JP2008196997A - Quantitative determination method for nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a quantitative determination method for nucleic acid for quantitatively and accurately determining each of a plurality of kinds of target nucleic acids contained in a nucleic acid sample, and to provide a quantitative determination device for nucleic acid used for the quantitative determination method. <P>SOLUTION: This quantitative determination method for quantitatively determining the plurality of kinds of target nucleic acids contained in the nucleic acid sample/nucleic acid quantitative determination device used for the quantitative determination method has (a) a process for labeling the plurality of kinds of target nucleic acids, respectively, using two kinds of ligands different in combinations in every kind of target nucleic acids; (b) a process for detecting and determining quantitatively one kind from among the plurality of kinds of target nucleic acids, by using two kinds of receptors coupled specifically with one kind of the ligand used for labeling of one kind of the target nucleic acid in the process (a); and (c) a process for correcting the value obtained in the process (b), by using an affinity of the receptor used for the detection in the process (b) to each of the respective kinds of ligands used in the process (a). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleic acid quantification method for quantifying a plurality of types of target nucleic acids contained in a nucleic acid sample with high accuracy, and a nucleic acid quantification apparatus used for the quantification method.

  The method for detecting and quantifying target nucleic acids present in biological samples is not only important in the analysis of proteins expressed and functioning in vivo at the nucleic acid molecule level, particularly in the study of protein expression control. It is also extremely important in genetic diagnosis such as detection of mutant genes of genetic diseases, diagnosis of cancer, detection of virus-related genes, and the like. In particular, in detection and quantification of nucleic acids in genetic diagnosis and the like, rapid processing is required to process many samples, while high accuracy is required because it is used as a definitive diagnosis. However, conventional methods for detecting nucleic acids are complicated and involve many steps, and the accuracy of detection and quantification is not sufficient. For this reason, improvement of the detection and quantification method of a nucleic acid which has high precision while maintaining rapidity is desired.

  A typical method for detecting a target nucleic acid present in a sample is a hybridization method. The nucleic acid hybridization method is a method for detecting a very small number of target nucleic acids from among many types of nucleic acids using a probe having a sequence complementary to the base sequence of the target nucleic acid to be detected. Various methods have been disclosed as applied nucleic acid detection methods. For example, (1) a method of synthesizing an oligonucleotide on a matrix using a photolithographic technique has been disclosed (for example, see Non-Patent Document 1). A DNA chip in which many types of DNA probes are synthesized on a solid phase by this method is very easy to operate compared to conventional hybridization methods, and can process many samples at once. In addition, since the B / F separation property is also good, this method can greatly improve the rapidity of nucleic acid detection.

However, depending on the hybridization conditions, the hybridization method has a problem of false positive hybridization in which a nucleic acid other than the target nucleic acid and having a base sequence similar to the base sequence of the target nucleic acid is also detected. . In particular, since the base sequence is basically only a sequence consisting of four types of bases, false positive hybridization is more likely to occur than when a protein or the like is detected. In order to avoid such false positive hybridization, hybridization may be performed under the optimum conditions of each probe. However, when using a DNA chip on which the probe is extremely solid-phased, for each probe, It is impossible to give optimum conditions.
In addition, with conventional DNA chips, if the type of target nucleic acid to be detected changes, a probe having a base sequence complementary to them must be re-immobilized. Therefore, it has a drawback that it is not versatile enough to be applied to clinical tests.
In order to solve the problem of the hybridization method using a DNA chip, methods such as a fluorescent beads method, a qPCR (Polymerase Chain Reaction) method, a TaqMan method, and a sniplex method have been devised. Since it is based on a hybridization method, the problem of false positive hybridization has not been solved. Furthermore, since an expensive chip, a fluorescent reagent and a fluorescent reader are required, it is not economically preferable.

Therefore, in recent years, as an alternative to the hybridization method, a method conventionally used as a protein detection / quantification method has been applied to a nucleic acid detection / quantification method. As this method, for example, there is an immunoaggregation method (for example, see Non-Patent Document 2).
The immunoaggregation method is based on the principle that the antibody-sensitized latex beads are aggregated when mixed with the antigen, and as a result, the aggregate can be visually observed on the slide. In recent years, with the development of colloidal sub-nano-order latex beads (hereinafter referred to as latex particles), the protein has been quantified by measuring near-infrared light scattering by latex particles. A latex immunoturbidimetric method was developed based on this principle. Specifically, when the target protein is mixed with latex particles to which an antibody that binds to the target protein is bound, the latex particles are linked in a chain reaction, resulting in an aggregate. Since the aggregate has an apparently large particle size, light can be scattered in the near-infrared light region having a higher wavelength than single latex particles having a smaller particle size. At present, latex particles are mainly made of polystyrene, and there are also small particles having a particle size of about several tens of nanometers.

  Examples of nucleic acid detection methods applying immunoagglutination include (1) a double-chain gene amplified using a gene fragment to which an antigen or antibody is bound, and an antibody or antigen that recognizes the antigen or antibody bound thereto A gene detection method is disclosed in which a target gene is detected by reacting with a particle or a particle bound with a substance that specifically binds to the antigen or antibody, and measuring the degree of aggregation of the particle ( For example, see Patent Document 1.)

  In addition, there is an ELISA method as a known protein detection and quantification method that is the same as the immunoagglutination method. In this method, a first antibody that recognizes a target protein is solid-phased on the inner wall of a microwell plate such as styrene resin, and then the target protein is captured and washed with the first antibody. Next, the target protein is detected by causing the second antibody labeled with an enzyme or a fluorescent substance to recognize the target protein, and finally measuring the enzyme activity or fluorescence of the fluorescent substance labeled on the second antibody. To quantify. This method requires a step for B / F separation and is more complicated than immunoaggregation, but its sensitivity is high, so it is useful for detection and quantification of trace biological substances in clinical examinations. It is widely applied from inspection to fully automatic analysis.

  When applying the above immunoturbidimetric method or ELISA method to nucleic acid detection / quantification, the target nucleic acid is labeled with a ligand, and the target nucleic acid is detected using a receptor that specifically binds to the ligand. It will be quantified. In other words, it is based on the binding reaction between a ligand and a receptor, such as an antigen-antibody reaction, so if the affinity and specificity of the receptor to be used are high, the target substance can be detected and quantified with high accuracy. Is possible. In addition, since the sequence information of the target nucleic acid can be converted into a combination of ligands into a multiplex, unlike the method using the above-mentioned DNA chip, even if the type of target nucleic acid to be detected or quantified changes, There is no need for a specific detection probe for each nucleic acid. Furthermore, the immunoturbidimetric method and ELISA method are already widely used and the operation is simple. Therefore, nucleic acid detection and quantification methods using the immunoturbidimetric method and ELISA method can detect a wide variety of nucleic acids. It can be applied to targeted clinical tests.

However, in the immunoturbidimetric method or ELISA method, when the affinity and specificity of the receptor for the ligand are low, the substance other than the ligand and the receptor cross each other, so that accurate nucleic acid detection and quantification is impossible. There is a problem of becoming. Therefore, it is preferable to use a receptor with high affinity and specificity for the ligand for detection and quantification of the target nucleic acid.
Fodor et al. (1991) Science 251 (4995): p767-773. Singer et al. (1956) The American journal of medicine 21 (6): p888-892. Japanese Patent Laid-Open No. 9-304383

However, it is usually difficult to obtain a receptor having a sufficiently high affinity and specificity for a ligand such that the target substance can be quantified with high accuracy. In particular, in the case of low molecular weight ligands used for nucleic acid labeling, there are many substances that are very similar in three-dimensional structure, and therefore, highly specific receptors that do not cross other substances. It is difficult to get a body. When an antibody is used as a receptor, depending on the type of antibody, it is often possible to obtain only an antibody having a low affinity for an antigen because immunity induction by the antigen is difficult to occur.
For this reason, when there is only one type of target nucleic acid contained in the sample, the target nucleic acid can be detected and quantified with high accuracy by the method (1) above, but multiple types of target nucleic acids are included. In such a case, sufficient accuracy cannot be expected in detection and quantification of each target nucleic acid. Since the types of ligands that can bind to nucleic acids are not yet sufficient, when detecting and quantifying multiple types of target nucleic acids, a combination of ligands with very similar structures such as rhodamine and TAMRA must be used. This is because it is often impossible to use only a receptor having a sufficiently high affinity and specificity for a ligand.

  The present invention applies an immunoagglutination method or an ELISA method to detect and quantify a plurality of types of target nucleic acids, even when using a receptor with insufficient affinity and specificity for a ligand, It is an object of the present invention to provide a method capable of detecting and quantifying a target nucleic acid with high accuracy, and a nucleic acid quantification apparatus used in the method.

  As a result of diligent research to solve the above problems, the present inventors have found that when a plurality of types of target nucleic acids labeled with different types of ligands are detected and quantified using receptors for the respective ligands, By correcting the measured value using the affinity of each receptor for each ligand present in the nucleic acid sample, even if a receptor with insufficient affinity and specificity for the ligand is used, the target nucleic acid The present invention has been completed by finding that it is possible to detect and quantitate with high accuracy. Specifically, the affinity for the target nucleic acid of a receptor that recognizes and binds to the target nucleic acid to the approximate abundance of the target nucleic acid obtained by measuring absorbance, enzyme activity, fluorescence intensity, etc. Correction can be made by multiplying the receptor that recognizes and binds to the target nucleic acid divided by the sum of the affinity for all labeled target nucleic acids present simultaneously in the nucleic acid sample.

