JP2004154143A - Method for the separation and purification of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method for nucleic acid fragments capable of simply and rapidly carrying out free from needs of any specific technique, complicated operation and specific device, by using a small device, namely, an analytical method for nucleic acid fragments which is capable of space-saving and automating. <P>SOLUTION: The analytical method comprises following steps. Step (1), a step isolating and purifying nucleic acid fragment containing the target nucleic acid fragment including a step making a nucleic acid adsorbed onto a solid phase composed of an organic high polymer having a hydroxy group on a surface thereof, and desorbed from the solid phase. (2), a step allowing the target nucleic acid fragment, at least one primer complementary to a portion of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one polymerase to react with each other, to conduct polymerase elongation reaction with using the target nucleic acid fragment as a template and using 3' terminal of the primer as initiation site. And (3) a step detecting whether polymerase elongation reaction proceeds or whether the polymerase elongation reaction product hybridizes with another nucleic acid. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to a method for separating and purifying nucleic acids, a nucleic acid separation and purification unit, and a method for analyzing nucleic acids using them.

  Nucleic acids are used in various forms in various fields. For example, in the area of recombinant nucleic acid technology, it is required to use nucleic acids in the form of probes, genomic nucleic acids, and plasmid nucleic acids.

  In the diagnostic field, nucleic acids are used in various ways. For example, nucleic acid probes are routinely used for detection and diagnosis of human pathogens. Similarly, nucleic acids are used to detect genetic disorders. Nucleic acids are also used to detect food contaminants. In addition, nucleic acids are routinely used in the localization, identification and isolation of nucleic acids of interest for a variety of reasons ranging from genetic mapping to cloning and recombinant expression.

  In many cases, nucleic acids are available only in very small amounts, and the isolation and purification operations are cumbersome and time consuming. This often time-consuming and cumbersome operation tends to lead to the loss of nucleic acids. The purification of sample nucleic acids from serum, urine and bacterial cultures also adds the risk of contamination and false positive results.

As one of the widely known purification methods, there is a method in which a nucleic acid is adsorbed on the surface of silicon dioxide, silica polymer, magnesium silicate, etc. and purified by subsequent operations such as washing and desorption (for example, Japanese Patent Publication No. 7-51065). Publication). Although this method is excellent in separation performance, there are problems such as difficulty in industrial mass production of adsorption media having the same performance, inconvenience in handling, and difficulty in processing into various shapes.
Japanese Patent Publication No. 7-51065

  An object of the present invention is to provide a method for separating and purifying a nucleic acid by adsorbing a nucleic acid in a specimen to a solid phase surface and then desorbing the nucleic acid through washing or the like. Another object of the present invention is separation and purification of nucleic acids using a solid phase that has excellent separation performance, good washing efficiency, is easy to process, and can produce a large amount of substantially the same separation performance. It is to provide a method and a nucleic acid separation and purification unit suitable for carrying out the method. Still another object of the present invention is to provide a nucleic acid analysis method using the above-described nucleic acid separation and purification method. Still another object of the present invention is to provide a method for analyzing a nucleic acid fragment that can be carried out easily and quickly using a small apparatus without the need for special techniques, complicated operations, and special apparatuses, i.e., An object of the present invention is to provide a method for analyzing nucleic acid fragments that can be automated in a small space.

  As a result of diligent investigations to solve the above problems, the present inventors have used an organic polymer having a hydroxyl group on the surface as the solid phase in a method for separating and purifying nucleic acid including a process of adsorbing and desorbing nucleic acid on the solid phase. Then, it was found that a nucleic acid with high purity can be separated from a sample solution containing nucleic acid by using a nucleic acid separation and purification unit in which the solid phase is accommodated in a container having two openings. Furthermore, by detecting pyrophosphoric acid generated during the polymerase extension reaction using the nucleic acid separated by the above method using a dry analytical element, it is excellent in simplicity and speed without requiring a special device. It was found that nucleic acid can be analyzed. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a method for separating and purifying nucleic acid, comprising the step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface.
Preferred embodiments of the method for separating and purifying nucleic acids are listed below.
A method for separating and purifying nucleic acid, wherein the organic polymer having a hydroxyl group on the surface is a saponified product of acetylcellulose;
A method for separating and purifying nucleic acid, wherein the organic polymer having a hydroxyl group on the surface is a surface saponified product of triacetylcellulose;
A method for separating and purifying nucleic acid, wherein the surface saponification rate of acetylcellulose is 5% or more;
A method for separating and purifying nucleic acid, wherein the surface saponification rate of acetylcellulose is 10% or more;
A method for separating and purifying nucleic acid, wherein acetylcellulose is a porous membrane;
A method for separating and purifying nucleic acid, wherein acetylcellulose is a non-porous membrane;
A method for separating and purifying nucleic acid, wherein the beads are coated with acetylcellulose;
A method for separating and purifying nucleic acid, wherein a nucleic acid in a sample solution is adsorbed and desorbed on a solid phase comprising an organic polymer having a hydroxyl group on the surface;
A method for separating and purifying nucleic acid, wherein the sample solution is a solution obtained by treating a specimen containing cells or viruses with a nucleic acid solubilizing reagent and adding a water-soluble organic solvent;
A method for separating and purifying nucleic acid, wherein the nucleic acid solubilizing reagent is a guanidine salt, a surfactant and a proteolytic enzyme;
Nucleic acid is adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface, then the solid phase is washed with a nucleic acid washing buffer, and then the solid phase is adsorbed onto the solid phase using a solution capable of desorbing the nucleic acid adsorbed on the solid phase. A method for separating and purifying nucleic acid, comprising a step of desorbing the adsorbed nucleic acid;
A method for separating and purifying nucleic acid, wherein the nucleic acid washing buffer is a solution containing 20 to 100% by weight of methanol, ethanol, isopropanol or n-propanol;
A method for separating and purifying nucleic acid, wherein the solution capable of desorbing the nucleic acid adsorbed on the solid phase is a solution having a salt concentration of 0.5 M or less;
A method for separating and purifying nucleic acid, comprising adsorbing and desorbing nucleic acid using a nucleic acid separation and purification unit containing a solid phase composed of an organic polymer having a hydroxyl group on the surface in a container having at least two openings;
(a) a solid phase composed of an organic polymer having a hydroxyl group on the surface; (b) a container having at least two openings containing the solid phase; and (c) a pressure coupled to one opening of the container. A method for separating and purifying nucleic acid, wherein nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit including a difference generator;
A method for separating and purifying nucleic acid, comprising the following steps.
(a) preparing a sample solution containing a nucleic acid using a specimen, and inserting one opening of the nucleic acid separation and purification unit into the sample solution containing the nucleic acid,
(b) A solid phase composed of an organic polymer having a hydroxyl group on the surface by sucking a sample solution containing nucleic acid by reducing the pressure in the container using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit. Contacting with,
(c) A step of bringing the container into a pressurized state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and discharging the sample solution containing the aspirated nucleic acid out of the container;
(d) inserting one opening of the nucleic acid separation and purification unit into the nucleic acid washing buffer;
(e) Using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, the inside of the container is evacuated and the nucleic acid washing buffer is sucked and contacted with a solid phase composed of an organic polymer having a hydroxyl group on the surface. The process of
(f) a step of bringing the container into a pressurized state using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit, and discharging the aspirated nucleic acid washing buffer to the outside of the container;
(g) inserting one opening of the nucleic acid separation and purification unit into a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface;
(h) Depressurize the inside of the container using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit to desorb the nucleic acid adsorbed on the solid phase composed of an organic polymer having a hydroxyl group on the surface. Aspirating the liquor and bringing it into contact with the solid phase; and
(i) Using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit, pressurize the container to desorb the nucleic acid adsorbed on the solid phase composed of an organic polymer having a hydroxyl group on the surface. Discharging the liquid from the container;
A method for separating and purifying nucleic acid, comprising the following steps.
(a) preparing a sample solution containing nucleic acid using a specimen and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized using the pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening. Contacting with a solid phase comprising an organic polymer having a hydroxyl group on
(c) injecting a nucleic acid washing buffer into the one opening of the nucleic acid separation and purification unit;
(d) The inside of the container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the injected nucleic acid washing buffer is discharged from the other opening, whereby a hydroxyl group is formed on the surface. Contacting with a solid phase comprising an organic polymer having
(e) a step of injecting a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface and discharging it out of the container.
According to another aspect of the present invention, there is provided a nucleic acid separation and purification unit in which a solid phase composed of an organic polymer having a hydroxyl group on the surface thereof is contained in a container having at least two openings.
According to another aspect of the present invention, (a) a solid phase comprising an organic polymer having a hydroxyl group on the surface,
(b) a container having at least two openings for containing the solid phase; and
(c) a pressure difference generator coupled to one opening of the container;
A nucleic acid separation and purification unit is provided.
Preferably, the pressure difference generator is detachably coupled to one opening of the container.
Preferably, the pressure difference generating device is a syringe.
Preferably, the pressure difference generator is a pipettor.
Preferably, the pressure difference generator is a pump.

According to still another aspect of the present invention, there is provided a method for introducing a hydroxyl group into acetyl cellulose, wherein the beads are coated with acetyl cellulose and then surface saponified.
According to still another aspect of the present invention, there is provided a bead having on its surface an acetylcellulose membrane having a hydroxyl group introduced by surface saponification.
According to still another aspect of the present invention, (1) a step of separating and purifying a nucleic acid fragment containing a target nucleic acid fragment by the method of the present invention described above;
(2) The target nucleic acid fragment, at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one polymerase are reacted, and the target nucleic acid fragment is used as a template. Performing a polymerase extension reaction starting from the 3 ′ end of the primer; and
(3) A step of detecting the presence or absence of progress of the polymerase extension reaction or detecting the presence or absence of hybridization between the polymerase extension reaction product and another nucleic acid:
A method for analyzing a nucleic acid is provided.
According to still another aspect of the present invention, (1) a means for extracting and purifying a nucleic acid comprising the above-described nucleic acid separation and purification unit of the present invention, (2) a reaction means for performing a polymerase extension reaction, and (3) A method for analyzing a nucleic acid according to the present invention, comprising: a means for detecting the presence or absence of progress of a polymerase extension reaction, or a means for detecting the presence or absence of hybridization between a polymerase extension reaction product and another nucleic acid. An analytical device is provided.

