JP2010200656A - Method for detecting pyrophosphoric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and highly sensitive method for specifically detecting and determining pyrophosphoric acid, and to provide a method for detecting a nucleic acid utilizing the method. <P>SOLUTION: The method for detecting the pyrophosphoric acid includes the steps of: subjecting a sample to a measuring system containing adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphoric acid (Pi), thiamin diphosphate (TPP), flavin adenine dinucleotide (FAD), magnesium ion (Mg<SP>2+</SP>), pyruvate dehydrogenase (PDH), and electronic mediator; and measuring an electrical current value at the measuring system. The electric current value exhibits the concentration of the pyrophosphoric acid in the sample. The method for detecting the nucleic acid includes detecting the pyrophosphoric acid formed according to a PCR using a DNA primer. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting pyrophosphate. Furthermore, the present invention relates to a method for detecting a target nucleic acid simply and with high sensitivity.

In recent years, the molecular diagnostic market including in vitro diagnostic agents has been growing rapidly, and in the medical field, it has become possible to treat diseases at the molecular level by analyzing disease-related genes.
In genetic testing, it is used not only for cancer areas that have even been called genetic diseases, but also for the purpose of investigating the risk of developing infectious diseases, Alzheimer's disease, lifestyle-related diseases, and the effects and safety of drug treatments. For example, warfarin, an anticoagulant for the treatment and prevention of thromboembolism, has been approved by the FDA to determine the optimal prescription amount after examining the genotype.

  As described above, especially in the above-described genetic tests, the above-described constitutional diagnosis and infectious disease diagnosis prior to drug treatment require in-situ diagnosis. It has been.

  Real-time PCR, which is currently the mainstream in genetic testing methods, crushes cells in body fluids typified by blood, extracts DNA, and uses PCR (Polymerase Chain Reaction) to target regions using purified DNA as a template. ) Amplification by subjecting to reaction (Patent Document 1). In the detection method using an intercalator, the presence or absence of amplification of the target nucleic acid is detected by detecting the fluorescence intensity that binds the double-stranded DNA to a fluorescent dye that intercalates with the amplified double-stranded DNA, such as SYBER GREEN I. And its amount can be measured. However, in this method, each step (DNA extraction and amplification) needs to be performed by human hands at the present time, and it takes a lot of time and labor, and light emission using an intercalator has a carcinogenic problem. This method requires great care and is not suitable for medical use.

  On the other hand, a technique for specifically detecting pyrophosphate generated in the process of DNA elongation reaction by an enzymatic reaction has been studied.

  There is a method of detecting by generating light in luciferase-luciferin reaction after allowing ATP sulfurylase to act on pyrophosphate (Patent Document 2).

  However, this method has a problem that the apparatus becomes large in terms of performing light detection.

On the other hand, a method for electrochemically detecting pyrophosphate without using light detection has been studied.
As a method for determining a DNA base sequence, Japanese Patent Application Laid-Open No. 2008-233050 (Patent Document 3) presents the following method. The first method is a reaction in which pyrophosphate is produced in the presence of phosphoenolpyruvate (PEP) and adenosine monophosphate (AMP) by the catalytic action of pyruvate dikinase (PPDK). The generated pyruvic acid is catalyzed by lactate dehydrogenase (LDH), and the potential due to the redox reaction is detected. The second method is a method in which PPi is produced in the presence of fructosyl 6-phosphate by the catalytic action of fructosyl 6-phosphate kinase to produce glyceraldehyde phosphate, and the redox potential is measured through an enzymatic reaction. The amount of pyrophosphoric acid is measured by measuring the amount of acid produced. These methods are excellent in that pyrophosphate can be detected electrochemically. However, in the disclosed method, it is necessary to immobilize the electrochemically active substance on the electrode surface using an insulating molecule on the electrode. The problem remains in that it requires a lot of operation.
US Pat. No. 6,174,670 International Publication No. 92/16654 Pamphlet JP 2008-2333050 A Japanese Patent No. 3866277 Japanese Patent Application Laid-Open No. 64-037300

  The present invention aims to solve the above-mentioned conventional problems, and specifically detects and quantifies pyrophosphate by combining two enzyme reactions of pyruvate phosphate dikinase and pyruvate dehydrogenase. An object is to provide a simple and highly sensitive method.

  In order to solve the above problems, the present invention converts pyrophosphate into a redox substance using an enzyme, and electrically detects the redox substance.