That is, the present invention is a method for quantifying a plurality of types of target nucleic acids contained in a nucleic acid sample, and (a) two types of combinations of different types of target nucleic acids for each type of target nucleic acid. And (b) specifically binding one type of target nucleic acid among a plurality of types to the ligand used for labeling the one type of target nucleic acid in step (a). (C) the affinity of the receptor used for detection in step (b) to each of the different types of ligands used in step (a). And a step of correcting the value obtained by the step (b) using the property.
In addition, the present invention is a method for quantifying a plurality of types of target nucleic acids contained in a nucleic acid sample, and (a) two types of combinations of different types of target nucleic acids for each type of target nucleic acid. And (b) specifically binding one type of target nucleic acid among a plurality of types to the ligand used for labeling the one type of target nucleic acid in step (a). A step of detecting and quantifying using two types of receptors, and (d) used for labeling the one type of target nucleic acid in step (a) of the receptor used for detection in step (b). Correcting the value obtained in step (b) using the ratio between the affinity for the ligand and the sum of the affinity of the receptor for each type of ligand used in step (a). And having There is provided a method of quantifying a nucleic acid, characterized.
The present invention is also a method for quantifying each of a plurality of types of target nucleic acids contained in a nucleic acid sample, wherein (a ′) a plurality of types of target nucleic acids is common to all types of target nucleic acids. Labeling with one ligand and two types of ligands consisting of different second ligands in all types of target nucleic acids, and (b) one type of target nucleic acid among a plurality of types, detecting and quantifying using two types of receptors that specifically bind to each of the first ligand and the second ligand used in the labeling of the one type of target nucleic acid in a); (D ′) Of the two types of receptors used for detection in step (b), the affinity of the receptor that specifically binds to the second ligand for the second ligand, Specifically binds to other ligands A step of correcting the value obtained by the step (b) by using the ratio of the receptor to the sum of the affinity for all kinds of ligands other than the first ligand present in the nucleic acid sample. And providing a nucleic acid quantification method characterized by comprising:
In addition, the present invention includes (e) the receptor contained in the nucleic acid sample after the step (b) and before the step (c), the step (d) or the step (d ′). And a step of measuring affinity for each of the various types of ligands. A method for quantifying nucleic acids, comprising:
The present invention also provides a nucleic acid quantification method, wherein the affinity is the reciprocal of the Kd value (equilibrium dissociation constant).
The present invention also provides a nucleic acid quantification method characterized in that the step of measuring affinity is performed using a method of analyzing a fluorescence signal.
The present invention also provides a nucleic acid quantification method characterized in that the method for analyzing the fluorescence signal is an FCS (Fluorescence Correlation Spectroscopy) analysis method.
The present invention also provides a nucleic acid quantification method characterized in that the method for analyzing the fluorescence signal is FIDA (fluorescence intensity distribution analysis) analysis method.
In the present invention, the ligand comprises a fluorescent substance, a hydrophilic organic compound, biotin (biotin), glutathione (Glutathione), DNP (dinitrophenol), digoxigenin, digoxin, and a sugar comprising two or more sugars. From the group consisting of a chain, a polypeptide comprising 6 or more amino acids, auxin, gibberellin, steroid, GST (glutathione-S-transferase), MBP (maltose binding protein), avidin, streptavidin, protein, and analogs thereof The present invention provides a nucleic acid quantification method characterized by being a selected compound.
In the present invention, the ligand comprises a fluorescent substance, a hydrophilic organic compound, biotin (biotin), glutathione (Glutathione), DNP (dinitrophenol), digoxigenin, digoxin, and a sugar comprising two or more sugars. From the group consisting of a chain, a polypeptide consisting of 6 or more amino acids, auxin, gibberellin, steroid, GST (glutathione-S-transferase), MBP (maltose binding protein), avidin, streptavidin, protein, and analogs thereof The present invention provides a method for quantifying a nucleic acid, which is a compound that is selected and has a linker bound to the nucleic acid.
In the present invention, the fluorescent substance may be FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine), Alexa dye series (manufactured by Molecular Probes), Cy dye series. The present invention provides a nucleic acid quantification method characterized by being a fluorescent substance selected from the group consisting of (GE Healthcare Bioscience).
The present invention also provides a nucleic acid quantification method, wherein the polypeptide comprising 6 or more amino acids is a polypeptide selected from the group consisting of a His tag, an HA tag, a Myc tag, and a Flag tag. To do.
In the step (a), the present invention provides a method of labeling with a ligand using PCR (Polymerase Chain Reaction) using two types of primers labeled with different ligands for one type of target nucleic acid. ) A nucleic acid quantification method characterized by being a method of amplification.
The present invention also provides a nucleic acid quantification method characterized in that the method of detecting and quantifying in the step (b) is an immunoturbidimetric method or an ELISA method.
Further, the present invention is an apparatus for quantifying each of a plurality of types of target nucleic acids in a nucleic acid sample labeled with two types of ligands having different combinations for each type of target nucleic acid, A quantification means for detecting and quantifying one type of target nucleic acid in the target nucleic acid using a receptor that specifically binds to a ligand used for labeling the one type of target nucleic acid; Input means for inputting the affinity of each of the types of ligands contained in the nucleic acid sample, the affinity of the receptor for the one type of target nucleic acid, And a correction means for correcting the value obtained by the quantification means using a ratio of the affinity to the total of each type of ligand contained in the nucleic acid sample. Toss And it provides a quantification device of a nucleic acid.
Further, the present invention is an apparatus for quantifying each of a plurality of types of target nucleic acids in a nucleic acid sample labeled with two types of ligands having different combinations for each type of target nucleic acid, A quantification means for detecting and quantifying one type of target nucleic acid in the target nucleic acid using a receptor that specifically binds to a ligand used for labeling the one type of target nucleic acid; Measuring means for measuring the affinity of the body for each type of ligand contained in the nucleic acid sample, input means for inputting the affinity obtained by the measuring means, and the acceptor Using the ratio of the affinity of the body to the one type of target nucleic acid and the sum of the affinity of the receptor to each of the types of ligands contained in the nucleic acid sample, And correcting means for correcting a value that has been, it is intended to provide a quantification device of a nucleic acid, comprising a.
In addition, the present invention provides a nucleic acid quantification apparatus, wherein the measurement means is a means that uses an FCS analysis method or a FIDA analysis method.
In addition, the present invention provides a nucleic acid quantification apparatus, wherein the quantification means is a means that uses an immunoturbidimetric method or an ELISA method.

By using the nucleic acid quantification method of the present invention, even if a receptor with insufficient affinity and specificity for a ligand or a receptor that crosses multiple types of ligands in a nucleic acid sample is used, the immune ratio By the turbidity method or ELISA method, it is possible to detect and quantify a plurality of types of target nucleic acids contained in a nucleic acid sample with high accuracy.
In addition, by using the nucleic acid quantification apparatus of the present invention, it is possible to quantify a plurality of types of target nucleic acids with high throughput and high accuracy.

  The target nucleic acid in the present invention means a nucleic acid having a specific base sequence that is a target for detection and quantification. The target nucleic acid may be naturally derived or synthesized. Moreover, either DNA and RNA may be sufficient and a nucleic acid analog may be sufficient. Examples of the target nucleic acid include genomic DNA, mRNA, hnRNA, synthetic DNA obtained by PCR amplification, cDNA synthesized from RNA using reverse transcriptase, and the like.

  The nucleic acid sample in the present invention is not particularly limited as long as it is a sample containing the target nucleic acid. The nucleic acid sample can be prepared from a biological sample collected from an animal or the like, a cultured cell solution, or the like. A biological sample or the like may be used as it is, or a nucleic acid solution extracted and purified from a biological sample or the like. Further, a biological sample or the like may be amplified by PCR or the like.

  The ligand in the present invention means one used for labeling a target nucleic acid, and is not particularly limited as long as it can be used for labeling a nucleic acid. The ligand may be one usually used for labeling proteins and the like. Examples of the ligand include a fluorescent substance, a hydrophilic organic compound, biotin (biotin), glutathione (Glutathione), DNP (dinitrophenol), digoxigenin, digoxinin, a sugar chain composed of two or more sugars, six or more There are polypeptides consisting of amino acids, auxins, gibberellins, steroids, proteins, and analogs thereof. Further, the ligand may be a compound in which a linker for a nucleic acid is bound in order to facilitate labeling of the nucleic acid.

  Since the detection sensitivity of the target nucleic acid is high, the ligand is preferably a fluorescent substance. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine), Alexa dye series (manufactured by Molecular Probes), Cy dye series (GE Healthcare). Bioscience).

  The ligand is preferably a sugar chain composed of 2 or more sugars or a polypeptide composed of 6 or more amino acids, since it has a high degree of freedom in designing chemical structures and it is easy to prepare a plurality of types. Moreover, since it is already widely used and is easy to obtain and use, it is preferably a His tag, an HA (hem agglutinin) tag, a Myc tag, and a Flag tag. There are many types of these general-purpose tags, but the amino acid sequences of the most widely used tags are shown in Table 1. Further, as a nucleic acid label, only one kind of these general-purpose tags may be used, or a combination of a plurality of kinds may be used.

  The receptor in the present invention means a substance that specifically binds to a ligand, and is not particularly limited as long as it specifically binds to the ligand. However, in the present invention, specifically binding means that it can be specifically bound to such an extent that it can be normally used for detection or purification of the ligand, and crosses with other substances. Also good. Examples of the receptor include an antibody of the ligand and a compound usually used for detection of the ligand.

The ligand antibody may be an unpurified antibody such as serum, or a polyclonal antibody, but is preferably a purified monoclonal antibody from the viewpoint of detection and quantification accuracy. In the nucleic acid quantification method of the present invention, an antibody having a higher affinity for an antigen bound to a protein or the like than the antigen alone may be used in order to bind a ligand bound to a target nucleic acid and a receptor.
The compound usually used for detection of the ligand is, for example, avidin or streptavidin when the ligand is biotin, GST (glutathione-S-transferase) when the ligand is glutathione, Examples of amylose such as maltose include MBP (maltose binding protein).

  By using the receptor exemplified above as a ligand and the ligand exemplified above as a receptor, the target nucleic acid can be detected and quantified. For example, avidin can be used as a ligand and biotin can be used as a receptor. However, the ligand is preferably a low molecular weight compound from the standpoint of easy labeling of nucleic acids.

