JP7801754B2 - METHOD FOR MEASURING TARGET NUCLEIC ACID SEQUENCE IN A TEST SAMPLE - Google Patents

Description

The present invention relates to a measurement device and a measurement method for measuring a target nucleic acid sequence in a test sample.

Real-time PCR using the TaqMan (registered trademark) probe method has been used in polymerase chain reactions (PCR), which have been conventionally employed as a method for definitively diagnosing viral infections such as the novel coronavirus (SARS-CoV-2) (see, for example, Patent Document 1 and Non-Patent Documents 1 and 2).

A TaqMan probe has a fluorescent substance at its 5' end and a quencher at its 3' end. In this state, the quencher is located near the fluorescent substance, suppressing the emission of fluorescence from the fluorescent substance even when excitation light is applied. When the target nucleic acid sequence is present, the primer and TaqMan probe bind to the target nucleic acid sequence during the annealing step of PCR. In the subsequent extension step, Taq polymerase synthesizes the gene, and when Taq polymerase reaches the TaqMan probe, it degrades the TaqMan probe due to its 5'->3' exonuclease activity. Once the TaqMan probe is degraded, the quencher no longer suppresses the emission of fluorescence from the fluorescent substance, and the fluorescent substance begins to emit fluorescence when excitation light is applied. As PCR amplification progresses, the fluorescence intensity increases.

On the other hand, electrochemical impedance spectroscopy (EIS) is known as a technique used to analyze diffusion processes in the liquid phase and chemical reactions at interfaces, as well as to analyze porous electrodes. (Hereinafter referred to as "EIS method"). EIS method has been reported to be used in a variety of fields, including corrosion resistance evaluation, evaluation of electrodeposition processes, performance evaluation of various sensors, performance evaluation of energy conversion devices such as batteries, and evaluation of the electrical properties of dielectric materials.

In light of this, a method has been proposed for detecting target DNA in a measurement sample using EIS in PCR, a DNA analysis and quantification method that amplifies and detects DNA strands (e.g., Patent Document 2 and Non-Patent Document 3).

This technique uses the EIS method to detect target substances present in a solution by measuring impedance. The DNA of the target substance is amplified by PCR, and during this process, a metal complex such as ruthenium is inserted into the double-stranded DNA, and an intermediary substance in the solution causes electron transfer with the electrode, i.e., an oxidation-reduction reaction. This oxidation-reduction reaction is measured by impedance. For example, if the target substance increases from its initial value, the impedance-changing substance that was originally present is taken up by the target substance, decreasing, causing a change in the charge mobility of the impedance-monitoring substance. The increase in the target substance can be detected by detecting this change in charge mobility in the form of an impedance response.

In Patent Document 2 and Non-Patent Document 3, after a predetermined measurement cycle is completed, the increase in charge transfer resistance (Rct) for each _cycle is confirmed, and it is determined that a significant increase in double-stranded DNA has occurred around the time when _Rct begins to increase rapidly.

_Rct is an indicator of the reluctance of a charge transfer reaction, as the rate of the charge transfer reaction is proportional to the current. If the charge transfer reaction is likely to occur, _Rct will be small, and if it is unlikely to occur, _Rct will be large. _{Rct is} roughly the diameter of the semicircular plot when the impedance obtained by the EIS method is plotted on a Cole-Cole plot, making it easy to understand intuitively from the Cole-Cole plot.

A Cole-Cole plot is a type of Nyquist plot in which the real part of the impedance is plotted on the horizontal axis and the imaginary part of the impedance is plotted on the vertical axis with negative values pointing upward. It depicts a characteristic locus depending on the configuration of the electrical circuit, making it easy to visually grasp the outline of the spectrum. Automatic measurements using the FRA (Frequency Response Analyzer) method, which is one of the EIS methods, generally have a function to create a Cole-Cole plot.

JP 2013-188164 A International Publication No. WO2019/026517

Proc Natl Acad Sci U S A. 1991 Aug 15; 88(16): 7276-7280. "Principles of Real-Time PCR," [online], Takara Bio Inc. [searched January 7, 2022], Internet <URL: https://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100009037> Special web column "Learning Applied Physics from the COVID-19 Pandemic," edited by the Japan Society of Applied Physics, [online], Japan Society of Applied Physics [searched January 7, 2022], Internet <URL: https://www.jsap.or.jp/columns-covid19/covid19_4-3-3>

The TaqMan probe method described in Patent Document 1 and Non-Patent Documents 1 and 2 requires a light source to generate excitation light and a light-receiving unit to detect only the fluorescence of the desired wavelength in order to perform fluorescence measurement, which poses the problem of making the equipment expensive and large.

Patent Document 2 and Non-Patent Document 3 describe a phenomenon in which, when the target substance, DNA, is amplified by PCR, a metal complex is incorporated into double-stranded DNA, resulting in a decrease in the redox reaction mediated by the intermediary substance, resulting in a decrease in signal. Therefore, since the amount of the target substance and the signal are inversely proportional, there is an inherent tendency for detection sensitivity to be low.

The present invention was made in light of these circumstances, and provides a measurement device and method for measuring target nucleic acid sequences that is inexpensive, compact, and has improved detection sensitivity.