  According to the method for separating and purifying nucleic acid of the present invention using a solid phase that is excellent in separation performance, good in washing efficiency, easy to process, and capable of mass-producing products having substantially the same separation performance, High purity nucleic acid can be separated from a sample solution containing Furthermore, operation becomes easy if the nucleic acid separation and purification unit of the present invention is used.

Embodiments of the present invention will be described below.
(1) Method for separating and purifying nucleic acid according to the present invention The method for separating and purifying nucleic acid according to the present invention comprises a step of adsorbing and desorbing a nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface.
In the present invention, the “nucleic acid” may be either single-stranded or double-stranded, and there is no molecular weight limitation.

  The organic polymer having a hydroxyl group on the surface is preferably a surface saponified product of acetylcellulose. The acetyl cellulose may be any of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose, but triacetyl cellulose is particularly preferable. In the present invention, surface saponified acetylcellulose is preferably used as the solid phase. Here, the surface saponification means that only the surface in contact with the saponification solution (for example, NaOH) is saponified. In the present invention, it is preferable that the solid phase structure remains acetylcellulose and only the surface of the solid phase is saponified. Thereby, the amount (density) of hydroxyl groups on the surface of the solid phase can be controlled by the degree of surface saponification treatment (surface saponification degree).

  In order to increase the surface area of the organic polymer having a hydroxyl group on the surface, it is preferable to form a film of the organic polymer having a hydroxyl group on the surface. Acetylcellulose may be a porous membrane or a non-porous membrane, but it is more preferable to make the membrane porous. When the solid phase is a porous membrane, it is preferable that the membrane structure remains acetylcellulose and only the surface of the structure is saponified. Thereby, the amount (density) of the spatial hydroxyl group can be controlled by the degree of surface saponification treatment (surface saponification degree) × pore diameter. Further, since the membrane structure is composed of acetylcellulose, a solid solid phase can be obtained. Here, saponifying acetylcellulose to introduce a hydroxyl group only on the surface means that the structure remains as acetylcellulose and the surface is celluloseized. When cellulose is used as a raw material, it cannot be made into a liquid, so that a porous membrane or a flat membrane cannot be produced industrially.

For example, a film of triacetylcellulose is commercially available from Fuji Photo Film under the trade name TAC base, and a microfilter FM500 (manufactured by Fuji Photo Film Co., Ltd.) is available as a porous film of triacetylcellulose.
Further, for example, it is also preferable to form a triacetyl cellulose film on the surface of polyethylene beads and saponify the surface to give a hydroxyl group on the surface. In this case, triacetyl cellulose is coated on the beads. The material of the beads is not limited to polyethylene as long as it does not contaminate the nucleic acid.

In order to increase the nucleic acid separation efficiency, a larger number of hydroxyl groups is preferred. For example, in the case of acetylcellulose such as triacetylcellulose, the surface saponification rate is preferably about 5% or more, more preferably 10% or more.
In order to saponify the surface of acetylcellulose, an object to be saponified is immersed in an aqueous sodium hydroxide solution. In order to change the surface saponification rate, the concentration of sodium hydroxide may be changed. The surface saponification rate is determined by quantifying residual acetyl groups by NMR.

  In the method for separating and purifying nucleic acid of the present invention, preferably, nucleic acid is adsorbed and desorbed using a nucleic acid separation and purification unit containing a solid phase composed of an organic polymer having a hydroxyl group on the surface in a container having at least two openings. It can be performed.

  More preferably, (a) a solid phase composed of an organic polymer having a hydroxyl group on the surface, (b) a container having at least two openings containing the solid phase, and (c) one opening of the container. Nucleic acid can be adsorbed and desorbed using a nucleic acid separation and purification unit including a combined pressure difference generator.

In this case, the first embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) inserting one opening of the nucleic acid separation and purification unit into a sample solution containing nucleic acid;
(b) A solid phase composed of an organic polymer having a hydroxyl group on the surface by sucking a sample solution containing nucleic acid by reducing the pressure in the container using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit. Contacting with,
(c) A step of bringing the container into a pressurized state using a pressure difference generator coupled to the other opening of the nucleic acid separation and purification unit, and discharging the sample solution containing the aspirated nucleic acid out of the container;
(d) inserting one opening of the nucleic acid separation and purification unit into the nucleic acid washing buffer solution;
(e) Using a pressure difference generator connected to another opening of the nucleic acid separation and purification unit, the container is evacuated and the nucleic acid washing buffer solution is sucked into a solid phase composed of an organic polymer having a hydroxyl group on the surface. Contacting,
(f) a step of bringing the container into a pressurized state using a pressure difference generator coupled to another opening of the nucleic acid separation and purification unit, and discharging the aspirated nucleic acid washing buffer solution out of the container;
(g) inserting one opening of the nucleic acid separation and purification unit into a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface;
(h) Depressurize the inside of the container using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit to desorb the nucleic acid adsorbed on the solid phase composed of an organic polymer having a hydroxyl group on the surface. Aspirating the liquor and bringing it into contact with the solid phase; and
(i) Using a pressure difference generator connected to the other opening of the nucleic acid separation and purification unit, pressurize the inside of the container to desorb the nucleic acid adsorbed on the solid phase composed of an organic polymer having a hydroxyl group on the surface. A process of discharging the liquid from the container.

The second embodiment of the method for separating and purifying nucleic acid of the present invention can include the following steps.
(a) preparing a sample solution containing nucleic acid using a specimen and injecting the sample solution containing the nucleic acid into one opening of the nucleic acid separation and purification unit;
(b) The container is pressurized using the pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the sample solution containing the injected nucleic acid is discharged from the other opening. Contacting with a solid phase comprising an organic polymer having a hydroxyl group on
(c) injecting a nucleic acid washing buffer into the one opening of the nucleic acid separation and purification unit;
(d) The inside of the container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and the injected nucleic acid washing buffer is discharged from the other opening, whereby a hydroxyl group is formed on the surface. Contacting with a solid phase comprising an organic polymer having
(e) a step of injecting a liquid capable of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface into the one opening of the nucleic acid separation and purification unit;
(f) The container is pressurized using a pressure difference generator coupled to the one opening of the nucleic acid separation and purification unit, and a liquid capable of desorbing the injected nucleic acid is discharged from the other opening, A step of desorbing nucleic acid adsorbed on a solid phase composed of an organic polymer having a hydroxyl group on the surface and discharging it out of the container.

  The method for separating and purifying nucleic acid using an organic polymer having a hydroxyl group on the surface will be described more specifically. In the present invention, the nucleic acid in the sample solution is preferably adsorbed on the solid phase by bringing the sample solution containing the nucleic acid into contact with the solid phase composed of an organic polymer having a hydroxyl group on the surface, and then adsorbed on the solid phase. Nucleic acids are desorbed from the solid phase using a suitable solution as described below. More preferably, the sample solution containing a nucleic acid is a solution obtained by adding a water-soluble organic solvent to a solution in which a nucleic acid is dispersed in a solution by treating a specimen containing cells or viruses with a solution that dissolves a cell membrane and a nuclear membrane. is there.

  The sample solution containing nucleic acid that can be used in the present invention is not limited, but in the diagnostic field, for example, whole blood collected as a specimen, plasma, serum, urine, stool, semen, saliva and other body fluids, or plants (or their) Particular), animals (or parts thereof), etc., or solutions prepared from biological materials such as lysates and homogenates thereof.

First, these specimens are treated with an aqueous solution containing a reagent that dissolves the cell membrane and solubilizes the nucleic acid. As a result, the cell membrane and the nuclear membrane are dissolved, and the nucleic acid is dispersed in the aqueous solution.
For cell membrane lysis and nucleic acid solubilization, for example, when the target sample is whole blood, (1) removal of red blood cells, (2) removal of various proteins, and (3) lysis of white blood cells and nuclear membranes Must be dissolved. (1) Removal of red blood cells and (2) removal of various proteins should be performed in order to prevent nonspecific adsorption to the solid phase and clogging of the porous membrane. Are required to solubilize the nucleic acids. In particular, (3) lysis of leukocytes and lysis of the nuclear membrane are important steps. In the method of the present invention, it is necessary to solubilize nucleic acids in this step. In the examples described below in the present specification, the above (1), (2) and (2) are obtained by incubating at 60 ° C. for 10 minutes with guanidine hydrochloride, Triton-X100 and protease K (manufactured by SIGMA) added. 3) is achieved at the same time.

Examples of the nucleic acid solubilizing reagent used in the present invention include a solution containing a guanidine salt, a surfactant and a proteolytic enzyme.
As the guanidine salt, guanidine hydrochloride is preferable, but other guanidine salts (guanidine isothiocyanate, guanidine thiocyanate) can also be used. The concentration of the guanidine salt in the solution is 0.5M to 6M, preferably 1M to 5M.

  As the surfactant, Triton-X100 can be used, but in addition to this, an anionic surfactant such as SDS, sodium cholate or sarcosine sodium, a nonionic surfactant such as Tween 20 or MegaFac, Various other amphoteric surfactants can also be used. In the present invention, it is preferable to use a nonionic surfactant such as polyoxyethylene octylphenyl ether (Triton-X100). The concentration of the surfactant in the solution is usually 0.05% to 10% by weight, particularly preferably 0.1% to 5% by weight.

  Protease K can be used as the proteolytic enzyme, but the same effect can be obtained with other proteases. Since protease is an enzyme, it is preferable to warm it, it is preferable to use it at 37 ° C to 70 ° C, particularly 50 ° C to 65 ° C.

  A water-soluble organic solvent is added to the aqueous solution in which nucleic acids are dispersed in this manner, and brought into contact with an organic polymer having a hydroxyl group on the surface. By this operation, the nucleic acid in the sample solution is adsorbed to the organic polymer having a hydroxyl group on the surface. In order to adsorb the nucleic acid solubilized by the operation described above in the present specification to a solid phase composed of an organic polymer having a hydroxyl group on the surface, a water-soluble organic solvent is mixed with the solubilized nucleic acid mixed solution. It is necessary that a salt be present in the obtained nucleic acid mixture.