  In the nucleic acid quantification method of the present invention, first, as step (a), a plurality of types of target nucleic acids contained in a nucleic acid sample are used, using two types of ligands having different combinations for each type of target nucleic acid, Label. By this step, the sequence information of the target nucleic acid is converted into the combination information of the ligand. At present, the types of ligands that can label nucleic acids are extremely limited, but by combining two types of ligands, fewer types of ligands can be used effectively, and more types of target nucleic acids can be detected and detected. Quantification is possible.

  When there are not many types of target nucleic acids contained in the nucleic acid sample, the first ligand common to all types of target nucleic acids and the second ligand that is different in all types of target nucleic acids 2 It is preferable to label each using a type of ligand. This is because correction described later and preparation of necessary reagents and the like are simplified.

  The method for labeling a target nucleic acid with a ligand is not particularly limited. For example, (i) a method of covalently binding a ligand to a base of a target nucleic acid using light or platinum, and (ii) two types of primers labeled with different ligands for one type of target nucleic acid. A method of PCR amplification, (iii) a method of synthesizing a target nucleic acid by PCR amplification or the like using a nucleotide to which a ligand is bound, and (iv) a nucleotide to which a ligand is bound using a terminal deoxyzyltransferase. There is a method of tailing at the 3 ′ end. In the methods (i), (iii), and (iv), one kind of ligand is labeled at a plurality of locations on the target nucleic acid, which is preferable from the viewpoint of detection sensitivity. The methods (ii) and (iii) are preferable because the detection sensitivity of the target nucleic acid is increased by PCR amplification. By using a primer to which a ligand is bound, each ligand is labeled on both ends of the target nucleic acid, so that the binding between each ligand and the receptor is less likely to be inhibited from each other. preferable.

  Next, as a step (b), using a receptor that specifically binds one type of target nucleic acid among a plurality of types to the ligand used for labeling the one type of target nucleic acid in step (a). Detect and quantify. That is, a plurality of types of target nucleic acids present in a nucleic acid sample are detected and quantified one by one. The method of detecting and quantifying is not particularly limited as long as it is a method that is usually used when detecting and quantifying a ligand-labeled substance using a receptor. Alternatively, a method using an ELISA method is preferable. This is because it is already widespread and easy to operate.

When using the immunoturbidimetric method, specifically, one type of target nucleic acid (hereinafter referred to as target nucleic acid 1) out of a plurality of types present in a nucleic acid sample is detected as follows. It can be quantified.
First, dispersible fine particles are prepared by binding a receptor that specifically binds to the ligand used for labeling the target nucleic acid 1 in the step (a). Only one type of receptor is bound to one dispersible fine particle. Therefore, the receptors of the two types of ligands (hereinafter referred to as ligand a and ligand b) used for labeling the target nucleic acid 1 (hereinafter referred to as receptor A and receptor B) are bound to each other. Types of receptor-bound dispersible fine particles (hereinafter referred to as receptor A-bound dispersible fine particles and receptor B-bound dispersible fine particles) are prepared.
The dispersible fine particles used in the present invention are not particularly limited as long as they are fine particles that can be dispersed in a solution, but latex particles made of polystyrene copolymerized with methacrylic acid are preferable in order to improve hydrophilicity and dispersibility. . In order to measure turbidity with a fully automatic immunoturbidimetric apparatus, the particle diameter of the dispersible fine particles is preferably 300 nm or less.

A method of binding a receptor to dispersible fine particles is widely used, and a method suitable for each type of dispersible fine particles can be used. For example, in the case of a plain type latex particle having no functional group on the surface, the receptor is adsorbed and bound to the latex particle by simply mixing the receptor and the latex particle. This is because the surface charge of the plain type latex particles is negatively charged due to methacrylic acid, and can be ionically bonded to a region having a positive charge of the receptor. However, there is a risk that the bound receptor is released, and it is generally considered that the sensitivity is low.
On the other hand, in the case of a functional group type latex particle designed so that a carboxyl group, an amino group or the like is exposed on the surface, a receptor can be bound by a method suitable for each functional group. For example, a method of bonding carboxylic acids with water-soluble carbodiimide known as EDAC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) method, EDC (1-ethyl-3- A typical method is a method in which (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are mixed in advance to bond a carboxylic acid and an amino group. In addition, there are a method of cross-linking amino groups using a linker having NHS bipolar, a method of binding a receptor with an activated aldehyde group or a tosyl group, and the like. In particular, it is preferable to bind the receptor using the EDAC method.

  Next, a suspension of the two types of receptor-bound dispersible fine particles prepared is added to and mixed with a nucleic acid sample containing a plurality of types of target nucleic acids labeled with a ligand in step (a). Since the target nucleic acid 1 binds to both of the two types of receptor-bound dispersible fine particles via the ligand, the dispersible fine particles aggregate. For example, when labeling the target nucleic acid 1 in step (a) is performed by PCR amplification using a primer bound with ligand a and a primer bound with ligand b, at one end of the target nucleic acid 1, One receptor A-binding dispersible fine particle is bonded to the receptor a via the ligand a, and one receptor B-bonding dispersible fine particle is bonded to the other end via the ligand b at the other end. Therefore, the larger the amount of the target nucleic acid 1 contained in the nucleic acid sample, the more aggregates are generated. In addition, since a plurality of receptors are usually bound to each receptor-bound / dispersible fine particle, the receptor-bound / dispersible fine particle and the target nucleic acid 1 are bound in a chain reaction to form a large aggregate.

  In order to prevent uneven binding of the ligand and the receptor, the mixing of the nucleic acid sample containing the target nucleic acid 1 and the receptor-bound dispersible fine particle suspension can be performed by using a vortex mixer or pipetting. It is preferable to carry out immediately, and it is preferable that the volume of each of the nucleic acid sample containing the target nucleic acid 1 and the receptor-bound dispersible fine particle suspension is not less than 20% of the other volume. The buffer composition of the receptor-bound dispersible fine particle suspension is preferably the same as that of the nucleic acid sample buffer, but if different, the receptor-bound dispersible fine particle is the same as the nucleic acid sample before mixing. It is preferable that the composition of the buffer of the receptor-bound dispersible fine particle suspension is the same as that of the nucleic acid sample by washing 2 to 3 times with the above buffer. The mixing of the nucleic acid sample containing the target nucleic acid 1 and the receptor-binding dispersible fine particle suspension can be performed under conditions suitable for the binding between the ligand and the receptor used. For example, when the ligand is an antigen and the receptor is an antibody, they can be mixed by reacting at 0 to 37 ° C. for 5 to 30 minutes.

  Next, using a spectrophotometer, the absorbance of the mixture of the nucleic acid sample containing the target nucleic acid 1 and the receptor-bound dispersible fine particle suspension is measured. When dispersible fine particles having a particle size of 300 nm or less are aggregated, the agglomerates enhance near-infrared light scattering. By measuring the luminous intensity of transmitted light with respect to near-infrared input light, The degree of aggregation of the dispersible fine particles can be measured. That is, the greater the amount of agglomerates present in the mixture, the greater the absorbance of the mixture at near infrared wavelengths. Since the amount of aggregates present in the mixed solution depends on the amount of target nucleic acid 1 present in the mixed solution, the higher the concentration of the target nucleic acid 1 in the mixed solution, the higher the absorbance of the mixed solution. Will grow. Therefore, the absorbance obtained by measuring the absorbance of the mixed solution and the calibration curve prepared by measuring the absorbance of a solution containing only the target nucleic acid 1 labeled with a ligand at each concentration, are present in the nucleic acid sample. The target nucleic acid 1 can be detected and quantified.

  Since the calibration curve mainly depends on the affinity and specificity of the ligand and the receptor, a calibration curve prepared using a nucleic acid other than the target nucleic acid 1 labeled with the same type of ligand as the target nucleic acid 1 is used. Can also be used. In addition, when a calibration curve prepared using the same type of ligand as the target nucleic acid 1 and the same type of receptor as that used for detection of the target nucleic acid 1 has already been prepared, the calibration curve is used. can do. However, it is preferable not to use a calibration curve using receptors or the like having different production lots when the receptor or the ligand has a large difference between production lots such as antibodies.

  As the buffer for measuring the absorbance, a commonly used buffer such as a phosphate buffer or TE buffer can be used. Further, for example, the OD value is set so that the absorbance of the mixed solution of the nucleic acid sample containing the target nucleic acid 1 and the receptor-bound dispersible fine particle suspension falls within an appropriate range of the dynamic range of the spectrophotometer in the OD value measurement. It is preferable to adjust the concentration of the receptor-bound dispersible fine particle suspension so as to be 0.01 to 1.

When using the ELISA method, specifically, the target nucleic acid 1 present in the nucleic acid sample can be detected and quantified as follows.
First, a receptor that specifically binds to any one of the two types of ligands used for labeling the target nucleic acid 1 is immobilized on the base.
The substrate material to which the receptor is immobilized is not particularly limited as long as it adsorbs protein, but polystyrene is preferable. This is because the amount of protein adsorbed is large, inexpensive, and frequently used. Moreover, it is preferable that it is a base | substrate with the active group couple | bonded with protein. Examples of the active group include an SH group, a succinimide group, and an NHS group. The shape of the substrate is not particularly limited, but a 96-well microplate is preferable from the viewpoint of operability. This is because by fixing the receptor near the bottom of each well, operations such as B / F separation and washing can be simplified.

  The method for fixing the receptor to the substrate is appropriately selected according to the type of the receptor and the substrate. For example, in a temperature range of 4 to 25 ° C., 0.1 μg to 1 mg of the receptor can be fixed to the substrate by contacting the substrate with the substrate for 1 second to 24 hours. After fixing the receptor, the substrate is preferably washed with a buffer to remove the non-immobilized receptor, and then the substrate surface on which the receptor is not fixed is blocked with a protein or the like. As the buffer, a commonly used buffer such as a phosphate buffer or a TE buffer can be used. The protein and the like are not particularly limited as long as they do not react with the receptor or ligand used in the present invention, but BSA (bovine serum albumin) is preferable.