In order to solve the above problems, the present invention provides a measurement method using a measurement device for measuring changes in impedance of a measurement object that may contain a target nucleic acid sequence, and for measuring the target nucleic acid sequence in a test specimen, the measurement device comprising: a container for containing the measurement object; at least one pair of electrodes provided in the container and in contact with the measurement object; an impedance measurement unit for measuring the impedance at the electrodes; and a temperature control unit for adjusting the temperature applied to the measurement object, wherein the measurement object is the test specimen, an impedance observation substance, and a probe having a sequence complementary to the target sequence of the target nucleic acid sequence, and the measurement object is a mixture containing an impedance change precursor modified with an impedance change substituent, a polymerase with 5'→3' exonuclease activity, and a primer, and wherein PCR (Polymerase Chain Reaction) is performed on the mixture, so that the impedance change precursor is decomposed by the polymerase with 5'→3' exonuclease activity to produce an impedance change substance.

In the measurement method , the measurement device may further include a determination unit that determines whether the impedance has crossed a predetermined threshold value.

In the measurement method , the impedance varying substance may enhance the charge mobility of the impedance observation substance, and the impedance observation substance may exhibit a charge on the electrode and not interact with anything other than the impedance varying substance.

In the measurement method , the impedance-changing substance may be modified with a compound having redox activity selected from the group consisting of methylene blue, Nile blue, anthraquinone derivatives, naphthalene derivatives, metal ammine complexes, and metal complexes containing bipyridine derivatives, phenanthroline derivatives, and/or dipyridophenazine derivatives as organic ligands.

In the measurement method , the impedance observation substance may be a metal complex forming a redox electrolyte selected from the group consisting of ferricyanide ion, ferrocyanide ion, ferrocene, and mixtures thereof.

In the measuring method , at least one of an absolute value of the impedance, a real part of the impedance, and an imaginary part of the impedance may be used as the impedance.

In the measurement method , an estimated value of charge transfer resistance calculated from the impedance may be used as the impedance.

In the measurement method , the Cole-Cole plot based on the measured values of the impedance may be approximated to a semicircle using two or more points on the Cole-Cole plot, and the diameter of the semicircle may be used as the estimated value of the charge transfer resistance; the difference in the real parts of the impedance using two or more points on the Cole-Cole plot may be used as the estimated value of the charge transfer resistance; the absolute value of the difference in the impedance using two or more points on the Cole-Cole plot may be used as the estimated value of the charge transfer resistance; the Cole-Cole plot may be approximated to an ellipse using three or more points on the Cole-Cole plot, and the diameter in the real axis direction of the ellipse may be used as the estimated value of the charge transfer resistance; or the Cole-Cole plot may be approximated to an arc using three or more points on the Cole-Cole plot, and the difference in the real parts of the arc when the imaginary part of the arc = 0Ω may be used as the estimated value of the charge transfer resistance.

The present invention also provides a measurement method for measuring a target nucleic acid sequence in a test specimen by combining a PCR (Polymerase Chain Reaction) method with an EIS (Electrochemical Impedance Spectroscopy) method, comprising: using an impedance change precursor, which is a probe having a sequence complementary to the target sequence of the target nucleic acid sequence and which has been modified with an impedance change substituent; carrying out a PCR reaction on a mixture containing the test specimen, an impedance observation substance, the impedance change precursor, and a polymerase having 5' exonuclease activity; inducing decomposition of the impedance change precursor into an impedance change substance by the polymerase; and measuring a change in impedance of the mixture observed as a result of the impedance change substance enhancing the charge mobility of the impedance observation substance by an EIS method.

In the measurement method, the impedance varying substance may enhance the charge mobility of the impedance observation substance, and the impedance observation substance may exhibit a charge on an electrode for measuring the impedance and not interact with anything other than the impedance varying substance.

In the measurement method, the impedance-changing substance may be modified with a compound having redox activity selected from the group consisting of methylene blue, Nile blue, anthraquinone derivatives, naphthalene derivatives, metal ammine complexes, and metal complexes containing bipyridine derivatives, phenanthroline derivatives, and/or dipyridophenazine derivatives as organic ligands.

In the measurement method, the impedance observation substance may be a metal complex that forms a redox electrolyte selected from the group consisting of ferricyanide ions, ferrocyanide ions, ferrocene, and mixtures thereof.

The measurement method may use at least one of the absolute value of the impedance, the real part of the impedance, and the imaginary part of the impedance as the impedance.

In the measurement method, the impedance may be an estimated value of charge transfer resistance calculated from the impedance.

In the measurement method, the Cole-Cole plot based on the impedance measurement values may be approximated to a semicircle using two or more points on the Cole-Cole plot, and the diameter of the semicircle may be used as the estimated value of the charge transfer resistance; the difference in the real parts of the impedances using two or more points on the Cole-Cole plot may be used as the estimated value of the charge transfer resistance; the absolute value of the difference in the impedances using two or more points on the Cole-Cole plot may be used as the estimated value of the charge transfer resistance; the Cole-Cole plot may be approximated to an ellipse using three or more points on the Cole-Cole plot, and the diameter of the ellipse in the real axis direction may be used as the estimated value of the charge transfer resistance; or the Cole-Cole plot may be approximated to an arc using three or more points on the Cole-Cole plot, and the difference in the real parts of the arc when the imaginary part of the arc = 0 Ω may be used as the estimated value of the charge transfer resistance.