A method for detecting pyrophosphate, comprising:
Adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD), magnesium ion ( Providing the sample to a measurement system comprising Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And measuring a current value in the measurement system,
Here, the current value indicates the concentration of pyrophosphate in the sample.
This is a method for detecting pyrophosphate.

  The electron mediator is ferricyanide, 1,2-naphthoquinone-4-sulfonic acid, 2,6-dichlorophenolindophenol, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate, methylene blue, galocyanine, thionine, It is at least one selected from the group consisting of phenazine methosulfate and Meldola Blue.

A method for detecting a nucleic acid comprising:
A sample is subjected to a reaction system containing a DNA primer having a sequence complementary to the sequence of the nucleic acid, DNA polymerase, and deoxynucleotide, and the DNA primer binds to a complementary sequence of the nucleic acid, and the extension reaction from the DNA primer Accompanied by a process wherein pyrophosphate is produced;
The samples were adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD) , Subjecting the sample to a measurement system comprising magnesium ion (Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And a step of measuring a current value in the measurement system, wherein the current value indicates a concentration of a nucleic acid having a specific sequence in the sample.

  The extension reaction from the DNA primer is a PCR reaction.

A method for typing an SNP sequence of DNA, wherein a sample is provided to a reaction system comprising an allele-specific primer, DNA polymerase, and deoxynucleotide, and the allele-specific primer is complementary to a complementary sequence of the target sequence of the nucleic acid. Binding and generating pyrophosphate upon extension reaction from the allele-specific primer;
The samples were adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD). , Subjecting the sample to a measurement system comprising magnesium ion (Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And a step of measuring a current value in the measurement system, wherein the generation of the current value is a means for determining the presence or absence of an SNP sequence in the sample.

  This is a method for typing an SNP sequence, wherein the extension reaction from the allele-specific primer is a PCR reaction.

  According to the pyrophosphate detection method of the present invention, pyrophosphate is catalyzed by pyruvate phosphate dikinase (PPDK) in the presence of phosphoenolpyruvate (PEP), adenosine monophosphate (AMP) and magnesium ion. In the reaction for generating pyruvic acid, the generated pyruvic acid is catalyzed by pyruvic acid dehydrogenase (PDH), and the current due to the redox reaction is detected.

(Embodiment 1)
Hereinafter, the pyrophosphate detection method of the present invention will be described.

In Embodiment 1 of the present invention, simple quantitative detection of pyrophosphate is performed using an enzyme reaction. Embodiment 1 of the present invention relates to a method for detecting pyrophosphate, which includes pyrophosphate phosphate dikinase (PPDK) (EC 2.7.9.1), adenosine monophosphate (AMP), phosphoenol. Pyruvate (PEP), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD), magnesium ion (Mg ²⁺ ), pyruvate dehydrogenase (PDH) (EC 1.2. 4.1) and a step of supplying a sample to a measurement system including an electron mediator, and a current value is measured in the measurement system.

  In the measurement system after providing the sample, the reactions shown by the following reaction formulas (Chemical Formula 1) to (Chemical Formula 3) occur. These will be described in order.

When pyrophosphate (PPi), adenosine monophosphate (AMP), phosphoenolpyruvate (PEP) and magnesium ion (Mg ²⁺ ) are mixed and pyruvate phosphate dikinase (PPDK) is allowed to act, pyruvate, adenosine Triphosphate (ATP) and phosphate (Pi) are produced. Next, the following reaction takes place starting from the generated pyruvic acid.

When pyruvate, phosphate (Pi) and electron mediator are mixed and pyruvate dehydrogenase acts in the presence of thiamine diphosphate (TPP), flavin adenine nucleotide (FAD) and magnesium ion (Mg ²⁺ ) Acetyl phosphate and carbon dioxide (CO ₂ ) are produced. At that time, the oxidized electron mediator is converted into a reduced form.

  Finally, when a voltage is applied to the electrode, the reduced electron mediator emits electrons on the electrode and returns to the oxidized form. This reaction is represented by the following reaction formula (III). By detecting the electrons emitted at this time as a charge amount, a current value depending on pyrophosphate can be measured.

  An example of the pyrophosphate detection device of the present invention is shown in FIG.

More specifically, the electrochemical measurement is performed using the measuring apparatus system shown in FIG.
This measuring apparatus system is configured as follows. In the figure, reference numeral 10 denotes a measurement cell, in which a stirring bar 7 is placed and fixed on a stirrer machine 8. The measurement electrode 3, the counter electrode 4, and the reference electrode 5 are set in the measurement cell 10 by the electrode fixing device 9. As the measurement electrode 3, a noble metal electrode such as a gold electrode or a carbon electrode is used. The counter electrode 4 is composed of a platinum electrode. The reference electrode 5 is composed of a silver / silver chloride electrode, and the surface is coated with silver chloride. These electrodes are respectively connected to an electrochemical measurement system 20 in which a potentiostat, a function generator, and the like are integrated, and control and data recording are performed by a personal computer 21.