  That is, by destroying the hydration structure of water molecules present around the nucleic acid, the nucleic acid is solubilized in an unstable state. When nucleic acid in this state is brought into contact with a solid phase composed of an organic polymer having a hydroxyl group on the surface, the polar group on the nucleic acid surface interacts with the polar group on the solid phase surface, and the nucleic acid is adsorbed on the solid surface. It is thought to do. In the method of the present invention, the nucleic acid can be brought into an unstable state by mixing a water-soluble organic solvent with the solubilized nucleic acid mixture and the presence of a salt in the obtained nucleic acid mixture.

  Examples of the water-soluble organic solvent used here include ethanol, isopropanol, and propanol. Among them, ethanol is preferable. The concentration of the water-soluble organic solvent is preferably 5% by weight to 90% by weight, and more preferably 20% by weight to 60% by weight. The concentration of ethanol added is particularly preferably as high as possible without causing pseudo-collects.

  Salts present in the resulting nucleic acid mixture include various chaotropic substances (guanidinium salts, sodium iodide, sodium perchlorate), sodium chloride, potassium chloride, ammonium chloride, sodium bromide, potassium bromide, bromide. Calcium, ammonium bromide and the like are preferred. In particular, a guanidinium salt is particularly preferable because it has the effects of cell membrane lysis and nucleic acid solubilization.

  Next, an organic polymer having a hydroxyl group on the surface to which the nucleic acid has been adsorbed is brought into contact with the nucleic acid washing buffer solution. This solution has a function of washing out impurities in the sample solution adsorbed on the organic polymer having a hydroxyl group on the surface together with the nucleic acid. Therefore, it is necessary to have a composition in which nucleic acids are not desorbed from an organic polymer having a hydroxyl group on the surface, but impurities are desorbed. The nucleic acid washing buffer solution is composed of an aqueous solution containing a main agent, a buffering agent, and, if necessary, a surfactant. As the main agent, an aqueous solution of about 10 to 100% by weight (preferably about 20 to 100% by weight, more preferably about 40 to 80% by weight) such as methanol, ethanol, isopropanol, n-isopropanol, butanol, and acetone is used as a buffering agent. Examples of the surfactant include the above-described buffer and surfactant. Among these, a solution containing ethanol, Tris and Triton-X100 is preferable. Preferred concentrations of Tris and Triton-X100 are 10 to 100 mM and 0.1 to 10% by weight, respectively.

  Next, the organic polymer having a hydroxyl group on the surface after the washing is brought into contact with a solution capable of desorbing the nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface. Since the target nucleic acid is contained in this solution, it is recovered and provided for subsequent operation, for example, amplification of the nucleic acid by PCR (polymerase chain reaction). The solution capable of desorbing nucleic acids preferably has a low salt concentration, and particularly preferably a solution having a salt concentration of 0.5 M or less. As this solution, purified distilled water, TE buffer or the like can be used.

(2) Nucleic acid separation and purification unit of the present invention The nucleic acid separation and purification unit of the present invention is a nucleic acid separation and purification unit in which a solid phase composed of an organic polymer having a hydroxyl group on the surface is contained in a container having at least two openings. .
There is no particular limitation on the material of the container, and it is sufficient that an organic polymer having a hydroxyl group can be accommodated on the surface and at least two openings can be provided. However, plastic is preferable from the viewpoint of ease of manufacture. For example, it is preferable to use a transparent or opaque resin such as polystyrene, polymethacrylic ester, polyethylene, polypropylene, polyester, nylon, and polycarbonate.

  A conceptual diagram of the container is shown in FIG. Basically, it has a solid phase container, the solid phase can be stored in the container, and the solid phase does not go out of the container when sucking and discharging sample liquid etc. For example, what is necessary is just to join a syringe. For this purpose, it is preferred that the container is initially divided into two parts and can be integrated after the solid phase is accommodated. Further, in order to prevent the solid phase from going out of the housing unit, a mesh made of a material that does not contaminate DNA can be placed above and below the solid phase.

  There is no particular limitation on the shape of the organic polymer having a hydroxyl group on the surface accommodated in the container, and in the case of a circle, a square, a rectangle, an ellipse, a membrane, a cylinder, a scroll, or an organic compound having a hydroxyl group on the surface. Although any shape such as a polymer-coated bead may be used, a highly symmetrical shape such as a circle, a square, a cylinder, or a scroll shape and a bead are preferable from the viewpoint of production suitability.

  One opening of the container is inserted into a sample solution containing nucleic acid, the sample solution is brought into contact with an organic polymer having a hydroxyl group on the surface by suction from the other opening, and then discharged, and then the nucleic acid washing buffer The target nucleic acid can be obtained by sucking and discharging the solution, and then sucking and discharging the solution capable of desorbing the nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface and collecting the discharged liquid. it can.

  A target nucleic acid can be obtained by sequentially immersing an organic polymer having a hydroxyl group on a surface in a sample solution containing a nucleic acid, a nucleic acid washing buffer solution, and a solution capable of desorbing a nucleic acid adsorbed on the organic polymer having a hydroxyl group on the surface. Can be obtained.

  The nucleic acid separation and purification unit of the present invention comprises: (a) a solid phase comprising an organic polymer having a hydroxyl group on the surface; (b) a container having at least two openings that accommodates the solid phase; and (c) the container It is preferable that the pressure difference generator connected to one opening is included. Hereinafter, the nucleic acid separation and purification unit will be described.

  The container is usually prepared in a manner divided into a main body that contains a solid phase composed of an organic polymer having a hydroxyl group on the surface and a lid, and each is provided with at least one opening. One is used as an inlet and an outlet for a sample solution containing nucleic acid, a nucleic acid washing buffer solution, and a solution capable of desorbing nucleic acid adsorbed on a solid phase (hereinafter referred to as “sample solution etc.”), and the other is in a container. Is connected to a pressure difference generator capable of causing the pressure to be reduced or increased. The shape of the main body is not particularly limited, but it is preferable to make the cross section circular in order to facilitate the manufacture and to facilitate the diffusion of the sample solution and the like over the entire surface of the solid phase. It is also preferable to make the cross section square in order not to generate solid-state cutting waste.

  The lid needs to be joined to the main body so that the inside of the container can be depressurized and pressurized by the pressure difference generator, but if this state can be achieved, the joining method can be arbitrarily selected. For example, use of an adhesive, screwing, fitting, screwing, fusion by ultrasonic heating and the like can be mentioned.

  The internal volume of the container is determined only by the amount of the sample solution to be processed, but is usually represented by the volume of the solid phase accommodated. That is, it is preferable that the thickness is about 1 mm or less (for example, about 50 to 500 μm) and the size is such that about 1 to 6 solid phases having a diameter of about 2 mm to 20 mm can be accommodated.

  The end face of the solid phase is preferably in close contact with the inner wall surface of the container to the extent that the sample solution or the like does not pass through.

  Under the solid phase facing the opening used for the inlet of the sample solution or the like, a space is provided without being in close contact with the inner wall of the container so that the sample solution or the like diffuses as evenly as possible over the entire surface of the solid phase.

  It is preferable to provide a member having a hole in the center on the solid phase facing the other opening, that is, the opening coupled to the pressure difference generator. This member suppresses the solid phase and has the effect of efficiently discharging the sample solution and the like, and has a funnel-shaped or bowl-shaped inclined surface so that the liquid collects in the central hole. preferable. The size of the hole, the angle of the inclined surface, and the thickness of the member can be appropriately determined by those skilled in the art in consideration of the amount of the sample solution to be processed, the size of the container for storing the solid phase, and the like. It is preferable to provide a space between the member and the opening for storing the overflowed sample solution or the like and preventing the sample solution from being sucked into the pressure difference generator. The size of this space can also be appropriately selected by those skilled in the art. In order to efficiently collect nucleic acids, it is preferable to aspirate a sample solution containing an amount of nucleic acid larger than the entire solid phase.

  In addition, in order to prevent the sample solution and the like from concentrating only in the portion directly under the opening being sucked and to allow the sample solution and the like to pass through the solid phase relatively uniformly, the space between the solid phase and this member It is preferable to provide a space. For this purpose, it is preferable to provide a plurality of protrusions from the member toward the solid phase. The size and number of the protrusions can be appropriately selected by those skilled in the art, but it is preferable to keep the solid phase opening area as large as possible while maintaining the space.

  Needless to say, if the container is provided with three or more openings, it is necessary to temporarily block the extra openings in order to allow suction and discharge of the liquid accompanying decompression and pressurization operations. Absent.

  The pressure difference generator first sucks a sample solution containing nucleic acid by reducing the pressure in the container containing the solid phase. Examples of the pressure difference generating device include a pump capable of suction and pressurization such as a syringe, a pipetter, or a peristaltic pump. Of these, a syringe is suitable for manual operation, and a pump is suitable for automatic operation. Also, the pipetter has the advantage that it can be easily operated by one hand. Preferably, the pressure difference generator is detachably coupled to one opening of the container.

  Next, a nucleic acid purification method using the above-described nucleic acid separation and purification unit will be described. First, one opening of the nucleic acid separation and purification unit is inserted into a sample solution containing nucleic acid. Next, the inside of the purification unit is depressurized using a pressure difference generator connected to the other opening, and the sample solution is sucked into the container. By this operation, the sample solution comes into contact with the solid phase, and the nucleic acid in the sample solution is adsorbed on the solid phase. At this time, it is preferable to suck an amount of the sample solution that comes into contact with almost the entire solid phase. However, if the sample solution is sucked into the pressure difference generator, the device is contaminated.

  After sucking an appropriate amount of the sample solution, the inside of the unit container is pressurized using a pressure difference generator, and the sucked liquid is discharged. It is not necessary to leave an interval before this operation, and it may be discharged immediately after suction.

  Next, the nucleic acid washing buffer solution is sucked into the container by the same decompression-pressurization operation as described above, and discharged from the container to wash the inside of the container. This solution has a function of washing away the sample solution remaining in the container and washing out impurities in the sample solution adsorbed on the solid phase together with the nucleic acid. Therefore, it is necessary to have a composition that does not desorb nucleic acids from the solid phase but desorbs impurities. The nucleic acid washing buffer solution is composed of an aqueous solution containing a main agent, a buffering agent, and, if necessary, a surfactant. The main agent is about 10 to 90% (preferably about 50 to 90%) such as methyl alcohol, ethyl alcohol, butyl alcohol, acetone and the like. 90%) of the aqueous solution, the buffering agent and the surfactant include the above-described buffering agent and surfactant. Among these, a solution containing ethyl alcohol, Tris and Triton-X100 is preferable. Preferred concentrations of Tris and Triton-X100 are 10-100 mM and 0.1-10%, respectively.