  Next, a target nucleic acid is bound to a receptor immobilized on the substrate via a ligand. Specifically, by adding a nucleic acid sample to the substrate, the target nucleic acid 1 and the receptor in the nucleic acid sample can be brought into contact with each other to bind the receptor and the target nucleic acid. Conditions such as buffer, temperature, and time for binding the receptor and the target nucleic acid are appropriately selected according to the type of the receptor and the ligand. For example, in the temperature range of 4-25 ° C., the nucleic acid sample is contacted with the substrate for 1 second to 24 hours, and then the substrate is washed with a buffer to remove unbound nucleic acid, thereby allowing the receptor and target to be removed. Nucleic acids can be bound. For quantification, the amount of the target nucleic acid 1 present in the nucleic acid sample is preferably smaller than the amount of the receptor immobilized on the substrate, so the concentration of the nucleic acid sample added to the substrate is adjusted by dilution or the like. It is preferable.

Next, using the modified receptor modified with the detection substance, the target nucleic acid bound to the receptor immobilized on the substrate is detected and quantified. The modified receptor is for detecting a receptor of a type different from the receptor immobilized on the substrate among the two types of receptors that specifically bind to each of the two types of ligands labeled with the target nucleic acid. It is modified with a substance. The detection substance is, for example, an enzyme or a fluorescent substance. The enzyme and the like are not particularly limited as long as they are usually used in biochemical techniques, but the enzyme is preferably alkaline phosphatase (AP) or horseradish peroxidase (HRP), and the fluorescent substance is FITC. Rhodamine is preferred. However, when the ligand labeled with the target nucleic acid is a fluorescent substance, it is preferably modified with an enzyme.
Specifically, by adding the modified receptor to the substrate, the target nucleic acid bound to the receptor immobilized on the substrate is bound to the modified receptor via a ligand, and then the substrate is bonded to the substrate. Wash with buffer to remove the unbound modified receptor. After that, according to the detection substance used for modification, the enzyme activity and fluorescence intensity are measured appropriately, and the target nucleic acid is detected and quantified by using a calibration curve prepared in advance as in the case of immunoturbidimetry. To do. The conditions such as buffer, temperature, and time when adding the modified receptor to the substrate are appropriately selected according to the kind of the receptor and the ligand, as in the case of binding the receptor and the target nucleic acid. The In addition, conditions and signal readers for detection, such as selection of a substrate for measuring enzyme activity and selection of an excitation wavelength for measuring fluorescence intensity, are appropriately selected according to the substance to be detected.

Next, as step (c), the affinity of the receptor used for detection in step (b) to each of the various types of ligands used in step (a) is obtained by step (b). Correct the value.
In addition to ligands that specifically bind to the receptor, many receptors bind to other ligands having a three-dimensional structure similar to the ligand, or substances that are not three-dimensionally similar to the ligand May bind nonspecifically. Because of such crossing property, if the value obtained in step (b) is used as it is, it will be calculated more apparently than the actual amount of target nucleic acid. Even when using a receptor having a target, the target nucleic acid can be more accurately quantified.

  Specifically, the affinity of the receptor used for detection in step (b) to the ligand used for labeling the one type of target nucleic acid in step (a), and step (a) of the receptor The value obtained in the step (b) is corrected using the ratio of the affinity to each of the various types of ligands used in step (b). The affinity of the receptor for the ligand is preferably the reciprocal of the Kd value because it is widely used for expressing the affinity of the two substances and the measurement is simple.

For example, in a nucleic acid sample in which target nucleic acid 1 labeled with ligand a and ligand b, target nucleic acid 2 labeled with ligand c and ligand d, and target nucleic acid 3 labeled with ligand e and ligand f are present. When the target nucleic acid 1 is quantified by the immunoturbidimetric method after adding the receptor A-binding dispersible fine particles and the receptor B-binding dispersible fine particles, the generated aggregates are the target nucleic acid 1 and the dispersible fine particles. In addition to these, there are those composed of the target nucleic acid 2 and dispersible fine particles, and those composed of the target nucleic acid 3 and dispersible fine particles. Here, the affinity of the receptor A for the ligand a, that is, the reciprocal of the Kd value is [Kd (Aa)] ⁻¹ , the reciprocal of the Kd value for the ligand b is [Kd (Ab)] ⁻¹ , and the Kd value for the ligand c is The reciprocal is [Kd (Ac)] ⁻¹ , the reciprocal of the Kd value for ligand d is [Kd (Ad)] ⁻¹ , the reciprocal of the Kd value for ligand e is [Kd (Ae)] ⁻¹ , and the Kd value for ligand f [Kd (Af)] ⁻¹ , the reciprocal of the Kd value for the ligand a of the receptor B is [Kd (Ba)] ⁻¹ , and the reciprocal of the Kd value for the ligand b is [Kd (Bb)] ^−1. , The reciprocal of the Kd value for the ligand c is [Kd (Bc)] ⁻¹ , the reciprocal of the Kd value for the ligand d is [Kd (Bd)] ⁻¹ , and the reciprocal of the Kd value for the ligand e is [Kd (Be)] ^{− 1,} the inverse of the Kd values for the ligand f [Kd (B )] When ^-1, the value represented by the following formula (1) as the correction coefficient, to correct over the value of the concentration of the target nucleic acid 1 obtained from the actual absorbance the correction factor in step (b) Thus, the target nucleic acid 1 can be quantified more accurately.

{[Kd (Aa)] ⁻¹ + [Kd (Ab)] ⁻¹ } / {[Kd (Aa)] ⁻¹ + [Kd (Ab)] ⁻¹ + [Kd (Ac)] ⁻¹ + [Kd (Ad)] ⁻¹ + [Kd (Ae)] ⁻¹ + [Kd (Af)] ⁻¹ } + {[Kd (Ba)] ⁻¹ + [Kd (Bb)] ⁻¹ } / {[Kd ( Ba)] ⁻¹ + [Kd (Bb)] ⁻¹ + [Kd (Bc)] ⁻¹ + [Kd (Bd)] ⁻¹ + [Kd (Be)] ⁻¹ + [Kd (Bf)] ⁻¹ }
... (1)

  Using two or more types of target nucleic acids contained in a nucleic acid sample, a first ligand common to all types of target nucleic acids and a second ligand that is different in all types of target nucleic acids When each is labeled, among the two types of receptors used for detection in step (b), the affinity of the receptor that specifically binds to the second ligand to the second ligand Using the ratio of the receptor that specifically binds to the second ligand to the sum of the affinity for each of all types of ligands other than the first ligand present in the nucleic acid sample, The value obtained by (b) can be corrected. This is because a receptor that specifically binds to the first ligand is commonly used in detection and quantification of all types of target nucleic acids. That is, since all types of target nucleic acids are labeled with the first ligand, the affinity of the receptor that specifically binds to the first ligand to each type of target nucleic acid is approximately equal, It can be considered that the receptors that specifically bind to the respective second ligands have substantially the same affinity for each type of target nucleic acid via the first ligand. Therefore, in the correction, the affinity of the receptor that specifically binds to the first ligand to the ligand other than the first ligand, and the receptor other than the receptor that specifically binds to the first ligand. This is because the affinity for the first ligand is assumed to be negligibly small.

  For example, a nucleic acid sample containing target nucleic acid 1 labeled with ligand a and ligand b, target nucleic acid 2 labeled with ligand a and ligand c, and target nucleic acid 3 labeled with ligand a and ligand d When the target nucleic acid 1 is quantified by immunoturbidimetry by adding receptor A-binding dispersible fine particles and receptor B-binding dispersible fine particles, the value represented by the following formula (2) is corrected. The target nucleic acid 1 can be quantified more accurately by using the coefficient and correcting the correction coefficient by multiplying the value of the concentration of the target nucleic acid 1 obtained from the actual absorbance in the step (b).

[Kd (Bb)] ⁻¹ / {[Kd (Bb)] ⁻¹ + [Kd (Bc)] ⁻¹ + [Kd (Bd)] ⁻¹ } (2)

  In order to use it for the correction in the step (c), it is preferable to measure the affinity of the receptor used for the detection in the step (b) with respect to each type of ligand contained in the nucleic acid sample. The method for calculating the affinity of the receptor for the ligand, that is, the reciprocal of the Kd value of the receptor and the ligand is not particularly limited, and can be determined by a conventional method. Examples of the method include a method of analyzing a fluorescent signal and a method of analyzing scattered light. The method of analyzing the scattered light is a method of irradiating the substance with light and analyzing the fluctuation of the scattering, and the change in the size of the substance can be monitored even without fluorescent labeling. Since detection sensitivity is good, a method of analyzing a fluorescent signal is preferable. Since analysis per molecule is possible, the FCS analysis method (for example, refer to JP-A-2001-272404) and the FIDA analysis method are particularly preferable.

When the Kd values of the receptor and the ligand are measured using the FCS analysis method, first, one molecule of the ligand is bound to the 5 ′ end of one base chain, and the 5 ′ of the other base chain. A double-stranded nucleic acid is prepared in which one molecule of fluorescent substance is bonded to the end.
The double-stranded nucleic acid can be obtained, for example, by PCR amplification using a primer having the ligand bound to the 5 ′ end and a primer having the fluorescent substance bound to the 5 ′ end, It can be prepared by hybridizing and annealing a synthetic single-stranded nucleic acid in which one molecule of ligand is bound and a synthetic single-stranded nucleic acid in which one molecule of the fluorescent substance is bound to the 5 ′ end. . The produced double-stranded nucleic acid is preferably purified and confirmed by mass spectrometry to be a double-stranded nucleic acid in which 99% or more of the ligand and the fluorescent substance are bound to each other.

  The fluorescent substance is not particularly limited as long as it is usually used in a method for analyzing a fluorescent signal. Examples of the fluorescent material include rhodamine green and TAMRA. However, in the case where the ligand is a fluorescent material, it is preferable to use a fluorescent material that does not interfere with the wavelength of the ligand. For example, when the ligand is Alexa546 (manufactured by Molecular Probes), it is preferable to use Alexa647 (manufactured by Molecular Probes) or the like having a longer absorption wavelength than the ligand.

