JP2003061658A - Method for analyzing target nucleic acid fragment and kit for analyzing target nucleic acid fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing a target nucleic acid fragment by which the analysis can simply and rapidly be carried out by using a small- sized device and a kit for analyzing the target nucleic acid fragment using the method for analyzing the same. SOLUTION: This method for analyzing the target nucleic acid fragment is to analyze pyrophosphoric acid produced during a polymerase chain elongation reaction based on a specific base sequence of the target nucleic acid fragment. The kit for analyzing the target nucleic acid fragment is used for carrying out the method for analyzing the same.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a target nucleic acid fragment having a specific nucleotide sequence, which is effective for clinical examination of infectious diseases caused by viruses, bacteria, etc., and examination of genetic diseases caused by genetic characteristics of individuals. And a kit for analyzing nucleic acid fragments using the method. More specifically, the present invention simply detects the presence or absence of the target nucleic acid fragment by detecting whether or not the polymerase extension reaction using the target nucleic acid as a template has proceeded by a colorimetric method, preferably using a dry analytical element. The present invention relates to a method for analyzing the abundance or the base sequence of a target nucleic acid fragment and a nucleic acid fragment analysis kit using the method.

[0002]

2. Description of the Related Art Conventionally, in clinical tests for infectious diseases caused by viruses, bacteria, etc., it is possible to identify pathogens such as viruses, bacteria, etc. by culturing specimens using body fluids such as blood, feces, sputum etc. as samples. Has been done. However, these methods also have problems that it takes a very long time to culture the specimen, and that the culture itself may not be successful depending on the type of virus or bacterium. Also, in terms of requiring a special technique for culturing a sample, it is not always a method that can obtain satisfactory results quickly and simply.

In addition, viruses utilizing the antigen-antibody reaction,
Methods for identifying pathogens such as bacteria are also used. This method can automate the inspection, and is a good method in terms of speed and simplicity. However, in an antigen detection method that detects a pathogen as an antigen, the pathogen cannot be detected in some cases due to an insufficient amount of the pathogen present in the sample, and there is a sensitivity problem. There is also a problem that it is difficult to determine the antigenic site unique to the type of pathogen. On the other hand, in the antibody detection method for detecting the antibody produced in the body due to the infection of the pathogen, there is a problem that it takes time from the infection of the pathogen to the production of the antibody, and the period cannot be detected.

On the other hand, a method of detecting a nucleic acid fragment (target nucleic acid fragment) having a base sequence unique to the type of pathogen such as virus or bacterium by utilizing the complementarity of the base sequences directly detects the pathogen. It is a method that enables identification, and is widely used as a genetic test method such as a DNA probe method or a PCR (polymerase chain reaction) method. For example, the HCV (hepatitis C virus) gene test method is effective as a method for directly knowing the amount of HCV in the study of INF administration in the treatment of interferon (INF) for hepatitis C and the monitoring of healing. .

It is expected that the genetic characteristics (genotype) of pathogens such as viruses and bacteria will be further clarified in the future, and new therapeutic agents utilizing the genetic characteristics will be developed. In that case, it is very important not only to identify the pathogen but also to know the genetic characteristics of the pathogen. The genetic test method is exactly a test method that can meet the demand.

On the other hand, not only the identification of the pathogen but also the genetic test method can directly detect the genetic characteristics of the individual. Therefore, it is possible to detect the mutation of the gene causing the genetic disease, cancer, diabetes, etc. It can also be used to detect genetic factors that influence the susceptibility to diseases such as lifestyle-related diseases. In particular, after the entire nucleotide sequence of the human genome has been determined, as a post-genome study, the relationship between genetic features and diseases will be clarified more than ever, and therapeutic drugs that utilize genetic features will be developed. I can expect to go. With the progress of post-genome research, it is expected that the demand for genetic test methods will increase in the future.