  Next, a solution capable of desorbing the nucleic acid adsorbed on the solid phase is introduced into the container by the same decompression-pressurization operation as described above, and discharged from the container. Since this effluent contains the target nucleic acid, it can be recovered and provided for subsequent operation, for example, amplification of the nucleic acid by PCR (polymerase chain reaction).

  FIG. 2 is a cross-sectional view of an example of the nucleic acid separation and purification unit of the present invention. However, the pressure difference generator is not shown. A container 1 that contains a solid phase is composed of a main body 10 and a lid 20 and is made of transparent polystyrene. The main body 10 contains a surface saponified triacetyl cellulose film as a solid phase 30. Further, an opening 101 for sucking a sample solution or the like is provided. A bottom surface 102 continuing from the opening is formed in a funnel shape, and a space 121 is provided between the solid phase 30. In order to support the solid phase 30 and maintain the space 121, a frame 103 integrated with the bottom surface 102 is provided.

  The main body has an inner diameter of 20.1 mm, a depth of 5.9 mm, and a length from the bottom surface 102 to the opening 101 of about 70 mm. The built-in solid phase 30 has a diameter of 20.0 mm, a thickness of about 50 to 500 μm, and an example of the thickness is 100 μm.

  In FIG. 2, a funnel-shaped pressing member 13 is provided on the upper part of the solid phase. A hole 131 is provided at the center of the pressing member 13, a group of protrusions 132 are provided below, and a space 122 is provided between the solid member 30. The diameter of the upper portion of the wall 104 is made larger than the diameter of the solid phase so that the sample solution or the like is not easily leaked from between the solid phase 30 and the wall 104 of the main body 10, and the end of the pressing member 13 is placed on the step 105. Yes.

  The lid 20 is joined to the main body 10 by ultrasonic heating. An opening 21 that couples the pressure difference generator is provided in a substantially central portion of the lid 20. Between the lid 20 and the pressing member 13, a space 123 for holding a sample solution or the like flowing out from the hole 131 is provided. The volume of the space 123 is about 0.1 ml.

(3) Nucleic acid analysis method of the present invention The nucleic acid analysis method of the present invention comprises (1) a step of separating and purifying a nucleic acid fragment containing a target nucleic acid fragment by the above-described method of the present invention;
(2) The target nucleic acid fragment, at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one polymerase are reacted, and the target nucleic acid fragment is used as a template. Performing a polymerase extension reaction starting from the 3 ′ end of the primer; and
(3) A step of detecting the presence or absence of progress of the polymerase extension reaction or detecting the presence or absence of hybridization between the polymerase extension reaction product and another nucleic acid:
It is characterized by including.

According to a preferred embodiment of the present invention, the presence or absence of the progress of the polymerase extension reaction is detected by detecting pyrophosphoric acid produced in association with the polymerase extension reaction.
In a more preferred embodiment, pyrophosphoric acid is analyzed using a colorimetric method, and more preferably, pyrophosphoric acid is detected using a dry analytical element. In the nucleic acid analysis method according to the present invention, the presence or abundance of the target nucleic acid fragment can be detected, or the base sequence of the target nucleic acid fragment can be detected. Here, the detection of the abundance is a concept including quantification of the target nucleic acid fragment. Specific examples of detection of the base sequence of the target nucleic acid fragment include detection of mutation or polymorphism of the target nucleic acid fragment. FIG. 3 is a conceptual diagram illustrating an embodiment of the present invention.

A first preferred embodiment of the method for analyzing a target nucleic acid fragment according to the present invention is listed below.
(I) Pyrophosphate is detected using a dry analytical element for quantifying pyrophosphate, characterized by comprising a reagent layer containing xanthosine or inosine, pyrophosphatase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a color former. .
(B) A polymerase selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and reverse transcriptase (reverse transcriptase) is used.

Furthermore, in another aspect of the present invention, when detecting the presence or absence of the progress of the polymerase extension reaction by detecting pyrophosphate generated in association with the polymerase extension reaction, the detection of pyrophosphate is carried out by using pyrophosphate as an enzyme. After being converted into inorganic phosphorus, it can be carried out using a dry analytical element for quantitative determination of inorganic phosphorus, which is equipped with a reagent layer containing xanthosine or inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a color former. Preferred forms in this case are listed below.
(Ii) Pyrophosphatase is used as an enzyme that converts pyrophosphate.
(B) A polymerase selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and reverse transcriptase (reverse transcriptase) is used.

Hereinafter, the nucleic acid analysis method of the present invention will be described in more detail.
(A) Target nucleic acid fragment: The target nucleic acid fragment to be analyzed in the present invention is a polynucleotide having a known base sequence, and is isolated from all living things such as animals, microorganisms, bacteria, plants, and the like. Genomic DNA fragments can be targeted. In addition, RNA fragments or DNA fragments that can be isolated from viruses, and cDNA fragments synthesized using mRNA as a template can also be targeted. It is desirable that the target nucleic acid fragment is purified as much as possible, and extra components other than the nucleic acid fragment are removed. For example, when targeting a genomic DNA fragment isolated from the blood of an animal (eg, human) or when targeting a nucleic acid (DNA or RNA) fragment of an infected bacterium or virus present in the blood, The destroyed white blood cell membrane, hemoglobin eluted from the red blood cells, and other general chemicals present in the blood must be sufficiently removed. In particular, hemogrombin inhibits the subsequent polymerase extension reaction. In addition, pyrophosphate and phosphate present as general biochemical substances in the blood interfere with accurate detection of pyrophosphate generated by the polymerase extension reaction.

(B) Primer complementary to the target nucleic acid fragment: The primer complementary to the target nucleic acid fragment used in the present invention has a base sequence complementary to the target site where the base sequence of the target nucleic acid fragment is known It is an oligonucleotide. A primer complementary to the target nucleic acid fragment hybridizes to a target site of the target nucleic acid fragment, so that a polymerase extension reaction proceeds using the target nucleic acid as a template from the 3 'end of the primer. That is, the point in the present invention is whether the primer recognizes the target site of the target nucleic acid fragment and specifically hybridizes. The preferable base number of the primer used in the present invention is 5 to 60 bases. Particularly preferred is 15 to 40 bases. When the number of bases of the primer is too small, not only the specificity of the target nucleic acid fragment with the target site is lowered, but also the hybrid itself with the target nucleic acid fragment cannot be formed stably. On the other hand, when the number of bases in the primer is too large, double strands are formed between the primers or in the primer by hydrogen bonding between the bases, and the specificity also decreases.

When detecting the presence of a target nucleic acid fragment using the method of the present invention, it is possible to use a plurality of primers complementary to each site for different sites of the target nucleic acid fragment. Thus, by recognizing the target nucleic acid fragment at a plurality of sites, the specificity is improved in detecting the presence of the target nucleic acid fragment. When a part of the target nucleic acid fragment is amplified (for example, PCR method), a plurality of primers can be designed according to the amplification method.
When detecting the base sequence of a target nucleic acid fragment using the method of the present invention, particularly when detecting the presence or absence of a mutation or polymorphism, it can handle the mutation or polymorphism so as to include the target mutation or polymorphism. Design primers with the type of base to be used. By doing so, the presence or absence of mutation or polymorphism in the target nucleic acid fragment causes a difference in the presence or absence of primer hybridization to the target nucleic acid fragment, and as a result, it can be detected as a difference in polymerase extension reaction. In addition, by setting a portion corresponding to the mutation or polymorphism near the 3 ′ end of the primer, a difference occurs in the recognition of the polymerase reaction site, and as a result, it can be detected as a difference in the polymerase extension reaction.

(C) Polymerase: When the target nucleic acid is DNA, the polymerase used in the present invention is a double-stranded portion formed by hybridization of a primer with a portion denatured into a single strand of the target nucleic acid fragment. It is a DNA polymerase that catalyzes a complementary extension reaction using deoxynucleoside triphosphate (dNTP) as a starting material and a target nucleic acid fragment as a template in the 5 ′ → 3 ′ direction. Specific examples of the DNA polymerase used include DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and the like. DNA polymerases can be selected or combined depending on the purpose. For example, when a part of the target nucleic acid fragment is amplified (for example, PCR method), it is effective to use Taq DNA polymerase having excellent heat resistance. In the case of amplifying a part of a target nucleic acid fragment using the amplification method (LAMP method: Loop-mediated Isotropic Amplification of DNA) described in “BIO INDUSTRY, Vol. 18, No. 2, 2001” Bst DNA polymerase is used as a strand displacement type DNA polymerase that has no nuclease activity in the 5 ′ → 3 ′ direction and catalyzes an extension reaction while releasing double-stranded DNA on the template as single-stranded DNA. It is effective. In addition, depending on the purpose, DNA polymerase α, T4 DNA polymerase, and T7 DNA polymerase having hexokinase activity in the 3 ′ → 5 ′ direction can be used in combination.

  Further, when the genomic nucleic acid or mRNA of the RNA virus is the target nucleic acid fragment, it is possible to use reverse transcriptase having reverse transcription activity. Further, reverse transcriptase and Taq DNA polymerase can be used in combination.

(D) Polymerase extension reaction: In the polymerase extension reaction of interest in the present invention, the target nucleic acid fragment as described in (A) above is specifically high in a part of the target nucleic acid fragment denatured into a single strand. Starting from the 3 ′ end of a primer complementary to the target nucleic acid fragment as described in (B) above, the deoxynucleoside triphosphate (dNTP) is used as a material and described in (C). All of the complementary nucleic acid extension reactions that proceed with the target nucleic acid fragment as a template using such a polymerase as a catalyst. This complementary nucleic acid extension reaction means that a continuous extension reaction occurs at least twice (for two bases).

  Examples of typical polymerase extension reactions and amplification reactions of target sites of target nucleic acid fragments accompanied by polymerase extension reactions are shown below as examples. In the simplest case, the target nucleic acid fragment is used as a template and the polymerase extension reaction in the 5 'to 3' direction is carried out only once. This polymerase extension reaction can be carried out under isothermal conditions. In this case, the amount of pyrophosphate produced as a result of the polymerase extension reaction is proportional to the amount of the initial target nucleic acid fragment. That is, this method is suitable for quantitatively detecting the presence of the target nucleic acid fragment.