The measurement device and method of the present invention allow for the device to be inexpensive and compact while maintaining the advantages of conventional PCR. Furthermore, detection sensitivity can be improved compared to conventional PCR detection methods using the EIS method.

1 is a perspective view schematically showing an example of a basic configuration of a measurement device according to a preferred embodiment of the present invention. 1 is a side cross-sectional view schematically showing an example of a basic configuration of a measurement device according to a preferred embodiment of the present invention. 1 is a diagram showing an impedance measuring unit and a determining unit in an example of a basic configuration as a preferred embodiment of a measuring device of the present invention. FIG. FIG. 2 is a diagram illustrating an example of an impedance measuring unit. FIG. 2 is a schematic diagram of a probe (impedance change precursor) contained in the object to be measured according to the present invention. FIG. 2 is a schematic diagram of a primer contained in the object to be measured according to the present invention. 1 is a schematic diagram of a target nucleic acid sequence that is a target substance of the present invention. FIG. 1 is a schematic diagram showing how a primer and a probe bind to a target nucleic acid sequence during the annealing step of PCR. FIG. 1 is a schematic diagram showing how a polymerase synthesizes a complementary strand in the extension step of PCR. FIG. 1 is a schematic diagram showing how the extension reaction progresses and the polymerase decomposes the probe. FIG. 10 is a schematic diagram showing how a probe is disassembled. FIG. 10 is a graph showing the relationship between the number of PCR cycles and impedance. FIG. 1 is a diagram showing an example of a general equivalent circuit of an object to be measured, which is an object to be measured by the EIS method. FIG. 1 is a schematic diagram illustrating an example of a Cole-Cole plot in an EIS method according to a preferred embodiment. FIG. 10 is a schematic diagram illustrating a method for determining charge transfer resistance R _ct in a Cole-Cole plot according to a preferred embodiment, in which two frequencies, high and low, are approximated to semicircles and the diameter of the semicircles is taken as the charge transfer resistance R _ct estimated from impedance. FIG. 10 is a schematic diagram illustrating that, in a Cole-Cole plot according to a preferred embodiment, the charge transfer resistance R _ct estimated from the impedance is the difference between the real parts of the impedances measured at two frequencies, high and low, or the absolute value of the impedance difference. FIG. 1 is a schematic diagram illustrating a Cole-Cole plot according to a preferred embodiment, in which an ellipse is approximated at three frequencies, and the diameter of the ellipse in the real axis direction is taken as the charge transfer resistance R _ct estimated from the impedance. FIG. 10 is a schematic diagram illustrating a Cole-Cole plot according to a preferred embodiment, in which three frequencies are approximated to arcs, and the difference between the real parts of the arcs at the point where the imaginary part of the arc is 0Ω is taken as the charge transfer resistance R _ct estimated from the impedance.

Below, preferred embodiments of the measuring device and measuring method of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following description, and various modifications and alterations are possible for those skilled in the art based on the gist of the invention as set forth in the claims or disclosed in the detailed description. Such modifications and alterations also fall within the scope of the present invention.

All documents cited herein are incorporated by reference in their entirety.

<Configuration of measuring device>
FIG. 1 is a perspective view schematically illustrating an example of the basic configuration of a preferred embodiment of a measuring device 1 of the present invention. The measuring device 1 includes a container 3 that contains a measurement object 2 (see FIG. 2 ) and at least one pair of electrodes 4 that are provided in the container 3 and contact the measurement object 2. FIG. 2 is a side cross-sectional view of the measuring device 1. The measurement object 2 is contained in the container 3. By containing a measurement object 2 in each container 3, contamination of the test sample can be prevented. A temperature control unit 6 that adjusts the temperature applied to the measurement object 2 is provided on the electrode 4, for example, via a seal unit 5. The measuring device 1 includes an impedance measurement unit 7 that measures the impedance Z of the measurement object 2 at the electrode 4, and a determination unit 8 that determines whether the impedance Z crosses a predetermined threshold value _Zth . The measuring device 1 measures changes in the impedance Z of the measurement object 2, which may contain a target nucleic acid sequence 9 (see FIG. 7 ), over time to determine the presence or absence of the target nucleic acid sequence 9.

To more strongly express the charge transfer fluctuation of the impedance-changing substance B (see Figure 11), electrode 4 preferably has compound C, which has affinity for impedance-changing substance B, disposed on its solution-side surface. This affinity-containing compound C is disposed so as to modify the surface of electrode 4, and is preferably present, for example, as a thin film layer. If a structure can be adopted in which impedance-changing substance B remains within the thin film layer of compound C, fluctuations in the amount of impedance-changing substance B present within the thin film layer will also cause fluctuations in membrane resistance, which can be detected as a sudden fluctuation in impedance Z. It should be noted that fluctuations in the amount of impedance-changing substance B can be detected as a fluctuation in impedance Z not only when it remains within the thin film layer, but also when it passes through the thin film layer and reaches electrode 4.

The affinity compound C is preferably a compound that has minimal interaction with PCR reaction solution components, amplification components, biological substances, etc., and examples include polymers and SAM membranes. Functional groups such as hydroxyl groups, ether groups, aldehyde groups, carbonyl groups, carboxyl groups, nitro groups, phosphate groups, and sulfonic acid groups are arranged on the structural surface. In the case of polymer materials, functional groups are arranged on the main chain, side chains, or both. Specific examples include polyvinyl alcohol, polyethylene glycol, polyester, polyacrylic acid, polyacrylamide, and polysaccharides.