However, the current genetic testing methods require special techniques, complicated operations, and special equipment, and the facilities that can carry out genetic testing methods are limited to large-scale testing centers. Has been. In the examination of infectious diseases caused by viruses, bacteria, etc., and also in the examination of individual genetic characteristics, it is more effective if the guideline for diagnosis and treatment can be determined on the spot as quickly as possible. For that purpose, a new genetic test method is required that can be performed by anyone with a simple operation and the test results can be obtained quickly.

[0008] A genetic test method utilizing detection of progress of polymerase extension reaction using a target nucleic acid fragment as a template has been developed so far for the purpose of improving convenience and speed. When amplifying a specific nucleic acid region of a target nucleic acid fragment by PCR, a method of detecting in real time the process of generation of an amplification product as a change in fluorescence intensity (Real Tim)
e PCR method), the amplification product is electrophoresed after PCR,
Since the step of analyzing the result is not required, it is a good method in terms of rapidity, and the TaqMan probe method (P
E Biosystems), and Molecul
It is commercialized as the ar Beacon method (Stratagene). However, these methods
T (fluorescence resonance e)
In this method, it is necessary to prepare a device capable of measuring changes in fluorescence intensity and a special hybridization probe labeled with a combination of a fluorescent dye and its quencher. There is a problem in point, and it is still out of the special technology.

A method of amplifying a specific nucleic acid region of a target nucleic acid fragment by PCR in the presence of an intercalating fluorescent substance and detecting a change in fluorescence intensity at that time (IM-P
CR: intercalation monitor
ng PCR method) "Medical History Vol.173, N
No. 12, 1995 ”. This method
The Real Time PCR method is good in that it does not require a special hybridization probe, but it also requires an apparatus capable of measuring changes in fluorescence intensity for its implementation. In addition, since the intercalating fluorescent substance binds to all the nucleic acid fragments existing in the system regardless of the presence or absence of PCR amplification of the specific nucleic acid region of the target nucleic acid fragment, there is a problem in specificity.

On the other hand, an oligonucleotide primer having nuclease resistance is hybridized with a specific region of the target nucleic acid fragment, and the extension reaction and the decomposition reaction are repeated in the presence of deoxynucleoside triphosphate (dNTP), DNA polymerase and nuclease, A method for detecting the produced pyrophosphoric acid or deoxynucleoside monophosphoric acid is disclosed in JP-A-7-231799. A method for detecting a target nucleic acid fragment by detecting pyrophosphate generated by the polymerase extension reaction is as follows:
It is excellent in that it enables detection of target nucleic acid fragments by detecting general chemical substances that are by-products of the polymerase extension reaction.

However, a method for simply detecting pyrophosphoric acid has not been known so far, and in the above publication,
Adenosine triphosphate (ATP) produced by reacting pyrophosphate with adenosine-5'-phosphosulfate and adenosine triphosphate (ATP) sulfurylase is only described as a method for detecting luminescence generated upon reaction with luciferin in the presence of luciferase. Not not. This method has a problem in terms of simplicity in that a device capable of measuring luminescence is required. In addition, by using a nuclease-resistant primer and using a DNA polymerase and a nuclease in combination and repeating the polymerase reaction and the nuclease reaction, it is possible to carry out under conditions where the extension reaction does not proceed substantially continuously. Then, it is different from the present invention.

[0012]

SUMMARY OF THE INVENTION The present invention is a target that can be implemented simply and quickly by anyone using a small device without the need for special techniques, complicated operations, and special devices. An object is to provide a method for analyzing a nucleic acid fragment. In addition, in order to achieve these goals,
Another object is to provide a method for analyzing a target nucleic acid fragment that can be automated in a space-saving manner. Further, it is an object to provide an analysis kit for a target nucleic acid fragment using these analysis methods.

[0013]

[Means for Solving the Problems] In order to solve the above problems, the present inventor uses a dry analytical element to generate pyrophosphate generated in a polymerase extension reaction based on a specific base sequence of a target nucleic acid fragment. The present invention has been completed by finding that it is possible to analyze a target nucleic acid fragment excellent in simplicity and speed without requiring a special device by detecting.