  Next, a mixed solution is prepared by mixing a solution containing a certain amount of the receptor and a solution containing the double-stranded nucleic acid at each concentration. By this mixing, the receptor and the double-stranded nucleic acid are bound. As the buffer of the mixed solution, a commonly used one such as a phosphate buffer or TBS (Tris buffered saline) can be used. Conditions such as the mixing temperature and time can be appropriately selected according to the receptor and the ligand. For example, the receptor and the double-stranded nucleic acid can be bound by mixing at room temperature for 30 minutes or mixing at 4 ° C. overnight.

  The translational diffusion time and the degree of fluorescence polarization are measured by analyzing the fluorescence signal of each mixed solution. The calculated diffusion time and fluorescence polarization degree vary depending on the concentration of the double-stranded nucleic acid. For example, when the concentration of the double-stranded nucleic acid is low, since the proportion of the receptor bound to the double-stranded nucleic acid is small, the average translational diffusion time is short and the degree of fluorescence polarization is small. On the other hand, as the concentration of the double-stranded nucleic acid increases, the proportion of the receptor bound to the double-stranded nucleic acid increases, so that the average translational diffusion time increases and the degree of fluorescence polarization increases. Thus, a calibration curve is prepared by plotting the translational diffusion time and fluorescence polarization degree at various concentrations of the double-stranded nucleic acid, with the translational diffusion time and the like as the vertical axis and the double-stranded nucleic acid concentration as the horizontal axis. You can also.

Specifically, first, the mixed solution is irradiated with excitation light capable of exciting the fluorescent material, and fluorescence from the fluorescent material is detected. The optical system used for detection may be, for example, a normal optical system having a detector for detecting fluorescence. A laser or the like can be used as a light source for exciting the fluorescent material, and the wavelength may be any wavelength from ultraviolet to visible and infrared. The excitation light is narrowed down through the objective lens and irradiated to the mixed solution. Fluorescence from the fluorescent material is collected by a condenser lens, and noise is removed by a pinhole. Only the fluorescence of a specific wavelength can be taken out by passing through the optical filter. The fluorescence is detected by a detector and signal analysis is performed. An autocorrelation function analysis is performed based on the detected fluorescence, and the time (translational diffusion time) in which one molecule of fluorescent substance stays in the confocal region can be calculated.
In the autocorrelation function analysis, a change in size can be monitored, and a change in the size of the molecule due to molecular interaction or decomposition can be detected. That is, when the receptor in the mixed solution binds to the double-stranded nucleic acid via the ligand, the molecular weight increases. Therefore, using the fluorescence from the fluorescent substance as an indicator, the receptor in the mixed solution And the binding ratio of the double-stranded nucleic acid can be measured.
Assuming that the binding between the ligand and the receptor follows the Michaelis-Menten equation, the translational diffusion time is divided by the vertical axis, and the translational diffusion time (μsec) is divided by the concentration (nM) of the labeled PCR amplified target nucleic acid solution. When the Eadie-Hofste plot is performed on the horizontal axis, the slope of the approximate straight line of the plot becomes the -Kd value of each labeled PCR amplified target nucleic acid. This is because the Km value corresponds to the Kd value.

On the other hand, similarly to the FCS analysis method, a mixed solution of a solution containing a certain amount of the receptor and a solution containing the double-stranded nucleic acid at each concentration is prepared, and the fluorescence signal of each mixed solution is calculated using the FIDA analysis method. Can also be used to measure the Kd values of the receptor and the ligand.
The fluorescence intensity and the number of molecules per molecule can be determined by the FIDA analysis method. Furthermore, the rotational diffusion state of the molecule can be expressed by the degree of fluorescence polarization P (FIDA-pol). Since the fluorescence polarization degree P reflects the size of the molecule, it is possible to know the change in the size due to the interaction or decomposition of the molecule as in the autocorrelation function analysis. Therefore, similar to the FCS analysis method, the Kd values of the receptor and the ligand are calculated from the slope of the approximate straight line created by plotting the Eadee-Hofstee using the fluorescence polarization degree P at various concentrations of the double-stranded nucleic acid. Can be calculated.

  For the FCS analysis method and the FIDA analysis method, the apparatus used for the excitation of the fluorescent substance, the detection of the fluorescence, and the FCS / FIDA analysis is a commercially available single molecule fluorescence analysis system such as the MF20 / MF10S apparatus (Olympus Corporation). Can be done using.

  The nucleic acid quantification method of the present invention is separately performed for each type of target nucleic acid contained in the nucleic acid sample, thereby quantifying all types of target nucleic acids contained in the nucleic acid sample with high accuracy. Can do.

  The nucleic acid quantification apparatus of the present invention is an apparatus used in the nucleic acid quantification method of the present invention, wherein one type of target nucleic acid among a plurality of types of target nucleic acids is used as a ligand used for labeling the one type of target nucleic acid. Quantitative means for detecting and quantifying using a receptor that specifically binds to the receptor, and inputting the affinity of the receptor for each type of ligand contained in the nucleic acid sample A ratio of the affinity of the receptor to the one type of target nucleic acid and the sum of the affinity of the receptor to each type of ligand contained in the nucleic acid sample. And a correcting means for correcting the value obtained by the quantifying means. If the affinity of each of the receptors for each ligand is unknown in advance, the affinity of each of the receptors for each type of ligand contained in the nucleic acid sample before the input means. And measuring means for measuring. By using the nucleic acid quantification apparatus of the present invention, it is possible to quickly and easily quantify a target nucleic acid with high accuracy for a plurality of types of target nucleic acids in a nucleic acid sample.

As the quantification means, a widely used immunoturbidimetric measuring apparatus and immunoassay apparatus can be used. As the input means and the correction means, it can be performed by a method, but it is preferable to use a general-purpose personal computer or the like. By using an automatic analyzer that connects all of the quantification means, the input means, and the correction means to automate all the steps, the affinity value for each ligand of each receptor is input and a nucleic acid sample to be measured is obtained. By simply setting in the analyzer, high-precision target nucleic acid quantification can be performed easily and with high throughput.
A commercially available single-molecule fluorescence analysis system can be used as the measuring means.
In addition, a sample used for preparing a calibration curve for measuring the affinity of each receptor for each ligand by using an automatic analyzer in which the measurement means such as a single molecule fluorescence analysis system is incorporated in the automatic analyzer. By simply setting each nucleic acid sample to be measured in the analyzer, the target nucleic acid can be quantified more rapidly and simply.

  FIG. 1 shows one embodiment of the nucleic acid quantification apparatus of the present invention. In this embodiment, an immunoturbidimetric apparatus is used as the quantification means, a personal computer is used as the input means, and an FCS measurement apparatus is used as the measurement means. Moreover, the flowchart of the procedure in this nucleic acid quantification apparatus is shown in FIG. The nucleic acid quantification apparatus of the present invention is not limited to this one aspect.

Specifically, first, calibration curve data created using the same type of ligand as the target nucleic acid for quantification and the same type of receptor as the receptor used to detect the target nucleic acid is stored. (Step 1). When the calibration curve data is not saved, the creation of the calibration curve is started. When the calibration curve data is saved, the creation of the calibration curve can be omitted.
Next, a ligand-labeled target nucleic acid solution having a known concentration and containing only the target nucleic acid for quantification is prepared as a calibration curve preparation solution (step 2). Each of the prepared ligand-labeled target nucleic acid solutions and receptor-binding fine particles are mixed to prepare a mixed solution in which an aggregate is formed. The measurement cell 2 containing the mixed solution is set in the turbidity measuring instrument 1, and A800 (absorbance at 800 nm) of the mixed solution is measured (step 3). The obtained measurement result is input to the computer 3 to create a calibration curve (step 4). The calibration curve may be created in the computer 3 or by a conventional method. The created calibration curve data is input to the computer 3 and stored. If there are other target nucleic acids for which quantitative measurement is performed simultaneously and there are target nucleic acids for which calibration curve data created using the same type of ligand and receptor as the target nucleic acid are not stored, repeat this procedure. Finally, calibration curve data is similarly stored for all types of target nucleic acids to be quantified (step 5).

Next, it is determined whether or not the Kd value measured using the same type of ligand as the target nucleic acid for quantification and the same type of receptor as that used for quantification of the target nucleic acid is stored (step) 6). When the Kd value is not stored, the measurement of the Kd value is started. When the Kd value is stored, the measurement of the Kd value can be omitted.
In order to measure the Kd value, a receptor 7 used for quantification of a target nucleic acid and a ligand-labeled target nucleic acid solution of each concentration used for preparing a calibration curve were mixed to produce an agglomerate 7. Is adjusted (step 7). After the sample 7 is placed in the measurement container 6, the measurement container 6 is set at a predetermined position in the dark room 5 of the FCS measurement device 4, and the fluorescence of the sample 7 is measured by the objective lens 8 and the CCD camera 9. , FCS measurement is performed, and FCS measurement data is input to the computer 10 (step 8). By plotting the FCS measurement data on the Eadee-Hofstee (step 9), the inclination (−Kd value) of each approximate line is calculated (step 10), and input to the computer 3 and stored (step 11). Thereafter, the calculated Kd value is substituted into the calculation formula (1) or (2) to calculate each correction coefficient (step 12). The plot, the calculation of the Kd value, the correction coefficient, and the like may be performed within the computer 10 or may be performed using a method. Note that the FCS measurement data may be automatically input to the computer 10 by connecting the FCS measurement device 4 and the computer 10. By connecting the computer 3 and the computer 10, the calculated correction coefficients are calculated. The computer 3 may be automatically input.