  When the amount of the target nucleic acid is small, it is preferable to amplify the target portion of the target nucleic acid by some means using a polymerase extension reaction. Various methods developed and invented so far can be used for amplification of the target nucleic acid. The most common and widespread method for amplifying a target nucleic acid is a PCR (polymerase chain reaction) method. In the PCR method, the temperature of the reaction solution is periodically controlled to denature (step of denaturing nucleic acid fragments from double strands to single strands) → annealing (primers are applied to nucleic acid fragments denatured into single strands). This is a method of amplifying a target portion of a target nucleic acid fragment by repeating a periodic step of hybridization) → polymerase (Taq DNA polymerase) extension reaction → denature. Finally, the target site of the target nucleic acid fragment can be amplified up to 1 million times the initial amount. Therefore, the amount of pyrophosphate accumulated in the polymerase extension reaction during the amplification process of the PCR method increases, and detection becomes easy.

  A cycling assay method using exonuclease described in JP-A-5-130870 is one of the methods for amplifying a target site of a target nucleic acid fragment using a polymerase extension reaction. This method is a method in which 5 ′ → 3 ′ exonuclease is allowed to act together with a polymerase extension reaction starting from a primer specifically hybridized to a target site of a target nucleic acid fragment, so that the primer is decomposed in the reverse direction. A new primer hybridizes in place of the degraded primer, and the extension reaction by the DNA polymerase proceeds again. This elongation reaction by polymerase and the degradation reaction by exonuclease that removes the previously elongated strand are sequentially and periodically repeated. Here, the elongation reaction by polymerase and the degradation reaction by exonuclease can be carried out under isothermal conditions. In this cycling assay method, the amount of pyrophosphate accumulated in the repeated polymerase extension reaction increases, and detection becomes easy.

  As a method for amplifying a target site of a target nucleic acid fragment developed in recent years, there is the LAMP method. In this method, at least four kinds of primers that complementarily recognize at least six specific sites of a target nucleic acid fragment, no nuclease activity in the 5 ′ → 3 ′ direction, and double-stranded DNA on a template are 1 This is a method of amplifying a target site of a target nucleic acid fragment as a special structure under isothermal conditions by using a strand displacement type Bst DNA polymerase that catalyzes an extension reaction while releasing it as a double-stranded DNA. The amplification efficiency of this LAMP method is high, and the amount of pyrophosphate accumulated in the polymerase extension reaction is very large, which makes detection easy.

  When the target nucleic acid fragment is an RNA fragment, a reverse transcriptase having reverse transcription activity can be used, and an extension reaction can be performed using the RNA strand as a template. Furthermore, RT-PCR method can be used in which reverse transcriptase and Taq DNA polymerase are used in combination and a PCR reaction is carried out following an RT (reverse transcription) reaction. By detecting pyrophosphate generated by this RT reaction or RT-PCR reaction, the presence of the RNA fragment of the target nucleic acid fragment can be detected. This method is effective when detecting the presence of an RNA virus.

(E) Detection of pyrophosphoric acid (PPi): As a conventional method for detecting pyrophosphoric acid (PPi), the method shown in Formula 1 is known. In this method, pyrophosphate (PPi) is converted into adenosine triphosphate (ATP) by sulfurylase, and luminescence generated by adenosine triphosphate acting on luciferin by luciferase is detected. Therefore, an apparatus capable of measuring luminescence is required to detect pyrophosphoric acid (PPi) by this method.

The pyrophosphoric acid detection method suitable for the present invention is the method shown in Formula 2 or Formula 3. The method shown in Formula 2 or Formula 3 is obtained by converting pyrophosphate (PPi) to inorganic phosphorus (Pi) with pyrophosphatase, and reacting purine nucleoside phosphorylase (PNP) with inorganic phosphorus (Pi) with xanthosine or inosine. Xanthine or hypoxanthine is oxidized with xanthine oxidase (XOD) to produce uric acid, and hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process is used to produce a color former (pigment precursor) with peroxidase (POD). Color is developed, and this is colorimetric. Since the results shown in these formulas 2 and 3 can be detected by colorimetry, pyrophosphoric acid (PPi) can be detected visually or using a simple colorimetric measuring device.

  Pyrophosphatase (EC 3, 6, 1, 1) purine nucleoside phosphorylase (PNP, EC 2.4.2.1), xanthine oxidase (XOD, EC 1.2.3.2) and peroxidase (POD, EC 1.11.1. 7) can use a commercially available thing. The color former (that is, the dye precursor) may be any one that generates a dye by hydrogen peroxide and peroxidase (POD). For example, a composition that generates a dye by oxidation of a leuco dye (eg, US Pat. No. 4,089) Triarylimidazole leuco dyes described in JP, 747 et al., Diarylimidazole leuco dyes described in JP-A-59-193352 (EP 0126241A), etc .; when oxidized, a dye is produced by coupling with other compounds And the like (for example, 4-aminoantipyrines and phenols or naphthols) can be used.

(F) Dry analytical element: A dry analytical element that can be used in the present invention is an analytical element composed of one or a plurality of functional layers, and is detected in at least one layer (or across a plurality of layers). A reagent is contained, and the produced dye produced by the reaction in the layer is colorimetrically determined from the outside of the analytical element by reflected light or transmitted light.

  In order to perform quantitative analysis using such a dry analytical element, a certain amount of liquid sample is spotted on the surface of the development layer. The liquid sample developed in the development layer reaches the reagent layer, where it reacts with the reagent and develops color. After spotting, the dry analytical element is kept at a certain temperature for an appropriate time (incubation), and the color development reaction proceeds sufficiently. For example, the reagent layer is irradiated with illumination light from the transparent support side and reflected in a specific wavelength range. The amount of reflected light is measured to determine the reflection optical density, and quantitative analysis is performed based on a calibration curve obtained in advance.

  Since dry analytical elements are stored and stored in a dry state until detection, there is no need to prepare the reagent at the time of use, and generally the reagent stability is higher in the dry state. It is easier and faster than the so-called wet method that must be prepared at the time of use. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.

(G) Dry analytical element for quantifying pyrophosphoric acid: The dry analytical element for quantifying pyrophosphoric acid that can be used in the present invention can have the same layer structure as that of various known dry analytical elements. The dry analytical element includes a support, a developing layer, a detection layer, a light shielding layer, an adhesive, in addition to the reagent for performing the reaction of Formula 2 or Formula 3 in the item (E) (detection of pyrophosphoric acid (PPi)) A multilayer including a layer, a water absorbing layer, an undercoat layer and other layers may be used. As such dry analytical elements, for example, Japanese Patent Laid-Open No. 49-53888 (corresponding US Pat. No. 3,992,158), Japanese Patent Laid-Open No. 51-40191 (corresponding US Pat. No. 4,042,335), and Japanese Patent Laid-Open No. There are those disclosed in Japanese Patent Application Laid-Open No. 55-164356 (corresponding US Pat. No. 4,292,272) and Japanese Patent Application Laid-Open No. 61-4959 (corresponding EPC Publication No. 0166365A).

The dry analytical element that can be used in the present invention includes a reagent for converting pyrophosphoric acid into inorganic phosphorus and a reagent layer containing a reagent group that performs a color reaction according to the amount of inorganic phosphorus. An element is mentioned.
In this dry analytical element for quantifying pyrophosphate, it can be carried out as described above in this specification until pyrophosphoric acid (PPi) is enzymatically converted into inorganic phosphorus (Pi) using pyrophosphatase. By using the following “quantitative method of inorganic phosphorus” (and combinations of reactions used in them) known in the field of biochemical testing, a color development reaction corresponding to the amount of inorganic phosphorus (Pi) can be performed. it can.

Note that when “inorganic phosphorus” is described, there are cases where “Pi” and “HPO ₄ ²⁻ , H ₂ PO ₄ ¹⁻ ” are both described as phosphoric acid (phosphate ions). In the example of the reaction shown below, it is expressed as “Pi”, but it may be expressed as “HPO ₄ ²⁻ ” for the same reaction formula.

  As a quantitative method for inorganic phosphorus, an enzyme method and a phosphorus molybdate method are known. Hereinafter, an enzyme method and a phosphomolybdate method as inorganic phosphorus determination methods will be described.

A. There are a method using peroxidase (POD) and a method using glucose-6-phosphate dehydrogenase (G6PDH) depending on the enzyme used in the last “color reaction” in a series of reactions for quantitative detection of the enzyme method Pi. Hereinafter, specific examples of these methods will be described.

(1) Example of method using peroxidase (POD) (1-1)
Inorganic phosphorus (Pi) is reacted with inosine by purine nucleoside phosphorylase (PNP), and the resulting hypoxanthine is oxidized by xanthine oxidase (XOD) to generate uric acid. Using hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process, 4-aminoantipyrine (4-AA) and phenol are oxidatively condensed with peroxidase (POD) to form a quinoneimine dye. To color.

(1-2)
Pyruvate is oxidized with pyruvate oxidase (POP) in the presence of inorganic phosphorus (Pi), cocarboxylase (TPP), flavin adenine dinucleotide (FAD), and Mg ²⁺ to produce acetylacetic acid. Using hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process, 4-aminoantipyrine (4-AA) and phenol are oxidized by peroxidase (POD) as in the case of (1-1) above. Condensation forms a quinoneimine dye, which is colorimetric.

  The final color reaction in the above (1-1) and (1-2) can be performed using a known “Trinder reagent” as a hydrogen peroxide detection reagent. In this reaction, phenol acts as a “hydrogen donor”. Phenols used as “hydrogen donors” are classic, and various improved “hydrogen donors” are currently used. Specific examples of such hydrogen donors include N-ethyl-N-sulfopropyl-m-anilysine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline. N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N- (2 -Hydroxy-3-sulfopropyl) -m-anilysine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy- - sulfopropyl) -3,5-dimethylaniline, N- ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, and the like N- sulfopropyl aniline.

(2) Method using glucose-6-phosphate dehydrogenase (G6PDH) (2-1)
Inorganic phosphorus (Pi) and glycogen are reacted with phosphorylase to produce glucose-1-phosphate (G-1-P). The resulting glucose-1-phosphate is converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase (PGM). In the presence of glucose-6-phosphate and nicotiamide adenine dinucleotide (NAD), NAD is reduced to NADH with glucose-6-phosphate dehydrogenase (G6PDH), and this is colorimetric.