A known method can be used to apply compound C to electrode 4. It is also possible to form compound C on electrode 4 using known techniques such as electropolymerization, photopolymerization, thermal polymerization, and radical polymerization. The thin film layer is preferably made of a material with a porous structure that allows impedance observation substance A to enter and exit and impedance variation substance B to adsorb and desorb, and is preferably 0.001 to 10 μm thick.

There are no particular restrictions on the material of electrode 4, as long as it is an electrode for electrochemical measurements. However, if compound C is disposed on the surface of electrode 4, it is preferable that the electrode be a carbon electrode made of graphite, carbon nanotubes, carbon black, etc., a gold electrode, or a conductive oxide electrode such as ITO.

As shown in FIG. 3, the impedance measurement unit 7 measures the impedance Z of the object under test 2 at a specific frequency, for example, using the four-terminal method. This specific frequency may be a single frequency, or multiple frequencies may be switched between for multiple measurements. As shown in FIG. 4, for example, the impedance measurement unit 7 has a signal source 10 that applies a signal of a specific frequency, and a current-voltage measurement means 11 that measures the current and voltage (both vector values) at that frequency, and the impedance Z can be calculated as voltage/current. The above-mentioned FRA, for example, can be used to measure the impedance Z. The impedance measurement unit 7 may be included in the measurement device 1, or may be an external device to the measurement device 1. In this embodiment, the impedance Z is measured, for example, for each PCR cycle.

The determination unit 8 includes, for example, a CPU (Central Processing Unit) as a processing means (not shown) and a memory as a storage means, and uses a software program stored in the memory to input the measured value of impedance Z and output the presence or absence of the target nucleic acid sequence 9 in the test sample depending on whether or not the impedance Z crosses a predetermined threshold value _Zth , or to output the quantification result of the target nucleic acid sequence 9 by a method described below.

<Object to be measured>
The object to be measured 2 is a mixture containing a test sample that may contain a target nucleic acid sequence 9, an impedance observation substance A, an impedance change precursor as a probe 12 (see Figure 5), a polymerase with 5' → 3' exonuclease activity, and a primer 13 (see Figure 6).

(Target nucleic acid sequence)
The measurement device 1 and measurement method of this embodiment can be suitably used for compounds in which the target nucleic acid sequence 9 in the test sample is a nucleic acid or a nucleic acid structure within the molecule, and are particularly suitable for monitoring the increase in double-stranded DNA, which is an amplified product of nucleic acid by PCR.

When detecting infection with an RNA virus, a test sample is collected from the test specimen (host). RNA is extracted from the collected test sample, and cDNA is synthesized and used as template DNA.

(Impedance observation material)
The impedance monitoring substance A is a substance that generates a charge on the electrode 4 and is the subject of impedance measurement, and preferably does not interact with substances other than the impedance varying substance B, or if it does, the interaction is extremely small. Fluctuations in the mobility of the impedance monitoring substance A in the solution are recognized as fluctuations in impedance on the electrode 4.

The impedance monitoring substance A is, for example, a metal complex that forms a redox electrolyte selected from the group consisting of ferricyanide ion, ferrocyanide ion, ferrocene, and mixtures thereof.

(probe)
As shown in Figure 5, probe 12 is a nucleotide having a base sequence complementary to the target base sequence of target nucleic acid sequence 9, and is an impedance-changing precursor modified with impedance-changing substituent B'. (In Figure 5, a ruthenium complex is shown as an example of impedance-changing substituent B'.)

The impedance varying substituent B' is a group derived from a compound that varies the charge mobility of the impedance observation substance A. The compound that varies the charge mobility of the impedance observation substance A may be a compound that enhances the charge mobility of the impedance observation substance A, or a compound that reduces the charge mobility of the impedance observation substance A. However, from the viewpoint of sensitivity, a compound that enhances the charge mobility of the impedance observation substance A is preferred. Examples of compounds that vary the charge mobility of the impedance observation substance A include compounds having redox activity selected from the group consisting of methylene blue, Nile blue, anthraquinone derivatives, naphthalene derivatives, metal ammine complexes, and metal complexes containing bipyridine derivatives, phenanthroline derivatives, and/or dipyridophenazine derivatives as organic ligands. Examples of the metal complex that can be used include ruthenium complexes, such as ruthenium complexes with bipyridine or dipyridophenazine as a ligand, and bis(2,2'-bipyridine)dipyrido[3,2-a:2',3'-c]phenazineruthenium(II) (Ru(bpy) _2DPPZ complex). It is not preferable for the impedance variable substance B described below to intercalate into base pairs. Therefore, when the impedance variable substituent B' contains a fused ring in which three or more aromatic rings are fused, it is preferable for the fused ring moiety to be bonded to the nucleotide of the probe 12 via a linker. For example, when bis(2,2'-bipyridine)dipyrido[3,2-a:2',3'-c]phenazine ruthenium(II) (Ru(bpy) _2DPPZ complex) is used as the compound that changes the charge mobility of impedance observation substance A, it is preferable to bind the dipyrido[3,2-a:2',3'-c]phenazine moiety of the organic ligand to the nucleotide of probe 12 via a linker. Alternatively, a phenanthroline derivative or a dipyridophenazine derivative in which a bulky substituent has been introduced into the phenanthroline skeleton or the dipyridophenazine skeleton may be used as the organic ligand.