Next, in the same manner as when preparing the calibration curve, the ligand-labeled nucleic acid sample and the receptor-binding fine particles are mixed to measure A800 of the mixed solution in which an aggregate is formed, and the measurement result is input to the computer 3 Then, the concentration of the target nucleic acid contained in the nucleic acid sample is estimated based on the already prepared calibration curve. By repeating this procedure, the concentration is similarly estimated for all types of target nucleic acids to be quantified (step 13). Note that the measured A800 may be automatically input to the computer 3 by connecting the turbidity measuring instrument 1 and the computer 3.
Correction is performed by multiplying the estimated concentration of the target nucleic acid contained in the nucleic acid sample by a stored correction coefficient (step 14). The correction may be performed by a manual method, but is preferably performed in the computer 3. When it is performed in the computer 3, the corrected concentration of the target nucleic acid contained in the nucleic acid sample can be displayed on the screen of the computer 3 (step 15). The interface may be a single computer, that is, the computer 3 and the computer 10 may be the same computer.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

  A nucleic acid sample containing a total of three types of target nucleic acids: target nucleic acid 1 having the base sequence of SEQ ID NO: 1, target nucleic acid 2 having the base sequence of SEQ ID NO: 2, and target nucleic acid 3 having the base sequence of SEQ ID NO: 3. The three types of target nucleic acids were respectively detected and quantified using an immunoturbidimetric method. The three types of target nucleic acids are human genome-derived base sequences, the target nucleic acid 1 is the accession number IMS-JST164838, the target nucleic acid 2 is the accession number IMS-JST058048, and the target nucleic acid 2 is the accession number IMS. -It is registered in the gene polymorphism database (Japan Single Nucleotide Polymorphism database, JSNP) as JST005689.

1. Labeling of a plurality of types of target nucleic acids in the nucleic acid sample The three types of target nucleic acids in the nucleic acid sample were labeled using two types of ligands having different combinations for each type of target nucleic acid. Specifically, two types of ligands consisting of a first ligand (biotin) common to all types of target nucleic acids and a second ligand (DNP, FITC, and TAMRA) that are different in all types of target nucleic acids. Used to label each.

Using the target nucleic acid 1 as a template, the forward primer 1 having the base sequence of SEQ ID NO: 4 and the reverse primer 1 having the base sequence of SEQ ID NO: 5 that can be PCR-amplified with the target nucleic acid 1 are respectively modified with a ligand. Thus, a 5 ′ biotinylated forward primer 1 in which biotin was bound to the 5 ′ end and a 5 ′ DNP reverse primer 1 in which DNP was bound to the 5 ′ end were prepared.
The forward primer 2 having the base sequence of SEQ ID NO: 6 and the reverse primer 2 having the base sequence of SEQ ID NO: 7 that can be PCR-amplified using the target nucleic acid 2 as a template are each modified with a ligand. Thus, 5 ′ biotinylated forward primer 2 in which biotin was bound to the 5 ′ end and 5 ′ FITC-conjugated reverse primer 2 in which FITC was bound to the 5 ′ end were prepared.
Using the target nucleic acid 3 as a template, the forward primer 3 having the base sequence of SEQ ID NO: 8 and the reverse primer 3 having the base sequence of SEQ ID NO: 9 that can amplify the target nucleic acid 3 are each modified with a ligand. Thus, a 5 ′ biotinylated forward primer 3 in which biotin was bound to the 5 ′ end and a 5 ′ TAMRA-conjugated reverse primer 3 in which TAMRA was bound to the 5 ′ end were prepared.

  Next, multiplex PCR was performed to label the three types of target nucleic acids with ligands using the nucleic acid sample as a template. Specifically, 2 ng of 5 ′ biotinylated forward primer 1, 5 ′ DNP reverse primer 1, 5 ′ biotinylated forward primer 2, 5 ′ FITC was added to 10 μL of 10 × Buffer (manufactured by TaKaRa). Reverse primer 2, 5 ′ biotinylated forward primer 3, and 5 ′ TAMRA reverse primer 3 each to a final concentration of 0.4 μM and dNTP mix (manufactured by GE Healthcare Biosciences) to a final concentration of 0.2 mM. And a nucleic acid sample for labeling was prepared so that the final volume was 98 μL. 2 μL of Titanium Taq DNA polymerase (manufactured by TaKaRa) is added to the nucleic acid sample for labeling, denaturation at 94 ° C. for 2 minutes using a thermal cycler DNA Engine RTC-200 (manufactured by MJ Japan), and then 94 ° C. Multiplex PCR was performed under reaction conditions consisting of 25 cycles of 30 seconds at 68 ° C. for 30 seconds and finally extension at 68 ° C. for 2 minutes, and then the primers were removed. In the obtained labeled nucleic acid sample, a piece 5′-end biotin label-piece 5′-end DNP-labeled PCR amplified target nucleic acid 1, piece 5′-end biotin-labeled-piece 5′-end FITC-labeled PCR amplified target nucleic acid 2, and Piece 5 'end biotin labeled-Piece 5' end TAMRA labeled PCR amplified target nucleic acid 3 was contained.

2. Detection and quantification of labeled target nucleic acid A 5′-end biotin label contained in the labeled nucleic acid sample-a 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 is a receptor that specifically binds to biotin. Detection and quantification by immunoturbidimetry using goat anti-biotin antibody (manufactured by SIGMA) and goat anti-TAMRA antibody (affinity purified product, manufactured by ROCKLAND), which is a receptor that specifically binds to TAMRA did. Specifically, goat anti-biotin antibody-bound dispersible fine particles (hereinafter referred to as anti-biotin particles) and goat anti-TAMRA antibody-bound dispersible fine particles (hereinafter referred to as anti-TAMRA particles) are added to the labeled nucleic acid sample, and turbidity is observed. The degree was measured.

(2-1) Preparation of anti-biotin particles and anti-TAMRA particles First, anti-biotin particles are prepared. A solution prepared to 9 mL by adding water to 1 mL of MES (Wako) buffer (500 mM, pH 6.1) is placed in a 50 mL tube, and 1 mL of 10% CM-MP (300 nm carboxy-type latex particles, Ceradyn). After adding slurry, 8 mL of 1 mg / mL goat anti-biotin antibody phosphate buffer solution was added. The 50 mL-volume tube was mixed for 5 seconds at an intermediate rotational strength of a vortex mixer (VortexGenie 2, manufactured by Scientific Industries), and then 1 mL of 1.152 g / L EDAC (MW = 191.7, manufactured by Sigma) MES. Buffer solution (50 mM, pH 6.1) was added and immediately mixed for 5 seconds at medium rotational strength of a vortex mixer. Thereafter, the 50 mL tube was set in a rotator (MTR-103, manufactured by ASONE), and mixed at room temperature for 1 hour, centrifuged at 15,000 × g for 5 minutes at 4 ° C., and centrifuged CM-MP Centrifugation was stopped at the minimum deceleration condition to prevent soaking up to the supernatant side. After removing the supernatant, 10 mL of MES buffer (50 mM, pH 6.1) is added, and sonication is performed at room temperature for 30 to 4 seconds with a sonicator (VC-130, manufactured by Sonics) for 3 to 4 seconds. The CM-MP mass was dispersed by the above. Again, after the 50 mL tube was centrifuged, 10 mL of MES buffer was added to disperse the CM-MP mass. Then, after leaving still at 16 degreeC for 16 hours, it confirmed that there was no sediment visually. CM-MP dispersed in the MES buffer thus obtained is 1% anti-biotin particle slurry.
A 1% anti-TAMRA particle slurry was prepared in the same manner as the preparation of the 1% anti-biotin particle slurry except that the goat anti-TAMRA antibody was used instead of the goat anti-biotin antibody.

(2-2) Preparation of ligand-labeled nucleic acid with known concentration used for preparation of calibration curve To create a calibration curve, piece 5'-end biotin label-piece 5'-end TAMRA-labeled PCR amplified target nucleic acid 3 to be quantified A solution with a known concentration was prepared. Specifically, except that only the target nucleic acid 3 is used as a template and only the 5 ′ biotinylated forward primer 3 and the 5 ′ TAMRA-modified reverse primer 3 are used as primers, the same as in the case where the target nucleic acid in the nucleic acid sample is labeled. Then, PCR was performed. By this PCR, a solution containing only the piece 5′-end biotin-labeled-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 was obtained. By measuring the nucleic acid concentration of the solution and diluting with a phosphate buffer, the concentration of 1 to 100 nM of the known piece 5 ′ end biotin label-piece 5 ′ end TAMRA labeled PCR amplified target nucleic acid 3 A solution was prepared.

(2-3) Preparation of calibration curve 1 μL of 1% anti-biotin particle slurry and 1 μL of 1% anti-biotin particle slurry were added to 2 μL of each piece of 5′-end biotin labeled at each concentration−5 ′ end TAMRA-labeled PCR amplification target nucleic acid 3 solution. % Anti-TAMRA particle slurry and 16 μL of phosphate buffer were added, mixed using a vortex mixer, and then allowed to stand at room temperature for 5 minutes to form aggregates. Thereafter, 80 μL of a phosphate buffer was added and mixed using a vortex mixer, and then the entire amount was placed in a cell (manufactured by ASONE). The cell was set on a spectrophotometer (SPECTRA max PLUS384, manufactured by Molecular Devices), and A800 (absorbance at 800 nm) was measured. Piece 5 'terminal biotin labeling-Piece 5' terminal TAMRA labeling The concentration (nM) of the PCR amplified target nucleic acid 3 solution is plotted on the horizontal axis and the absorbance is plotted on the vertical axis, and the calibration curve is plotted by plotting the data obtained by the measurement. Created. The prepared calibration curve is shown in FIG. The calibration curve had a slope of 0.0012 and a vertical axis intercept of 0.0276.

(2-4) Quantification of piece 5'-end biotin label-piece 5'-end TAMRA-labeled PCR amplification target nucleic acid 3 in the labeled nucleic acid sample piece 5'-end biotin label-piece 5'-end TAMRA-labeled PCR amplification at each concentration Except that the labeled nucleic acid sample was used instead of the target nucleic acid 3 solution, A800 of the labeled nucleic acid sample was measured in the same manner as in (2-3) above, and was 0.078. From the obtained data and the calibration curve prepared in the above (2-3), as shown in FIG. 4, the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 in the labeled nucleic acid sample Was estimated to be 42 nM. From this concentration, the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 in the labeled nucleic acid sample can be quantified.