(2-2)
Inorganic phosphorus (Pi) and maltose are reacted using maltose phosphorylase (MP), and glucose-1-phosphate (G-1-P) is reacted. Hereinafter, similarly to the above (2-1), the resulting glucose-1-phosphate is converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase (PGM). In the presence of glucose-6-phosphate and nicotiamide adenine dinucleotide (NAD), NAD is reduced to NADH with glucose-6-phosphate dehydrogenase (G6PDH), and this is colorimetric.

B. Phosphomolybdate method Directly quantifies “phosphorus molybdate (H ₃ [PO ₄ Mo ₁₂ O ₃₆ ]), which is a complex of inorganic phosphorus (phosphate) and water-soluble molybdate ions under acidic conditions. And “reduction method” in which molybden blue (Mo (III)) is quantified from Mo (IV) to Mo (III) by a reducing agent following the reaction of the direct method. Examples of water-soluble molybdate ions include aluminum molybdate, cadmium molybdate, calcium molybdate, barium molybdate, lithium molybdate, potassium molybdate, sodium molybdate, and ammonium molybdate. Examples of typical reducing agents used in the reduction method include 1,2,4 aminonaphtholsulfonic acid, ferrous ammonium sulfate, ferrous chloride, stannous chloride-hydrazine, sulfate-p-methylamino. Phenol, N, N-dimethyl-phenylenediamine, ascorbic acid, malachite green and the like can be mentioned.

A dry analytical element in the case of using a light-transmissive water-impermeable support can practically have the following configuration. However, the content of the present invention is not limited to this.
(1) One having a reagent layer on a support.
(2) One having a detection layer and a reagent layer in this order on a support.
(3) One having a detection layer, a light reflection layer, and a reagent layer in this order on a support.
(4) One having a second reagent layer, a light reflecting layer, and a first reagent layer in this order on a support.
(5) One having a detection layer, a second reagent layer, a light reflection layer, and a first reagent layer in this order on a support.

  In the above (1) to (3), the reagent layer may be composed of a plurality of different layers. For example, the first reagent layer contains the enzyme pyrophosphatase required for the pyrophosphatase reaction shown in Formula 2 or Formula 3, the substrate xanthosine or the substrate inosine and the enzyme PNP required for the PNP reaction, and the second reagent layer contains the formula 2 or The enzyme XOD required for the XOD reaction shown in Formula 3 and the enzyme POD required for the POD reaction shown in Formula 2 or Formula 3 and the coloring dye (dye precursor) may be contained in the third reagent layer, respectively. Good. Alternatively, two reagent layers may be used, and the pyrophosphatase reaction and the PNP reaction may proceed in the first reagent layer, and the XOD reaction and the POD reaction may proceed in the second reagent layer. Alternatively, the pyrophosphatase reaction, the PNP reaction, and the XOD reaction may proceed in the first reagent layer, and the POD reaction may proceed in the second reagent layer.

  A water absorption layer may be provided between the support and the reagent layer or the detection layer. Moreover, you may provide a filtration layer between each layer. Further, a spreading layer may be provided on the reagent layer, and an adhesive layer may be provided therebetween.

  The support can be any of light-opaque (opaque), light-translucent (translucent), and light-transmissive (transparent), but is generally light-transmissive and water-impermeable. A support is preferred. Preferable materials for the light transmissive water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.

  When a porous layer is used as the reagent layer, the porous medium may be fibrous or non-fibrous. As the fibrous material, for example, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like can be used. Examples of non-fibrous materials include membrane filters made of cellulose acetate described in JP-A-49-53888, JP-A-49-53888, JP-A-55-90859 (corresponding to US Pat. No. 4,258). , 001) Japanese Patent Application Laid-Open No. 58-70163 (corresponding US Pat. No. 4,486,537) and the like may be any of a continuous void-containing granular structure layer composed of inorganic or organic fine particles. JP 61-4959 A (corresponding European published EP 0166365A), JP 62-116258 A, JP 62-138756 A (corresponding European published EP 0226465A), JP 62-138757 ( Corresponding European publication EP 0226465A), JP-A-62-138758 (corresponding European publication EP 0226465A) and the like are also suitable.

  The porous layer may be a spreading layer having a so-called metering action that spreads the liquid in an area approximately proportional to the amount of liquid supplied. Of these, a woven fabric, a knitted fabric and the like are preferable as the spreading layer. A woven fabric or the like may be subjected to glow discharge treatment as described in JP-A-57-66359. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770 (corresponding to: EP 0162301A), JP-A-62-219397 (corresponding to West German Patent Publication DE 3717913A), JP-A. A hydrophilic polymer or a surfactant as described in JP-A-63-112999 (corresponding: DE 3717913A) and JP-A-62-182652 (corresponding: DE 3717913A) may be contained.

  For example, after impregnating or applying the reagent of the present invention to a porous film made of paper, cloth, polymer or the like in advance, another water-permeable layer provided on the support, for example, a detection layer, is disclosed in JP-A-55. It is also a useful method to adhere by the method as described in Japanese Patent No. 1645356.

  The thickness of the reagent layer thus produced is not particularly limited, but when it is provided as a coating layer, a range of about 1 μm to 50 μm, preferably 2 μm to 30 μm is appropriate. When using a method other than coating, such as lamination by lamination, the thickness can vary greatly within a range of several tens to several hundreds of μm.

  When the reagent layer is composed of a water-permeable layer made of a hydrophilic polymer binder, examples of the hydrophilic polymer that can be used include the following. Gelatin and derivatives thereof (for example, phthalated gelatin), cellulose derivatives (for example, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymers and methacrylamide copolymers (for example, acrylamide or copolymers of methacrylamide and various vinyl monomers) Polymer), polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, sodium polyacrylate, copolymers of acrylic acid and various vinyl monomers, and the like.

The reagent layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21777 (corresponding US Pat. No. 3,992,158), JP-A-55-164356 (corresponding US Pat. No. 4,292,272), An aqueous solution or aqueous dispersion containing the reagent composition of the present invention and a hydrophilic polymer according to the method described in the specification of Kokai 54-101398 (corresponding US Pat. No. 4,132,528) or the like is used as a support or a detection layer. It can be provided by coating on another layer and drying. The dry thickness of the reagent layer containing a hydrophilic polymer as a binder is in the range of about 2 μm to about 50 μm, preferably about 4 μm to about 30 μm, and the coating amount is about 2 g / m ² to about 50 g / m ² , preferably about It is in the range of 4 g / m ² to about 30 g / m ² .

  In addition to the reagent composition of formula 2 or formula 3, the reagent layer has an enzyme activator, a coenzyme for the purpose of improving various properties such as coating properties, diffusibility of the diffusible compound, reactivity, and storage stability. In addition, surfactants, pH buffer compositions, fine powders, antioxidants, and other various additives made of organic or inorganic substances can be added. Examples of the buffering agent that can be contained in the reagent layer include “Chemical Handbook Basic” edited by the Chemical Society of Japan (Maruzen Co., Ltd., 1966), pages 1312-1320, R.C. M.M. C. Dawson et al, “Data for Biochemical Research” 2nd edition (Oxford at the Clarendon Press, 1969), “Biochemistry,” pages 467-477 (1966), “Analytical 104” There is a pH buffer system described in pages 300-310 (1980). Examples of pH buffer systems include buffers containing borate; buffers containing citric acid or citrate; buffers containing glycine; buffers containing bicine; buffers containing HEPES; Good buffering agents such as buffering agents. A buffer containing phosphate cannot be used for a dry analytical element for detecting pyrophosphate.

  The dry analytical element for quantification of pyrophosphoric acid that can be used in the present invention can be prepared by known methods described in the aforementioned patent specifications. The pyrophosphoric acid quantification dry analytical element is cut into small pieces such as a square having a side of about 5 mm to about 30 mm or a circle of about the same size. No. 56-142454 (corresponding U.S. Pat. No. 4,387,990), JP-A-57-63452, JP-A-58-32350, JP-T-58-501144 (corresponding international publication: WO083 / 00391). It is preferable to use it as a chemical analysis slide by placing it in a slide frame as described above in terms of manufacturing, packaging, transportation, storage, measurement operation, and the like. Depending on the purpose of use, it is used in a long tape form in a cassette or magazine, or a small piece is placed in a container with an opening, or a small piece is affixed or placed in an open card, or a cut piece is used. It can be used as it is.

  The dry analytical element for quantifying pyrophosphoric acid that can be used in the present invention can quantitatively detect pyrophosphoric acid, which is an analyte in a liquid sample, by the same operations as those described in the above-mentioned patent specifications. For example, an aqueous liquid sample solution in the range of about 2 μL to about 30 μL, preferably 4 μL to 15 μL is spotted on the reagent layer. The spotted analytical element is incubated at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably at a constant temperature in the range of about 30 ° C to about 40 ° C for 1 to 10 minutes. The color development or discoloration in the analytical element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of pyrophosphoric acid in the sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis can be performed with high accuracy by keeping the amount of liquid sample to be spotted, incubation time and temperature constant.

  The measuring operation is disclosed in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding to US Pat. No. 4,424,191). With the described chemical analyzer, highly accurate quantitative analysis can be performed with extremely easy operation. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.

  In the pyrophosphoric acid quantitative dry analytical element that can be used in the present invention, it is stored and stored in a dry state until analysis is performed, so there is no need to prepare a reagent at the time of use, and generally the dry state is better. Because of the high stability of the reagent, it is superior to the so-called wet method in which the reagent solution must be prepared at the time of use, and is superior in simplicity and speed. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.

  The dry analytical element for inorganic phosphorus determination that can be used in the second embodiment of the present invention can be prepared by removing pyrophosphatase from the reagent layer in the pyrophosphoric acid quantitative dry analytical element. It is also possible to use a dry analytical element described in JP-A-7-197. The dry analytical element for quantitative determination of inorganic phosphorus is the same as the dry analytical element for quantitative determination of pyrophosphoric acid in the layer configuration, production method and method of use, except that the reagent layer does not contain pyrophosphatase.

  (H) Kit: The target nucleic acid of the present invention is analyzed by analyzing at least one primer complementary to a part of the target nucleic acid fragment to be analyzed, at least one deoxynucleoside triphosphate (dNTP), at least one polymerase, and pyro. It can carry out using the kit containing each element of the dry analytical element for phosphoric acid determination.