Modification with impedance-varying substituent B' can be performed using well-known methods. The modification can be performed at either the 3' or 5' end of the nucleotide of probe 12, although modification at the 5' end of the nucleotide of probe 12 is preferred due to ease of modification and low cost. The 5' or 3' end of the nucleotide of probe 12 may be directly modified with a functional group possessed by a compound that varies the charge mobility of impedance observation substance A, or the 5' or 3' end of the nucleotide of probe 12 may be modified with a compound that varies the charge mobility of impedance observation substance A via a crosslinker, spacer, etc.

For example, by reacting the amino group in the base at the 5' end of a nucleotide having a sequence complementary to the target sequence of the target nucleic acid sequence 9 with a crosslinker such as N-succinimidyl S-acetylthioglycolate (SATA), and then reacting this with a compound that changes the charge mobility of the impedance observation substance A in which one hydrogen atom bonded to a carbon atom of an organic ligand is substituted with a bromine atom, an impedance change precursor modified with the impedance change substituent B' can be obtained.

The base sequence of probe 12 is determined appropriately depending on the target nucleic acid sequence 9, but the number of bases in probe 12 is preferably at least 20, and more preferably approximately 30. The upper limit of the number of bases in probe 12 is, for example, approximately 100 or less. For example, when the impedance-changing substituent B' is modified at the 5' end, a base sequence non-specific to the target sequence may be added to the 3' end to obtain a probe 12 with the desired number of bases.

The probe 12 may also be modified with fluorescent compounds or substituents to enable it to be used for optical detection as well as electrochemical detection.

(polymerase)
As the polymerase, a polymerase having 5' to 3' exonuclease activity can be used, and in particular, various heat-resistant polymerases such as Taq DNA polymerase, which are commonly used in PCR, can be used.

In addition to the above, the mixture liquid that is the object to be measured 2 may also contain, as needed, one or more of the buffer solutions, salts, and additives commonly used in PCR, at commonly used concentrations.

(Impedance-varying substances)
The probe 12 modified with the impedance-changing substituent B' has a large molecular weight and a small diffusion coefficient, and therefore does not affect the charge mobility of the impedance observation substance A, and the impedance Z of the object to be measured 2 does not change.

When a target nucleic acid sequence 9 is present in a mixture liquid, which is the object to be measured 2, and a PCR reaction is performed on the mixture liquid, first, in the annealing step of the PCR, the primer 13 and probe 12 complementarily bind to a predetermined region of the target base sequence 9, and then, in the extension step of the PCR, the probe 12 is degraded by the action of the polymerase to produce an impedance change substance B. This impedance change substance B has one base at the 5' or 3' end of the nucleotide of the probe 12 modified with an impedance change substituent B'. Compared to the impedance change precursor, its molecular weight is smaller and its diffusion coefficient is larger, resulting in a greater effect of enhancing or attenuating the charge mobility of the impedance change observation substance A.

(Primer)
As shown in FIG. 6, the primer 13 is a nucleotide having a base sequence complementary to the target base sequence of the target nucleic acid sequence 9, and can be designed by a known method as appropriate depending on the target nucleic acid sequence 9.

<Method for detecting a target nucleic acid sequence>
The probe 12 modified with the impedance-changing substituent B' has a large molecular weight and therefore a small diffusion coefficient, so it does not affect the charge mobility of the impedance observation substance A, and the impedance Z of the object to be measured 2 does not change.

In the annealing step of PCR, if a target nucleic acid sequence 9 is present in the object to be measured 2, the probe 12 and primer 13 complementarily bind to a specific region of the target nucleic acid sequence 9, as shown in Figure 8.

In the extension step of PCR, polymerase synthesizes a complementary strand during the extension reaction, as shown in Figure 9.

As the extension reaction progresses further, the polymerase reaches the probe 12, as shown in Figure 10, and decomposes the probe 12 into impedance-varying substance B.

As shown in Figure 11, when the probe 12 is decomposed, the impedance Z of the object to be measured 2 decreases due to the action of the impedance change substance B. More specifically, when the probe 12 modified with the impedance change substituent B' is decomposed, the number of bases per modification of the impedance change substituent B' becomes one base (impedance change substance B), and the molecular weight decreases, resulting in an increase in the diffusion coefficient. As the diffusion coefficient increases, the effect of, for example, enhancing the charge mobility of the impedance observation substance A becomes greater, and the impedance Z of the impedance observation substance A decreases. As shown in Figure 12, the impedance Z decreases further as PCR amplification progresses.

If the target nucleic acid sequence 9 is not present in the object to be measured 2, the probe 12 is not degraded, and therefore the impedance Z of the object to be measured 2 does not change even if the number of PCR cycles increases.

12, when the impedance Z crosses the predetermined threshold value _Zth , it can be determined that the target nucleic acid sequence 9 is present in the object to be measured 2. On the other hand, when the impedance Z does not cross the predetermined threshold value _Zth even after a predetermined number of PCR cycles, it can be determined that the target nucleic acid sequence 9 is not present in the object to be measured 2.