3. Measurement of the affinity of the receptor used for quantification of the target nucleic acid with respect to each of all kinds of second ligands (DNP, FITC, and TAMRA) contained in the labeled nucleic acid sample
(3-1) Preparation of ligand-labeled nucleic acid for use in calculating Kd value In order to calculate Kd value by FCS analysis method, Alexa647, which is a fluorescent substance, and target nucleic acid 3 labeled with the second ligand are used. Prepared by PCR amplification.

First, in place of 5 ′ biotinylated forward primer 3, except that 5 ′ Alexa647 forward primer 3 in which Alexa647 was bound to the 5 ′ end of forward primer 3 was used, everything was the same as in the above (2-2). PCR was performed. By the PCR, a solution containing the piece 5′-end Alexa647-labeled piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 was obtained. By measuring the nucleic acid concentration of the solution and diluting with TBS, a solution having a known concentration of 5'-end Alexa647-labeled piece 5'-end TAMRA-labeled PCR amplified target nucleic acid 3 having a concentration of 1 to 100 nM is prepared. did.
Next, in place of 5 ′ TAMRA-modified reverse primer 3, all except that 5′-FITC-modified reverse primer 3 in which FITC was bonded to the 5′-end of reverse primer 3 was used as a piece 5′-end Alexa647-labeled piece 5 In the same manner as for the 'terminal TAMRA-labeled PCR amplified target nucleic acid 3, a solution having a concentration of 1 to 100 nM, a known piece 5'-end Alexa647-labeled piece 5'-end FITC-labeled PCR amplified target nucleic acid 3 solution was prepared.
Furthermore, in place of the 5 ′ TAMRA-modified reverse primer 3, all of them were used except that the 5 ′ DNP-modified reverse primer 3 in which DNP was bound to the 5 ′ end of the reverse primer 3 was used. Similarly to the terminal TAMRA-labeled PCR amplified target nucleic acid 3, a solution having a known concentration of 5'-end Alexa 647-labeled piece 5'-end DNP-labeled PCR amplified target nucleic acid 3 having a concentration of 1 to 100 nM was prepared.

(3-2) Measurement of translational diffusion time or fluorescence polarization degree of labeled PCR amplified target nucleic acid 3 solution at each concentration 10 μL of 0.01 mg / mL goat anti-TAMRA antibody TBS solution and 10 μL of (3-1) above The prepared labeled PCR-amplified target nucleic acid 3 solutions having respective concentrations were mixed and then allowed to stand at 24 ° C. for 30 minutes to form aggregates. Then, this mixed solution was set in MF20 apparatus (Olympus Corporation), excited using the light source of 633 nm He-Ne laser, and the fluorescence of this mixed solution was measured. Thereafter, FCS measurement was performed for 10 seconds for each mixed solution, and the translational diffusion time was calculated. Furthermore, FIDA-pol measurement was performed for 1 second for each mixed solution, and the degree of fluorescence polarization was calculated.

(3-3) Calculation of Kd value The concentration (nM) of the labeled PCR amplification target nucleic acid 3 solution is plotted on the horizontal axis, the translational diffusion time (μsec) is plotted on the vertical axis, and the data obtained in (3-2) is plotted. A calibration curve was created. The prepared calibration curve is shown in FIG. A calibration curve can also be created by plotting the data obtained in (3-2) above with the horizontal axis of the concentration of the labeled PCR amplified target nucleic acid 3 solution and the vertical axis of fluorescence polarization.

  Data obtained in (3-2) above, with the translational diffusion time (μsec) divided by the concentration (nM) of the labeled PCR amplified target nucleic acid 3 solution on the horizontal axis and the translational diffusion time (μsec) on the vertical axis. Was subjected to Eadie-Hofste plot. FIG. 6 shows the result of the plot and the approximate straight line of each labeled PCR amplified target nucleic acid 3 solution.

From the result of FIG. 6, the approximate straight line of the piece 5′-end Alexa647-labeled piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 solution had a slope of −31.9 and a vertical axis intercept of 2120. Similarly, the piece 5′-end Alexa647-labeled piece 5′-end FITC-labeled PCR amplified target nucleic acid 3 solution has an approximate slope of −102, a vertical axis intercept of 1910, and a piece 5′-end Alexa647 labeled. -The approximate straight line of the 5'-end DNP-labeled PCR amplified target nucleic acid 3 solution had a slope of -124 and a vertical axis intercept of 1720.
That is, goat anti-TAMRA antibody and piece 5′-end Alexa647-labeled piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3, piece 5′-end Alexa647-labeled piece 5′-end FITC-labeled PCR amplified target nucleic acid 3, or piece 5 ′ The Kd value (unit: nM) of the terminal Alexa647-labeled-piece 5′-end DNP-labeled PCR amplified target nucleic acid 3 was 31.9, 102, or 124, respectively. That is, the affinity of goat anti-TAMRA antibody for TAMRA, FITC, or DNP (reciprocal of Kd value, unit: 1 / nM) was 0.0313, 0.00980, or 0.00807, respectively.

4). Correction of values obtained by immunoturbidimetric method In this example, three types of target nucleic acids in a nucleic acid sample are divided into a first ligand (TAMRA) common to all types of target nucleic acids and all types of targets. In the quantification of the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 because two kinds of ligands consisting of second ligands (DNP, FITC, and TAMRA) different in nucleic acid are used for labeling. Can be corrected by using the ratio of the affinity of the goat anti-TAMRA antibody to TAMRA and the sum of the affinities of all types of ligands other than TAMRA present in the labeled nucleic acid sample. it can. Here, the sum of the affinity (reciprocal of Kd value) of the goat anti-TAMRA antibody to TAMRA, FITC, or DNP is 0.0492, so the value obtained by dividing 0.0313 by 0.0492 is the correction coefficient. Yes, the correction coefficient can be corrected by multiplying the density obtained in (2-4). Therefore, the true concentration of the strip 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 contained in the labeled nucleic acid sample was 26.7 nM instead of the estimated 42 nM. .

  In place of the goat anti-TAMRA antibody, a goat anti-FITC antibody (manufactured by DAKO) or a goat anti-DNP antibody (manufactured by BETHYL) is used to obtain a piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplification target. Similarly to the nucleic acid 3, the true concentrations of the piece 5′-end biotin label-piece 5′-end FITC-labeled PCR amplified target nucleic acid 1 and piece 5′-end biotin-labeled-piece 5′-end DNP-labeled PCR amplified target nucleic acid 2 are determined. be able to.

In the same manner as in Example 1, using the nucleic acid sample containing the target nucleic acid 1, the target nucleic acid 2, and the target nucleic acid 3, the three types of target nucleic acids were detected and quantified using the ELISA method.
First, in the same manner as in Example 1, three types of target nucleic acids in the nucleic acid sample were labeled by PCR amplification, and a piece 5′-end biotin label-piece 5′-end FITC-labeled PCR amplified target nucleic acid 1, piece 5 ′ A labeled nucleic acid sample containing terminal biotin labeled-piece 5 'end DNP labeled PCR amplified target nucleic acid 2 and piece 5' end biotin labeled-piece 5 'end TAMRA labeled PCR amplified target nucleic acid 3 was obtained.
Further, in the same manner as in Example 1, a known 5′-end biotin-labeled-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 solution having a concentration of 1 to 100 nM for use in preparing a calibration curve. It was adjusted.

  The piece 5'-end biotin label-piece 5'-end TAMRA-labeled PCR amplified target nucleic acid 3 contained in the labeled nucleic acid sample was detected and quantified by ELISA. Specifically, after binding each labeled PCR amplified target nucleic acid to a solid phase coated with avidin via biotin, a goat anti-TAMRA antibody is used to bind the solid 5 ′ end biotin label to the solid phase. -The 5'-end TAMRA labeled PCR amplified target nucleic acid 3 was detected.

First, a calibration curve was created.
Each well of an avidin-coated microplate (Reacti-Bind ^™ Streptavidin HBC plate, manufactured by Pierce) was washed 3 times with 200 μL of Super Block Blocking Buffer 15500 (manufactured by Pierce), and then 100 μL of Super Block Block was added to each of the Block Block Blocks. Furthermore, 10 μL each of the known 5′-end biotin-labeled-piece 5′-end TAMRA-labeled PCR amplification target nucleic acid 3 solution at each concentration of 1 to 100 nM was mixed in one well. After the avidin-coated microplate was allowed to stand at room temperature for 2 hours, the solution in each well was removed, and the plate was washed 3 times with 200 μL of Super Block Blocking Buffer. Thereafter, 100 μL of a goat anti-TAMRA antibody solution in which a goat anti-TAMRA antibody was dissolved in a Super Block Blocking Buffer at 0.2 μg / mL was added to each well. The avidin-coated microplate was allowed to stand at room temperature for 30 minutes, and then the solution in each well was removed and washed with 200 μL of Super Block Blocking Buffer three times. Furthermore, 100 μL each of HRP-labeled anti-goat IgG antibody solution in which HRP-labeled anti-goat IgG antibody (manufactured by Pierce) was dissolved in Super Block Blocking Buffer at 0.2 μg / mL was added to each well at room temperature. Left for 30 minutes. After the avidin-coated microplate was allowed to stand at room temperature for 30 minutes, the solution in each well was removed, washed with 200 μL of Super Block Blocking Buffer three times, and then 100 μL of 1-step Turbo TMB ELISA (manufactured by Pierce) was added to each well. ) And left at room temperature for 15 minutes, and then A450 was measured using a spectrophotometer. Piece 5 'terminal biotin labeling-Piece 5' terminal TAMRA labeling The concentration (nM) of the PCR amplified target nucleic acid 3 solution is plotted on the horizontal axis and the absorbance is plotted on the vertical axis, and the calibration curve is plotted by plotting the data obtained by the measurement. Created. The prepared calibration curve is shown in FIG. The calibration curve had a slope of 0.0098 and a vertical axis intercept of 0.0181.