  An example of the measurement procedure is shown below.

  The measurement cell 10 is filled with the reaction solution 1 including the measurement system, and the reaction solution 1 is stirred by the stirrer 7. When a certain potential or higher is applied to the measurement electrode 3 with respect to the reference electrode 5 using the electrochemical measurement system 20, the mediator in the reaction solution is oxidized, and the measurement electrode 3, the counter electrode 4, Current flows between the two. The potential varies depending on the mediator species. A potential higher than the redox potential of the mediator species is applied to the measuring electrode 3. The current value at this time is measured using the electrochemical measurement system 20.

  Since the current value depends on the initial presence of pyrophosphate, pyrophosphate is detected based on this oxidation current value.

  The present invention relates to a reaction in which pyruvate phosphate dikinase is allowed to act on pyrophosphate, AMP, phosphoenolpyruvate, and magnesium ions to convert them into ATP, pyruvate, and phosphate, and pyruvate, phosphate, thiamine. Combining diphosphate, flavin adenine nucleotide, magnesium ion and electron mediator with pyruvate dehydrogenase to produce acetyl phosphate and carbon dioxide, and converting oxidized electron mediator to reduced form It is a feature.

  The pyrophosphate detection method and detection apparatus according to the present invention can detect nucleic acids and SNPs quickly, simply and with high sensitivity, and can be applied to fields dealing with genetic testing, such as the medical field and food field.

The figure which shows the pyrophosphate detection apparatus of this invention

1 Reaction Solution 2 Injection Tube 3 Measurement Electrode 4 Counter Electrode 5 Reference Electrode 7 Stir Bar 8 Stirrer Machine 9 Electrode Fixing Device 10 Cell 20 Electrochemical Measurement System 21 Personal Computer

Claims (6)

A method for detecting pyrophosphate, comprising:
Adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD), magnesium ion ( Providing the sample to a measurement system comprising Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And measuring a current value in the measurement system,
Here, the current value indicates the concentration of pyrophosphate in the sample.
Method for detecting pyrophosphate.   The electron mediator is ferricyanide, 1,2-naphthoquinone-4-sulfonic acid, 2,6-dichlorophenolindophenol, dimethylbenzoquinone, 1-methoxy-5-methylphenazinium sulfate, methylene blue, galocyanine, thionine, The method for detecting pyrophosphate according to claim 1, wherein the pyrophosphate detection method is at least one selected from the group consisting of phenazine methosulfate and Meldola Blue. A method for detecting a nucleic acid comprising:
A sample is subjected to a reaction system containing a DNA primer having a sequence complementary to the sequence of the nucleic acid, DNA polymerase, and deoxynucleotide, and the DNA primer binds to a complementary sequence of the nucleic acid, and the extension reaction from the DNA primer Accompanied by a process wherein pyrophosphate is produced;
The samples were adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD). , Subjecting the sample to a measurement system comprising magnesium ion (Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And a step of measuring a current value in the measurement system, wherein the current value indicates a concentration of the nucleic acid having a specific sequence in the sample.   The method for detecting a nucleic acid according to claim 3, wherein the extension reaction from the DNA primer is a PCR reaction. A method for typing an SNP sequence of DNA, wherein a sample is provided to a reaction system comprising an allele-specific primer, DNA polymerase, and deoxynucleotide, and the allele-specific primer is complementary to a complementary sequence of the target sequence of the nucleic acid. Binding and generating pyrophosphate upon extension reaction from the allele-specific primer;
The samples were adenosine monophosphate (AMP), phosphoenolpyruvate (PEP), pyruvate phosphate dikinase (PPDK), phosphate (Pi), thiamine diphosphate (TPP), flavin adenine dinucleotide (FAD). , Subjecting the sample to a measurement system comprising magnesium ion (Mg ²⁺ ), pyruvate dehydrogenase (PDH), and an electron mediator;
And a step of measuring a current value in the measurement system, wherein the generation of the current value is a means for determining the presence or absence of the SNP sequence in the sample.   The method for typing a DNA SNP sequence according to claim 5, wherein the extension reaction from the allele-specific primer is a PCR reaction.

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