  The form of the kit includes an opening capable of supplying a liquid containing a target nucleic acid fragment having at least a part of the base sequence, at least one primer complementary to a part of the target nucleic acid fragment, and at least one deoxynucleoside At least one reaction cell unit capable of holding triphosphate (dNTP) and at least one kind of polymerase, a detection unit capable of holding a dry analytical element for quantifying pyrophosphate, and the opening and reaction cell unit The cartridge may include a narrow tube or a groove that connects between the detection units and can move the liquid.

  As such a cartridge, the cartridge described in US Pat. No. 5,919,711 can be used. FIG. 4 shows an example of a kit in the form of a cartridge according to the present invention. In the kit 10, a sample solution containing the target nucleic acid can be supplied from the opening 31. The opening 31 is connected to the reaction cell 32 by a thin tube 41. The reaction cell 32 holds in advance at least one primer 81 complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate (dNTP) 82, and at least one polymerase 83. Further, the reaction cell 32 is connected to the detection unit 33 by a thin tube 42. A dry analysis element 51 is held in the detection unit 33 in advance. The sample solution in which the polymerase extension reaction has progressed in the reaction cell 32 moves through the thin tube 42 and is supplied onto the pyrophosphoric acid quantitative dry analysis element 51 of the detection unit 33 to detect pyrophosphoric acid generated by the polymerase extension reaction. In the kit 10, centrifugal force, electrophoresis, electroosmosis, or the like can be used to move the liquid between the opening 31 and the reaction cell 32 and between the reaction cell 32 and the detection unit 33. Further, the reaction cell 32, the thin tubes 41 and 42, and the detection unit 33 are preferably sealed by the base 21 and the lid 22.

  When the cartridge-type kit 10 as shown in FIG. 4 is used, as shown in FIG. 5, the temperature control units 61 and 62 of the reaction cell 32 and the detection unit 33, and the pyrophosphoric acid quantitative dry analysis element 51 are provided. It is desirable to use a device including detection units 71 and 72 that can detect the color development or color change of the light by reflected light.

  The kit in the form of a cartridge that can be used in the present invention is not limited to that shown in FIG. Reagents necessary for the polymerase extension reaction may be held in separate spaces. In that case, each reagent may be moved to the reaction cell during the reaction. A plurality of reaction cells may be used.

  When the pyrophosphoric acid produced by the polymerase extension reaction is detected using a dry analytical element for quantitative determination of inorganic phosphorus after pyrophosphoric acid is enzymatically converted to inorganic phosphorus, the target nucleic acid is previously stored in the first reaction cell. Holding at least one primer complementary to a portion of the fragment, at least one deoxynucleoside triphosphate (dNTP), and at least one polymerase, performing a polymerase extension reaction in the first reaction cell; The reaction solution in the first reaction cell is transferred to the second reaction cell that is connected to the first reaction cell by a capillary tube and pre-stored with pyrophosphatase. The produced pyrophosphoric acid is converted into inorganic phosphorus in the second reaction cell, and then the reaction solution in the second reaction cell is connected to the second reaction cell by a capillary. Have been moves to the detection portion in advance quantifying inorganic phosphorus the dry analytical element is held, it is also possible to detect the inorganic phosphorus.

  It is also possible to install a plurality of sets of “opening-capillary tube-reaction cell-capillary tube-detection unit” on one cartridge in parallel or in the radial direction of concentric circles. In this case, for example, by changing the base sequence of at least one primer complementary to a part of the target nucleic acid fragment held in the reaction cell according to the type of target nucleic acid, multiple types of target nucleic acids can be detected simultaneously. Kits that can be provided can be provided.

(4) Analysis apparatus of the present invention The present invention further provides an analysis apparatus for performing the above-described nucleic acid analysis method. The analyzer comprises (1) a means for extracting and purifying a nucleic acid containing the nucleic acid separation and purification unit described hereinabove, (2) a reaction means for performing a polymerase extension reaction, and (3) a polymerase. It is comprised from the means for detecting the presence or absence of progress of extension reaction, or detecting the presence or absence of hybridization with a polymerase extension reaction product and another nucleic acid.

  The means for nucleic acid extraction / purification is composed of the nucleic acid separation and purification unit described above in this specification. In addition, a space for setting a sample liquid, a space for setting a nucleic acid separation and purification unit, and a sample at a constant temperature. Means for incubating at 37 ° C. (for example, 37 ° C.), means for sucking and discharging the sample liquid or treatment liquid, and the like can be provided.

  The reaction means for performing the polymerase extension reaction is a means capable of performing a nucleic acid synthesis reaction such as PCR. Generally, a reaction vessel for carrying out the reaction and a reagent necessary for the reaction are contained in the reaction vessel. And dispensing means to be added therein. In addition, temperature adjusting means (such as a thermal cycler or an incubator) for adjusting the temperature in the reaction vessel can be provided. A nucleic acid fragment containing a target nucleic acid fragment purified by a nucleic acid separation and purification unit by dispensing means, at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one kind Polymerase is added to the reaction vessel, and a polymerase extension reaction starting from the 3 ′ end of the primer using the target nucleic acid fragment as a template is performed.

As a means for detecting the progress of the polymerase extension reaction, a dry analytical element for quantifying pyrophosphoric acid or a dry analytical element for quantifying inorganic phosphorus as described above in this specification can be used. In addition, a means for incubating the dry analytical element at a constant temperature and a reflection photometric device for measuring the color of the dry analytical element can be provided. In addition, as a means for detecting the presence or absence of hybridization between the polymerase extension reaction product and another nucleic acid, usual means used for detecting the presence or absence of hybridization can be used.
The analyzer of the present invention (especially when using an analytical element for quantifying pyrophosphate) can carry out the polymerase extension reaction in the next step using (part of) the separated and purified nucleic acid aqueous solution as it is, and after the polymerase extension reaction. (A part of the reaction solution) can be used for detection by the pyrophosphoric acid quantitative analysis element in the next step (that is, the reaction solution after the polymerase extension reaction can be spotted on the pyrophosphoric acid quantitative analysis element as it is. ) Is very suitable for system automation.
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
(1) Preparation of Nucleic Acid Separation / Purification Unit Container A nucleic acid separation / purification unit container having a portion for accommodating a solid phase for nucleic acid adsorption having an inner diameter of 7 mm and a thickness of 2 mm was prepared from high impact polystyrene.
(2) Preparation of nucleic acid purification solid phase and nucleic acid separation / purification unit The nucleic acid purification solid phase and comparative solid phase shown in Table 1 were prepared. In order to perform surface saponification, an object to be surface saponified was immersed in an aqueous solution of 0.02N to 2N sodium hydroxide for 20 minutes. The surface saponification rate varies depending on the concentration of sodium hydroxide. These solid phases were accommodated in the solid phase accommodating part of the container for nucleic acid separation and purification unit by the amount shown in Table 1 to prepare a nucleic acid separation and purification unit.

(3) Preparation of nucleic acid adsorption buffer solution and washing buffer solution Nucleic acid adsorption buffer solution and washing buffer solution having the formulation shown in Table 2 were prepared.

(4) Nucleic acid purification operation 200 μl of human whole blood was collected using a vacuum blood collection tube. To this, 200 μl of a nucleic acid adsorption buffer solution having a formulation shown in Table 2 and 20 μl of protease K were added and incubated at 60 ° C. for 10 minutes. After incubation, 200 μl of ethanol was added and stirred. After stirring, the tip of the disposable pipette tip connected to one opening of the nucleic acid separation / purification unit prepared in (1) is inserted into the whole blood sample processed as described above, and the other nucleic acid separation / purification unit. The fluid was inhaled and then drained using a syringe connected to the opening.

  Immediately after the discharge, the tip of the pipette tip was inserted into 1 ml of the nucleic acid washing buffer solution, and the liquid was sucked and then discharged to wash the inside of the nucleic acid separation and purification unit. After washing, the tip of a pipette tip was inserted into 200 μl of purified distilled water, and distilled water was aspirated and discharged, and the discharged liquid was collected.

(5) Determination of nucleic acid recovery amount and determination of purity The absorbance of the recovered effluent was measured to determine the nucleic acid recovery amount and purity. The amount recovered is quantified by light absorption measurement at a wavelength of 260 nm, and the purity of the nucleic acid is determined by the ratio of light absorption at 260 nm and 280 nm. If this ratio is 1.8 or more, the purity is judged to be good. The The results are shown in Table 3. From this result, it can be seen that when the surface saponification rate is 5% or more, the amount of recovered DNA is good and the purity is high.

Example 2 Purification of Nucleic Acid from Whole Blood Sample Using Triacetylcellulose Porous Membrane with 100% Surface Saponification Rate Triacetylcellulose porous membrane with 100% surface saponification rate (manufactured by Fuji Photo Film) as a nucleic acid adsorption solid phase Nucleic acids were purified from whole blood samples as in Example 1 using the solid phase E) of Example 1. Whole blood sample before purification (diluted 5 times and OD was measured) and OD after purification were measured. The results are shown in FIG. From the results of FIG. 6, it can be seen that components other than nucleic acids have been completely removed by the separation and purification method of the present invention.

Example 3: Amplification of nucleic acid The nucleic acid purified in Example 2 was used to amplify a nucleic acid by polymerase chain reaction. As a positive control, Human DNA manufactured by Clontech was used. The reaction solution of polymerase chain reaction (PCR) was purified water (36.5 μl), 10 × PCR buffer (5 μl), 2.5 mM dNTP (4 μl), Taq FP (0.5 μl), primer (2 μl), sample (Nucleic acid) 2 μl.
In PCR, one cycle of denaturation at 94 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute was repeated for 30 cycles. The following primers were used.

1) p53 exon 6
Forward: GCGCTGCCTCA GATACGGATG
Reverse: GGAGGGCCAC TGACAACCA

2) p53 exon 10
Forward: GATCCGTCAT AAAGTCAAAC (SEQ ID NO: 1)
Reverse: GGATGAGAAT GGAATCCCTAT (SEQ ID NO: 2)

3) ABO type gene exon 6
Forward: CACCTGCAGA TGTGGGGTGGC ACCCTGCCA (SEQ ID NO: 3)
Reverse: GTGGAATTCA CTCGCCACTG CCTGGGTCTC (SEQ ID NO: 4)

4) ABO type gene exon 7
Forward: GTGGCTTTCC TGAAGCTGTTC (SEQ ID NO: 5)
Reverse: GATGCCGTTG GCCTGGTCGA C (SEQ ID NO: 6)

  The result of electrophoresis of the PCR reaction product is shown in FIG. Invitrogen's 100 bp marker was used. From the results of FIG. 7, it was confirmed that the desired nucleic acid could be amplified by PCR using the nucleic acid separated and purified by the method of the present invention.