That is, the present invention provides a target nucleic acid fragment whose base sequence is known, at least one primer complementary to a part of the target nucleic acid fragment,
At least one deoxynucleoside triphosphate (dN
TP) and at least one polymerase, and detecting the presence or absence of progress of a polymerase extension reaction starting from the 3 ′ end of the primer using the target nucleic acid fragment as a template, and analyzing the target nucleic acid fragment. In the method, the method for analyzing a target nucleic acid fragment, which comprises detecting the presence or absence of progress of the polymerase extension reaction by detecting pyrophosphate produced in the polymerase extension reaction, and the method for performing the analysis method. An analysis kit for a target nucleic acid fragment is provided.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION A preferred embodiment of the method for analyzing a target nucleic acid fragment of the present invention is a method in which pyrophosphate is analyzed using a colorimetric method, and more preferably, the detection of pyrophosphate is carried out by a dry analytical element. There is a method to use. In the method for analyzing a target nucleic acid fragment according to the present invention, the presence or amount of the target nucleic acid fragment can be detected, or the base sequence of the target nucleic acid fragment can be detected. In addition,
The detection of the abundance referred to here is a concept including quantification of the target nucleic acid fragment. Specific examples of the detection of the base sequence of the target nucleic acid fragment include detection of mutation or polymorphism in the target nucleic acid fragment. FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention.

The first preferred embodiment of the method for analyzing a target nucleic acid fragment according to the present invention is listed below. (A) 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) The polymerase is DNA polymerase I, DNA
A polymerase selected from the group consisting of the Klenow fragment of polymerase I, Bst DNA polymerase, and reverse transcriptase is used.

Another aspect of the present invention is that 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 pyrophosphate. It is in a kit containing the elements of the dry analytical element for quantification.

Further, a second preferred form of the present invention is
A target nucleic acid fragment of which at least a part of the base sequence is known, 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 to produce the target. When the presence or absence of the progress of the polymerase extension reaction starting from the 3 ′ end of the primer using the nucleic acid fragment as a template is detected by detecting the pyrophosphate produced by the polymerase extension reaction,
For the detection of pyrophosphate, the enzymatic conversion of pyrophosphate to inorganic phosphate is followed by xanthosine or inosine, purine nucleoside phosphorylase, xanthine oxidase,
A method for analyzing a target nucleic acid fragment is characterized in that it is carried out using a dry analytical element for quantifying inorganic phosphorus, which comprises a reagent layer containing a peroxidase and a color former.

The preferred modes of the method for analyzing a target nucleic acid fragment according to the second mode of the present invention are listed below. (A) Pyrophosphatase is used as an enzyme that converts pyrophosphate. (B) The polymerase is DNA polymerase I, DNA
A polymerase selected from the group consisting of the Klenow fragment of polymerase I, Bst DNA polymerase, and reverse transcriptase is used.

Another aspect of the present invention is to provide 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, pyrophosphatase, And a dry analytical element for quantitative determination of inorganic phosphorus.

The embodiments of the present invention will be described in more detail below.

(A) Target nucleic acid fragment: The target nucleic acid fragment to be analyzed in the present invention is a polynucleotide of which at least a part of the base sequence is known, and all the organisms such as animals, microorganisms, bacteria and plants. Genomic DNA fragments isolated from RNA or DNA fragments that can be isolated from viruses,
Also, a cDNA fragment synthesized using mRNA as a template can be used. It is desirable that the target nucleic acid fragment be purified as much as possible and that excess components other than the nucleic acid fragment be removed. For example, when a genomic DNA fragment isolated from the blood of an animal (for example, a human) is targeted or a nucleic acid (DNA or RNA) fragment of an infectious bacterium or virus present in blood is targeted, the isolation process Destroyed white blood cell membranes, hemoglobin eluted from red blood cells, and other common chemicals present in blood must be well removed. In particular, hemoglobin inhibits the subsequent polymerase extension reaction. Pyrophosphate and phosphate present in blood as general biochemical substances interfere with accurate detection of pyrophosphate produced by polymerase extension reaction.