PCR conditions are not particularly limited, but for example, an initial denaturation step at 98°C for 2 minutes is followed by 40 cycles of a denaturation step at 98°C for 10 seconds, an annealing step at 55°C for 20 seconds, and an extension step at 68°C for 30 seconds. Impedance measurement is preferably performed after the extension step, and more preferably immediately after the extension step, i.e., after the extension step and before the next denaturation step. The temperature for impedance measurement can be selected as appropriate, and impedance measurement is preferably performed at a constant temperature for each PCR cycle.

The threshold value Z _th can be appropriately determined from the results of a preliminary experiment on a specific object 2 to be measured, for example, and can be applied to similar objects 2 to be measured thereafter.

<Quantitative measurement of target nucleic acid sequence>
Quantitative measurement of the target nucleic acid sequence 9 can be performed using methods similar to the well-known absolute and relative quantitation methods for real-time PCR. In the absolute quantitation method, for example, a calibration curve is plotted with the _CT value (the PCR cycle number when the amplification curve reaches the threshold) on the Y axis and the amount of standard on the X axis, and the amount of the target nucleic acid sequence 9 is quantified. In the relative quantitation method, for example, the _CT value (Y axis) is plotted against the logarithm of the dilution rate or template amount (X axis), and each calibration curve is created.

As described above, the measurement device 1 and measurement method of this embodiment, which are capable of detecting and quantitatively measuring the target nucleic acid sequence 9, make it possible to achieve large-scale continuous analysis and real-time analysis.

<Impedance measurement value>
13 is a diagram showing an example of a general equivalent circuit of a measurement target 2, which is an object to be measured by the EIS method. Such equivalent circuits are known in the prior art of the EIS method, and will be briefly explained below. Depending on the type of measurement target 2, the equivalent circuit may differ from that shown in the diagram.

The solution resistance _Rs is an electrical resistance component determined by the resistivity of the object to be measured 2, etc. The charge transfer resistance _Rct is a resistance due to a reaction of the object to be measured 2 at the interface of the electrode 4. The electric double layer capacitance _Cdl represents the capacitance component of the object to be measured 2. The Warburg impedance W represents the diffusion resistance.

Under conditions where a substance responsible for charge transfer in the object to be measured 2 is taken in, the progress of change in the chemical properties of the object to be measured 2 can be known, for example, by measuring the charge transfer resistance R _ct based on the measurement of the impedance Z of the object to be measured 2.

The impedance Z of such an equivalent circuit is approximately the solution resistance _Rs when measured at high frequencies of, for example, several kHz or more. This is because the equivalent circuit corresponds to a frequency range in which the electric double layer capacitance _Cdl can be approximated as a short circuit.

At frequencies where the absolute value of the charge transfer resistance _Rct is non-negligible relative to the absolute value of the capacitive reactance due to the electric double layer capacitance _Cdl , for example, in the frequency range of several hundred Hz to several Hz, a Cole-Cole plot shows a semicircular impedance. If a frequency near the apex of the semicircle is selected as the frequency at which measurement is performed, the imaginary part of the impedance will be approximately half of the charge transfer resistance _Rct . In other words, impedance can be measured using the imaginary part of the impedance measured at a single frequency. It is also possible to select multiple frequencies near the apex of the semicircle and perform measurements, and then average the values of the imaginary part of the impedance.

At low frequencies, the electric double layer capacitance C _dl approaches a frequency range where it can be approximated as an open circuit, and the impedance of the equivalent circuit approaches the sum of the solution resistance R _s and the charge transfer resistance R _ct . That is, by selecting such a low frequency as the measurement frequency, the real part of the impedance approaches the sum of the solution resistance R _s and the charge transfer resistance R _ct . In other words, impedance can be measured using the real part of the impedance measured at a single frequency. It is also possible to select multiple frequencies where the electric double layer capacitance C _dl can be approximated as an open circuit, perform measurements, and then average the real part of the impedance. Furthermore, it is also possible to use the absolute value of the impedance instead of the real part of the impedance, which may be particularly effective when the imaginary part of the impedance is not zero at the frequency.

For example, in the even lower frequency range of a few Hz or less, the impedance changes in such a way that the change due to the Warburg impedance W becomes dominant. In other words, as the frequency decreases, both the real and imaginary parts of the impedance increase.

14 is a schematic diagram showing an example of a Cole-Cole plot in the EIS method. As can be seen from this Cole-Cole plot, there is no significant difference between the absolute value of the impedance |Z| and the real part of the impedance _Zreal . The imaginary part of the impedance also shows a similar tendency.

15 is a schematic diagram showing how to determine the charge transfer resistance R _ct in a Cole-Cole plot when two frequencies, high and low, are approximated as semicircles and the diameter of the semicircles is used as the charge transfer resistance R _ct estimated from the impedance. In the figure, once the solution resistance R _s and two points a-jb on the semicircle whose center point is on the real axis are determined, b:(a-R _s )=(R _s +R _ct -a):b, the charge transfer resistance R _ct estimated from the impedance measured at two frequencies, high and low, f, can be determined using the following formula. The solution resistance R _s can be determined, for example, by measuring the impedance at a high frequency of 1 kHz or higher at least once to obtain an impedance equivalent to the solution resistance R _s . For example, the real part Z _real of the impedance measured at a frequency f=1 kHz can be used.

As mentioned above, when using either the absolute value, imaginary part, or real part of impedance, measurement results at one frequency or multiple frequencies can be used. On the other hand, this method using approximation to a semicircle requires measurement results at at least two frequencies (two points on the Cole-Cole plot). Furthermore, it is also possible to use measurement results at more frequencies to approximate to a semicircle and then take the average of these.