  Next, all the above except that the labeled nucleic acid sample was used instead of the known 5′-end biotin-labeled-piece 5′-end TAMRA-labeled PCR amplification target nucleic acid 3 solution at concentrations of 1 to 100 nM. When A450 in the labeled nucleic acid sample was measured in the same manner as the calibration curve, it was 0.35. From the obtained data and the prepared calibration curve, as shown in FIG. 8, the concentration of the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 in the labeled nucleic acid sample is 33. Estimated to be 9 nM. From the concentration, the piece 5'-end biotin label-piece 5'-end TAMRA-labeled PCR amplified target nucleic acid 3 in the labeled nucleic acid sample can be quantified.

  Based on the affinity (reciprocal of Kd value) for TAMRA, FITC, or DNP of the goat anti-TAMRA antibody obtained in Example 1 and the correction coefficient, the concentration estimated by the ELISA method was corrected in the same manner as in Example 1. did. As a result, the true concentration of the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 contained in the labeled nucleic acid sample is 21.6 nM instead of the estimated 33.9 nM. Met.

  By using the nucleic acid quantification method and nucleic acid quantification apparatus of the present invention, a plurality of types of target nucleic acids contained in a nucleic acid sample can be detected with high accuracy and rapidly by immunoturbidimetry or ELISA. Therefore, it can be used in fields such as clinical examinations that require extremely high accuracy.

It is the schematic which showed the one aspect | mode of the quantitative determination apparatus of the nucleic acid of this invention. In this embodiment, an immunoturbidimetric apparatus is used as the quantification means, a personal computer is used as the input means, and an FCS measurement apparatus is used as the measurement means. The flowchart of the procedure in the nucleic acid quantification apparatus shown in FIG. 1 is shown. S in the figure means a step. In Example 1, by plotting the data obtained by measurement with the concentration (nM) of the piece 5 ′ end biotin labeled-piece 5 ′ end TAMRA labeled PCR amplified target nucleic acid 3 solution on the horizontal axis and the absorbance on the vertical axis It is a created calibration curve. In Example 1, the outline | summary of the calculation method of the density | concentration of the piece 5 'terminal biotin label | marker-piece 5' terminal TAMRA label | marker PCR amplification target nucleic acid 3 estimated when A800 is 0.078 is shown. . In Example 1, it is a calibration curve created by plotting data obtained by measurement with the concentration (nM) of the labeled PCR amplification target nucleic acid 3 solution as the horizontal axis and the translational diffusion time (μsec) as the vertical axis. . In Example 1, assuming that the binding between each labeled PCR amplified target nucleic acid 3 and the goat anti-TAMRA antibody follows the Michaelis-Menten equation, the translational diffusion time (μsec) is set to the concentration of the labeled PCR amplified target nucleic acid 3 solution (nM ) Is an approximate straight line created by plotting Eadie-Hofstey on the data obtained by measurement, with the horizontal axis representing the value divided by) and the vertical axis representing the translational diffusion time (μsec). However, the horizontal axis is displayed on a logarithmic scale. In Example 2, by plotting the data obtained by measurement with the concentration (nM) of the piece 5′-end biotin label-piece 5′-end TAMRA-labeled PCR amplified target nucleic acid 3 solution on the horizontal axis and the absorbance on the vertical axis It is a created calibration curve. In Example 2, the outline | summary of the calculation method of the density | concentration of the piece 5 'terminal biotin label | marker-piece 5' terminal TAMRA label | marker PCR amplification target nucleic acid 3 estimated in case A450 is 0.35 is shown. .

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Immunoturbidimetry apparatus, 2 ... Measurement cell, 3 ... Computer, 4 ... FCS measurement apparatus, 5 ... Measurement container, 6 ... Measurement container, 7 ... FCS measurement sample, 8 ... Objective lens, 9 ... CCD Camera, 10 ... computer

Claims (18)

A method for quantifying each of a plurality of types of target nucleic acids contained in a nucleic acid sample,
(A) labeling a plurality of types of target nucleic acids with two types of ligands having different combinations for each type of target nucleic acid;
(B) One type of target nucleic acid among a plurality of types is detected using two types of receptors that specifically bind to the ligand used for labeling the one type of target nucleic acid in step (a). Quantifying the process,
(C) A step of correcting the value obtained in the step (b) using the affinity of the receptor used in the detection in the step (b) with respect to each type of ligand used in the step (a). When,
A method for quantifying a nucleic acid, comprising: A method for quantifying each of a plurality of types of target nucleic acids contained in a nucleic acid sample,
(A) labeling a plurality of types of target nucleic acids with two types of ligands having different combinations for each type of target nucleic acid;
(B) One type of target nucleic acid among a plurality of types is detected using two types of receptors that specifically bind to the ligand used for labeling the one type of target nucleic acid in step (a). Quantifying the process,
(D) The affinity of the receptor used for detection in step (b) to the ligand used for labeling the one type of target nucleic acid in step (a) and the receptor used in step (a) Correcting the value obtained by step (b) using the ratio of the total affinity to each of the obtained types of ligands;
A method for quantifying a nucleic acid, comprising: A method for quantifying each of a plurality of types of target nucleic acids contained in a nucleic acid sample,
(A ′) Labeling a plurality of types of target nucleic acids using a first ligand common to all types of target nucleic acids and two types of ligands consisting of different second ligands in all types of target nucleic acids And a process of
(B) Specifically binding one type of target nucleic acid among a plurality of types to each of the first ligand and the second ligand used for labeling the one type of target nucleic acid in step (a). Detecting and quantifying using two types of receptors,
(D ′) Of the two types of receptors used for detection in step (b), the affinity of the receptor that specifically binds to the second ligand for the second ligand, Using the ratio of the receptor that specifically binds to the ligand to the total affinity of all types of ligands other than the first ligand present in the nucleic acid sample, obtained by step (b). Correcting the obtained value;
A method for quantifying a nucleic acid, comprising: After the step (b), before the step (c), the step (d) or the step (d ′),
(E) measuring the affinity of the receptor for each type of ligand contained in the nucleic acid sample;
The method for quantifying a nucleic acid according to any one of claims 1 to 3, wherein:   The nucleic acid quantification method according to any one of claims 1 to 4, wherein the affinity is an inverse of a Kd value (equilibrium dissociation constant).   6. The method for quantifying a nucleic acid according to claim 4, wherein the step of measuring the affinity is performed using a method of analyzing a fluorescent signal.   The nucleic acid quantification method according to claim 6, wherein the method of analyzing the fluorescence signal is an FCS (Fluorescence Correlation Spectroscopy) analysis method.   The nucleic acid quantification method according to claim 6, wherein the method of analyzing the fluorescence signal is FIDA (Fluorescence Intensity Distribution Analysis) analysis method.   The ligand is a fluorescent substance, a hydrophilic organic compound, biotin (biotin), glutathione, DNP (dinitrophenol), digoxigenin, digoxin, a sugar chain composed of two or more sugars, six or more amino acids A compound selected from the group consisting of polypeptide, auxin, gibberellin, steroid, GST (glutathione-S-transferase), MBP (maltose binding protein), avidin, streptavidin, protein, and analogs thereof The method for quantifying a nucleic acid according to any one of claims 1 to 8.   The ligand is a fluorescent substance, a hydrophilic organic compound, biotin (biotin), glutathione (Glutathione), DNP (dinitrophenol), digoxigenin, digoxin, a sugar chain composed of two or more sugars, six or more amino acids Selected from the group consisting of a polypeptide consisting of auxin, gibberellin, steroid, GST (glutathione-S-transferase), MBP (maltose binding protein), avidin, streptavidin, protein, and analogs thereof, and nucleic acid The method for quantifying a nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid is a compound to which a linker is bound.   The fluorescent substance is FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine), Alexa dye series (manufactured by Molecular Probes), Cy dye series (GE Healthcare Bioscience) The method for quantifying a nucleic acid according to claim 9 or 10, wherein the method is a fluorescent substance selected from the group consisting of:   The method for quantifying a nucleic acid according to claim 9 or 10, wherein the polypeptide comprising 6 or more amino acids is a polypeptide selected from the group consisting of a His tag, an HA tag, a Myc tag, and a Flag tag.   In the step (a), the method of labeling with a ligand is a method of performing PCR (Polymerase Chain Reaction) amplification on one type of target nucleic acid using two types of primers each labeled with a different ligand. The method for quantifying a nucleic acid according to any one of claims 1 to 12, wherein:   The method for quantifying a nucleic acid according to any one of claims 1 to 13, wherein the method of detecting and quantifying in the step (b) is an immunoturbidimetric method or an ELISA method. An apparatus for quantifying each of a plurality of types of target nucleic acids in a nucleic acid sample labeled with two types of ligands having different combinations for each type of target nucleic acid,
Quantification means for detecting and quantifying one type of target nucleic acid among the plurality of types of target nucleic acids using a receptor that specifically binds to a ligand used for labeling the one type of target nucleic acid When,
An input means for inputting the affinity of each of the receptors for each type of ligand contained in the nucleic acid sample;
Using the ratio between the affinity of the receptor for the one type of target nucleic acid and the sum of the affinity of the receptor for each type of ligand contained in the nucleic acid sample, the quantification is performed. Correction means for correcting the value obtained by the means;
A nucleic acid quantification apparatus comprising: An apparatus for quantifying each of a plurality of types of target nucleic acids in a nucleic acid sample labeled with two types of ligands having different combinations for each type of target nucleic acid,
Quantification means for detecting and quantifying one type of target nucleic acid among the plurality of types of target nucleic acids using a receptor that specifically binds to a ligand used for labeling the one type of target nucleic acid When,
A measuring means for measuring the affinity of each of the receptors for each type of ligand contained in the nucleic acid sample;
Input means for inputting the affinity obtained by the measuring means;
Using the ratio between the affinity of the receptor for the one type of target nucleic acid and the sum of the affinity of the receptor for each type of ligand contained in the nucleic acid sample, the quantification is performed. Correction means for correcting the value obtained by the means;
A nucleic acid quantification apparatus comprising:   17. The nucleic acid quantification apparatus according to claim 16, wherein the measuring means is a means that uses an FCS analysis method or a FIDA analysis method.   The nucleic acid quantification apparatus according to any one of claims 15 to 17, wherein the quantification means is a means that uses an immunoturbidimetric method or an ELISA method.

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