Example 4: Detection of Pseudomonas aeruginosa in human whole blood by nucleic acid extraction-amplification (PCR) -detection (dry analytical element for pyrophosphate quantification) (experiment based on the examination of Pseudomonas aeruginosa sepsis)
(1) Preparation of human whole blood to which Pseudomonas aeruginosa was added Based on the culture solution of Pseudomonas Syringae cultured overnight in LB medium (Luria-Bertani medium), the concentration was changed by dilution with PBS. By adding the solution to whole human blood collected from EDTA, 0, 5 × 10 ⁵ , 5 × 10 ⁶ , 2.5 × 10 ⁶ , 5 × 10 ⁷ , and 1 × 10 ⁸ cells per mL Six levels of human whole blood including the number were prepared. Here, the number of cells is a value estimated using a spectrophotometer.

(2) Preparation of dry analytical element for quantitative determination of pyrophosphoric acid Composition shown in Table 4 on colorless transparent polyethylene terephthalate (PET) smooth film sheet (support) having a thickness of 180 μm and provided with a gelatin subbing layer The aqueous solution of (a) was applied so as to have the following coverage, and dried to provide a reagent layer.

  On this reagent layer, an adhesive layer aqueous solution having the composition (b) described in Table 5 below was applied so as to have the following coverage, and dried to provide an adhesive layer.

Next, water is supplied over the entire surface of the adhesive layer at a rate of 30 g / m ² to swell the gelatin layer, and a pure polyester bronze woven fabric is applied almost uniformly and lightly on the laminating layer. To provide a porous spreading layer.

  Next, an aqueous solution of the composition (c) shown in Table 6 below is applied almost uniformly from above the spreading layer so as to have the following coverage, dried, cut into 13 mm × 14 mm, and placed in a plastic mounting material. As a result, a dry analytical element for quantifying pyrophosphoric acid was prepared.

(3) Extraction and purification of nucleic acid from human whole blood Six levels of human whole blood prepared by adding Pseudomonas aeruginosa prepared in (1) above were used as samples, and from each of the methods described in Example 2 A nucleic acid aqueous solution was obtained by extracting and purifying the nucleic acid in the same manner. The amount of nucleic acid obtained from 6 levels of human whole blood (estimated using a spectrophotometer) was 20-30 ng / μl. The resulting aqueous nucleic acid solution is a mixed aqueous solution of human genomic nucleic acid and added Pseudomonas aeruginosa genomic nucleic acid, and the majority is occupied by human genomic nucleic acid.

(4) PCR amplification PCR amplification was carried out under the following conditions using the nucleic acid aqueous solution obtained by extraction and purification from the 6-level human whole blood sample in (3) as it was.

<Primer>
The following primer set having a sequence specific to the genomic nucleic acid of Pseudomonas aeruginosa (ice nucleation protein (Inak) N-terminal) was used.
Primer;
5′-GCGATGTCTGTAATGAACTCTCGACAAGC-3 ′ (SEQ ID NO: 7)
Primer (lower);
5′-GGTCTGCAAAATTCTGCGGCGCGTCTC-3 ′ (SEQ ID NO: 8)

  PCR amplification is performed by repeating 30 cycles of [denaturation: 94 ° C./minute, annealing: 55 ° C./minute, polymerase extension reaction: 72 ° C./minute] with the composition of the reaction solution shown below. Carried out.

<Composition of reaction solution>
10 x PCR buffer-5 μL
2.5 mM dNTP 4 μL
20 μM primer 1 μL
1 μL of 20 μM primer
Pyrobest 0.25μL
5 μL of nucleic acid sample solution obtained in (3)
Purified water 33.75 μL

(5) Detection Using Analytical Element for Pyrophosphoric Acid Quantification The solution after the PCR amplification reaction in (4) is spotted on the dry analytical element for quantifying pyrophosphoric acid prepared in (2) as it is. The reflection optical density (OD _R ) obtained by incubating the dry analytical element for quantitative determination of phosphoric acid at 37 ° C. for 5 minutes and measuring from the support side at a wavelength of 650 nm is shown in FIG. 8 and FIG.

From the results of Example 4, from a human whole blood containing Pseudomonas aeruginosa, a nucleic acid sample solution containing a target nucleic acid fragment obtained according to the method for separating and purifying nucleic acid of the present invention was analyzed with a sequence specific to the genomic nucleic acid of Pseudomonas aeruginosa PCR is performed using the primer set possessed, and the solution after the PCR amplification reaction is used as it is, and the produced pyrophosphoric acid is measured as a reflection optical density (OD _R ) using a dry analytical element for quantifying pyrophosphate. It can be seen that a reflection optical density (OD _R ) corresponding to the amount of Pseudomonas aeruginosa present in human whole blood is obtained.

FIG. 1 is a conceptual diagram of the nucleic acid separation and purification unit of the present invention. FIG. 2 is an example of the nucleic acid separation and purification unit of the present invention. However, the pressure difference generator to be coupled to the opening 21 is not shown. In FIG. 2, 1 is a container, 10 is a main body, 101 is an opening, 102 is a bottom surface, 103 is a frame, 104 is a wall, 105 is a step, 121 is a space, 122 is a space, 123 is a space, 13 is a pressing member, 131 Indicates a hole, 132 indicates a protrusion, 20 indicates a lid, 21 indicates an opening, and 30 indicates a solid phase. FIG. 3 is a conceptual diagram illustrating the analysis of the nucleic acid of the present invention. FIG. 4 is a perspective view showing an example of a kit in the form of a cartridge that can be used in the present invention. In FIG. 4, 10 is a kit, 21 is a substrate, 22 is a lid, 31 is an opening, 32 is a reaction cell, 33 is a detection unit, 41 and 42 are thin tubes, 51 is a dry analytical element, 81 is a primer, and 82 is deoxy. Nucleoside triphosphate (dNTP), 83 indicates a polymerase. FIG. 5 is a perspective view showing the system configuration when using the kit in the form of a cartridge of the present invention. In FIG. 5, 10 is a kit, 32 is a reaction cell, 33 is a detection unit, 61 is a temperature control unit, 62 is a temperature control unit, 71 is a detection unit, and 72 is a detection unit. FIG. 6 is a diagram showing the results of nucleic acid purification from a whole blood sample using a triacetylcellulose porous membrane having a surface saponification rate of 100%. FIG. 7 shows the results of electrophoresis of PCR reaction products using nucleic acids separated and purified according to the method of the present invention. FIG. 8 shows the relationship between the number of Pseudomonas aeruginosa added and the reflection optical density (OD _R ). FIG. 9 shows the relationship between the number of Pseudomonas aeruginosa added and the reflection optical density (OD _R ).

Claims (13)

(1) a step of separating and purifying a nucleic acid fragment containing a target nucleic acid fragment by a method for separating and purifying nucleic acid, comprising the step of adsorbing and desorbing nucleic acid on a solid phase comprising an organic polymer having a hydroxyl group on the surface;
(2) The target nucleic acid fragment, at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one polymerase are reacted, and the target nucleic acid fragment is used as a template. Performing a polymerase extension reaction starting from the 3 ′ end of the primer; and
(3) A step of detecting the presence or absence of progress of the polymerase extension reaction or detecting the presence or absence of hybridization between the polymerase extension reaction product and another nucleic acid:
A method for analyzing nucleic acid, comprising: The method for analyzing a nucleic acid according to claim 1, wherein the presence or absence of progress of the polymerase extension reaction is detected by detecting pyrophosphate generated in association with the polymerase extension reaction. The method for analyzing nucleic acid according to claim 2, wherein pyrophosphoric acid is detected using a colorimetric method. The method for analyzing nucleic acid according to claim 2 or 3, wherein pyrophosphoric acid is detected using a dry analytical element. The dry analytical element is a dry analytical element for quantifying pyrophosphoric acid, comprising a reagent layer containing a reagent that converts pyrophosphoric acid into inorganic phosphorus and a reagent group that performs a color reaction according to the amount of inorganic phosphorus. The nucleic acid analysis method as described. The method for analyzing nucleic acid according to claim 5, wherein the dry analytical element is a dry analytical element for quantifying pyrophosphate comprising a reagent layer containing xanthosine or inosine, pyrophosphatase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a color former. . The nucleic acid analysis according to any one of claims 1 to 6, wherein the polymerase is selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and reverse transcriptase (reverse transcriptase). Method. After the pyrophosphate is enzymatically converted to inorganic phosphorus, a pyroanalyte is then detected, and then a dry analytical element for quantitative determination of inorganic phosphorus is provided with a reagent layer containing xanthosine or inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a coloring agent. The method for analyzing nucleic acid according to claim 2 or 3, wherein the method is used. The method for analyzing nucleic acid according to claim 8, wherein the enzyme that converts pyrophosphate into inorganic phosphorus is pyrophosphatase. The nucleic acid analysis method according to claim 8 or 9, wherein the polymerase is selected from the group consisting of DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and reverse transcriptase (reverse transcriptase). The nucleic acid analysis method according to any one of claims 1 to 10, wherein the nucleic acid analysis is detection of the presence or abundance of the target nucleic acid fragment or detection of a base sequence of the target nucleic acid fragment. The method for analyzing a nucleic acid according to claim 11, wherein the detection of the base sequence of the target nucleic acid fragment is detection of a mutation or polymorphism in the target nucleic acid fragment. (1) Means for extracting and purifying a nucleic acid comprising a nucleic acid separation and purification unit containing a solid phase composed of an organic polymer having a hydroxyl group on the surface in a container having at least two openings, (2) Polymerase extension And (3) a means for detecting the presence or absence of progress of the polymerase extension reaction or a means for detecting the presence or absence of hybridization between the polymerase extension reaction product and another nucleic acid. An analyzer for performing the nucleic acid analysis method according to any one of 1 to 12.

Images