16 is a schematic diagram showing how the charge transfer resistance R ct is estimated from the impedance by using the difference in the real parts of the impedance measured at two frequencies, high and low, or the absolute value of the _impedance difference (absolute value of the vector difference) in a Cole-Cole plot. This method also requires measurement results at at least two frequencies (two points on the Cole-Cole plot). Furthermore, it is also possible to use measurement results at more frequencies and take the average of these.

FIG. 17 is a schematic diagram illustrating how a Cole-Cole plot is approximated to an ellipse at three frequencies, and the diameter of the ellipse in the real axis direction is used as the charge transfer resistance R _ct estimated from the impedance. Measurements of a specific object often result in a Cole-Cole plot that resembles a vertically squashed semicircle, so approximating to an ellipse can sometimes result in a more accurate approximation. This method requires measurement results at at least three frequencies (three points on the Cole-Cole plot). Alternatively, measurement results at more frequencies can be averaged. Similar approximations are possible even if the Cole-Cole plot in the actual measurement of a specific object resembles a vertically stretched semicircle (i.e., the diameter in the real axis direction is the minor axis).

FIG. 18 is a schematic diagram showing how a Cole-Cole plot is approximated to arcs at three frequencies, and the difference between the real parts of the arcs when the imaginary part of the arcs is 0 Ω is used as the charge transfer resistance R _ct estimated from the impedance. In a Cole-Cole plot where the center of the semicircle is close to being below the real axis, approximating the arc may result in a more accurate approximation. This method also requires measurement results at at least three frequencies (three points on the Cole-Cole plot). Furthermore, it is also possible to use measurement results at more frequencies and take their average.

Thus, there are five ways to calculate the charge transfer resistance R _ct estimated from the impedance, just shown as examples. There are eight ways to calculate the impedance Z of the object to be measured 2, including the real part, imaginary part, and absolute value of the impedance, just shown as examples, but the methods are not limited to these.

In the following examples, genomic DNA preparation, PCR, and other procedures can be performed using methods commonly known to those skilled in the art. See, for example, Molecular Cloning: A Laboratory Manual, 4th ed. (Michael R. Green and Joseph Sambrook).

<PCR and Impedance Measurement>
PCR is performed under the conditions shown in Table 1 (composition), Table 2 (reaction conditions), and Table 3 (sequence), for example. Genomic DNA containing the human β-actin gene locus is prepared from human cells and used as template DNA. PCR is performed using KOD FX (Toyobo Co., Ltd.) according to the manufacturer's instructions. PCR primers and impedance monitoring substances (ferricyanide and ferrocyanide) are commercially available.

The impedance change precursor (Ru complex modified probe) is prepared according to the method described in the following document, for example.
Jing-Tang Lin et al., “A Sulfhydryl-Reactive Ruthenium (II) Complex and Its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays”, PLOS ONE, Volume 7, Issue 4, April 2012

The target nucleic acid sequence is measured using a measuring device capable of measuring impedance (Figure 3). Impedance is measured using changes in the impedance monitoring substance as an indicator while PCR is being performed (Figures 12 and 14). Impedance is measured after the extension step in Table 2. For example, the impedance measurement conditions are a voltage of 100 mV and a frequency of 10 kHz to 0.1 Hz.

As another example, PCR can be performed under the conditions shown in Table 4 (composition), Table 5 (reaction conditions), and Table 6 (sequence). In Table 5, the annealing step and extension step are performed as a single step, but the present invention is equally applicable in this case. Genomic DNA derived from human cells containing the human β-actin locus is prepared from human cells and used as template DNA. PCR is performed using Premix Ex Taq (registered trademark) (Takara Bio Inc.) according to the manufacturer's instructions. Commercially available PCR primers and impedance monitoring substances (ferricyanide and ferrocyanide) are used. Other conditions are the same as in Example 1, except that impedance measurements are performed after the annealing and extension steps in Table 5. Impedance is measured during PCR to determine the target nucleic acid sequence.

The impedance is measured and the target nucleic acid sequence is determined while PCR is being performed in the same manner as in Example 2, except that in PCR, the nucleic acid sequence of the impedance-changing precursor is changed from that of SEQ ID NO: 7 to that of SEQ ID NO: 8.

The present invention has been described above based on embodiments and examples, but the present invention can be implemented in various modified forms. Various modified forms are also included within the scope of the present invention.

REFERENCE SIGNS LIST 1 Measuring device 2 Measurement object 3 Container 4 Electrode 5 Sealing unit 6 Temperature control unit 7 Impedance measurement unit 8 Determination unit 9 Target nucleic acid sequence
10 Signal source
11 Current and voltage measurement means
12 probes (impedance variation precursors)
13 Primer B Impedance change substance B' Impedance change substituent R _s Solution resistance R _ct Charge transfer resistance Z Impedance Z _th Threshold

Claims (15)

