concentration of the amplification product generated by PCR and the glucose concentration determined by the phosphatase chain reaction.

As a result, as shown in Figure 5, as the amplification product concentration (0 ~ 75 nM) was changed, it was confirmed that the glucose concentration in the solution changes linearly. Through the above experimental results, it was verified that quantitative detection of PCR amplification products is possible by using the target nucleic acid detection technology based on the autoglucometer proposed in the present invention.

Example 7: Validation of target nucleic acid detection technology selectivity using autoglucometer

When various types of nucleic acids were added, an experiment was conducted to confirm the change in glucose concentration in the solution. In the experimental group, when nucleic acid was not added (a), target nucleic acid (HBV gDNA) was added (b), non-specific nucleic acid E.coli gDNA was added (c) and non-specific nucleic acid Enterococcus faecium gDNA was added. Divided into cases (d).

As a result, as shown in FIG. 6, in the case of the solution (b) to which the target nucleic acid (HBV gDNA) was added, a significantly higher glucose concentration was measured compared to the solution (a) to which the nucleic acid was not added. However, in the case of the solution to which the non-specific nucleic acid ( Escherichia coli (c) and Enterococcus faecium (d) gDNA) was added, the glucose concentration similar to the solution (a) to which the nucleic acid was not added was measured. Through the above experimental results, the selectivity of the target nucleic acid detection technology based on the autoglucometer proposed in the present invention was verified.

According to the present invention, the target nucleic acid of the nucleic acid amplification product can be quickly and easily detected and quantified by using an inexpensive autonomous blood glucose meter that anyone can easily and conveniently use.

Since the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (4)

1. Method for detecting or quantifying target nucleic acid in a sample comprising the following steps:

   (a) nucleic acid for detection; dNTP; A target nucleic acid amplification primer; And amplifying the target nucleic acid using a sample containing the nucleic acid polymerase to obtain a target nucleic acid amplification reaction solution;

   (b) glucose in the target nucleic acid amplification solution; Phosphoenolpyruvate (PEP); Hexokinase; And adding a target nucleic acid detection reaction solution containing pyruvate kinase, and converting the glucose into glucose-6-phosphate; And

   (c) detecting or quantifying target nucleic acid in the sample by measuring the glucose concentration of the target nucleic acid detection reaction solution of step (b).
2. The method of claim 1, wherein the amplification of the target nucleic acid in step (a) is PCR reaction, RT-PCR, Gap-LCR ligase chain reaction, Gap-LCR, transcription-mediated amplification, self-maintaining sequence replication, consensus sequence priming. A method characterized by performing in a method selected from the group consisting of polymerase chain reaction and nucleic acid base sequence-based amplification.
3. The method of claim 1, wherein the measurement in step (c) is performed using a blood glucose meter.
4. The method according to claim 1, wherein when the concentration of glucose is higher than the result of using the control group without the target nucleic acid, it is determined that the target nucleic acid is present in the sample.

Images