A measurement method using a measurement device for measuring a change in impedance of a measurement object that may contain a target nucleic acid sequence, and measuring the target nucleic acid sequence in a test specimen, comprising:
The measuring device
a container for accommodating the object to be measured;
At least one pair of electrodes provided in the container and in contact with the object to be measured;
an impedance measuring unit that measures the impedance at the electrode;
a temperature control unit that adjusts the temperature applied to the object to be measured;
Equipped with
The object to be measured is
The test specimen;
an impedance observation material;
a probe having a sequence complementary to a target sequence of the target nucleic acid sequence, the probe being an impedance-changing precursor modified with an impedance-changing substituent;
a polymerase having 5' to 3' exonuclease activity;
A primer,
A mixture containing
The method comprises carrying out PCR (Polymerase Chain Reaction) on the mixed solution, whereby the impedance change precursor is decomposed by the polymerase having 5' to 3' exonuclease activity to generate an impedance change substance. The measurement method according to claim 1 , wherein the measurement device further comprises a determination unit that determines whether the impedance has crossed a predetermined threshold value. the impedance varying substance enhances the charge mobility of the impedance observation substance,
3. The measurement method according to claim 1, wherein the impedance observation substance generates a charge on the electrode and does not interact with any substance other than the impedance change substance. The measurement method according to any one of claims 1 to 3, wherein the impedance changing substance is modified with a compound having redox activity selected from the group consisting of methylene blue, Nile blue, anthraquinone derivatives, naphthalene derivatives, metal ammine complexes, and metal complexes containing bipyridine derivatives, phenanthroline derivatives, and/or dipyridophenazine derivatives as organic ligands . 5. The measurement method according to claim 1, wherein the impedance observation substance is a metal complex forming a redox electrolyte selected from the group consisting of ferricyanide ion, ferrocyanide ion, ferrocene, and mixtures thereof. 6. The measurement method according to claim 1, wherein at least one of an absolute value of the impedance, a real part of the impedance, and an imaginary part of the impedance is used as the impedance. The measurement method according to any one of claims 1 to 5, wherein an estimated value of charge transfer resistance calculated from the impedance is used as the impedance. a Cole-Cole plot based on the impedance measurement value is approximated to a semicircle using two or more points on the Cole-Cole plot, and the diameter of the semicircle is used as the estimated value of the charge transfer resistance;
the difference between the real parts of the impedances obtained using two or more points on the Cole-Cole plot is used as an estimate of the charge transfer resistance;
the absolute value of the difference in impedance between two or more points on the Cole-Cole plot is used as the estimated value of the charge transfer resistance;
The Cole-Cole plot is approximated to an ellipse using three or more points on the Cole-Cole plot, and the diameter of the ellipse along the real axis is used as the estimated value of the charge transfer resistance; or
8. The measurement method according to claim 7, wherein the Cole-Cole plot is approximated to an arc using three or more points on the Cole-Cole plot, and the difference between real parts of the arc when the imaginary part of the arc is 0 Ω is used as the estimated value of the charge transfer resistance. A method for measuring a target nucleic acid sequence in a test sample by combining a PCR (Polymerase Chain Reaction) method and an EIS (Electrochemical Impedance Spectroscopy) method, comprising:
a probe having a sequence complementary to a target sequence of the target nucleic acid sequence, using an impedance change precursor modified with an impedance change substituent;
carrying out a PCR reaction on a mixture containing the test specimen, an impedance monitoring substance, the impedance change precursor, and a polymerase having 5' exonuclease activity;
causing the polymerase to decompose the impedance-varying precursor into an impedance-varying substance;
A measurement method in which the impedance change of the mixed solution, which is observed when the impedance change substance enhances the charge mobility of the impedance observation substance, is measured by an EIS method. the impedance varying substance enhances the charge mobility of the impedance observation substance,
10. The measurement method according to claim 9, wherein the impedance observation substance generates a charge on an electrode for measuring the impedance and does not interact with any substance other than the impedance change substance. The measurement method according to claim 9 or 10, wherein the impedance-changing substance is modified with a compound having redox activity selected from the group consisting of methylene blue, Nile blue, anthraquinone derivatives, naphthalene derivatives, metal ammine complexes, and metal complexes containing bipyridine derivatives, phenanthroline derivatives, and/or dipyridophenazine derivatives as organic ligands. The measurement method according to any one of claims 9 to 11, wherein the impedance observation substance is a metal complex that forms a redox electrolyte selected from the group consisting of ferricyanide ion, ferrocyanide ion, ferrocene, and mixtures thereof. The measurement method described in any one of claims 9 to 12, wherein at least one of the absolute value of the impedance, the real part of the impedance, and the imaginary part of the impedance is used as the impedance. The measurement method described in any one of claims 9 to 12, wherein an estimated value of charge transfer resistance calculated from the impedance is used as the impedance. a Cole-Cole plot based on the impedance measurement value is approximated to a semicircle using two or more points on the Cole-Cole plot, and the diameter of the semicircle is used as the estimated value of the charge transfer resistance;
the difference between the real parts of the impedances obtained using two or more points on the Cole-Cole plot is used as an estimate of the charge transfer resistance;
the absolute value of the difference in impedance between two or more points on the Cole-Cole plot is used as the estimated value of the charge transfer resistance;
The Cole-Cole plot is approximated to an ellipse using three or more points on the Cole-Cole plot, and the diameter of the ellipse along the real axis is used as the estimated value of the charge transfer resistance; or
15. The measurement method according to claim 14, wherein the Cole-Cole plot is approximated to an arc using three or more points on the Cole-Cole plot, and the difference between real parts of the arc at an imaginary part of 0 Ω is used as the estimated value of the charge transfer resistance.