(B) Primer complementary to target nucleic acid fragment: The primer complementary to the target nucleic acid fragment used in the present invention is a base complementary to a target site where the base sequence of the target nucleic acid fragment is known. An oligonucleotide having a sequence. The primer complementary to the target nucleic acid fragment hybridizes to the target site of the target nucleic acid fragment, and the polymerase extension reaction proceeds from the 3'end of the primer as a starting point using the target nucleic acid as a template. That is, in the present invention, the point is whether or not the primer recognizes the target site of the target nucleic acid fragment and specifically hybridizes. The preferred number of bases of the primer used in the present invention is 5 to 60 bases. Particularly preferred is 15 to 40 bases. If the number of bases in 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 stably formed. If the number of bases in the primer is too large, hydrogen bonds between the primers or between the bases form a double strand due to hydrogen bonding between the bases, which also lowers the specificity.

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 different sites of the target nucleic acid fragment and complementary to each site. By recognizing the target nucleic acid fragment at a plurality of sites in this way, the specificity is improved in detecting the presence of the target nucleic acid fragment. Also,
When a part of the target nucleic acid fragment is amplified (for example, PCR method), it is possible to design a plurality of primers 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, the mutation or polymorphism is dealt with so that the target mutation or polymorphism part is included. Design a primer based on the type of base to be used. By doing so, the presence or absence of the hybridization of the primer to the target nucleic acid fragment is different depending on the presence or absence of the mutation or polymorphism of the target nucleic acid fragment, and as a result, it can be detected as the difference in the polymerase extension reaction. In addition, setting a portion corresponding to a mutation or polymorphism near the 3'end of the primer causes a difference in the recognition of the reaction site of the polymerase, 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 chain formed by the hybridization of a primer to the single-stranded denatured portion of the target nucleic acid fragment. Is a DNA polymerase that catalyzes a complementary extension reaction using a target nucleic acid fragment as a template, in the 5 ′ → 3 ′ direction starting from the portion of (1) and using deoxynucleoside triphosphate (dNTP) as a material. Specific examples of the DNA polymerase used include DNA polymerase I, Klenow fragment of DNA polymerase I, Bst
There are DNA polymerase and the like. DNA polymerases can be selected or combined depending on the purpose.
For example, a part of the target nucleic acid fragment is amplified (for example, PC
R method), it is effective to use Taq DNA polymerase having excellent heat resistance. In addition, "BIO
INDUSTRY, Vol. 18, No. 2,200
1 ”(LAMP method: Loop-
mediated Isothermal Amplif
When a part of the target nucleic acid fragment is amplified by using cationation of DNA), there is no nuclease activity in the 5 ′ → 3 ′ direction and the double-stranded DNA on the template is
It is effective to use Bst DNA polymerase as a strand displacement type DNA polymerase which catalyzes an elongation reaction while releasing it as a double-stranded DNA. In addition, depending on the purpose, it is also possible to use DNA polymerase α, T4 DNA polymerase, and T7 DNA polymerase which have hexokinase activity in the 3 ′ → 5 ′ direction in combination.

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

(D) Polymerase extension reaction: The polymerase extension reaction of interest in the present invention includes the above (A)
Of a primer complementary to the target nucleic acid fragment as described in (B) above, which hybridizes specifically to a part of the single-stranded denatured portion of the target nucleic acid fragment as described in A complementary nucleic acid extension that proceeds from the 3 ′ end as a starting material using deoxynucleoside triphosphate (dNTP) as a material, using the polymerase as described in (C) above as a catalyst and the target nucleic acid fragment as a template All reactions are included. This complementary nucleic acid extension reaction means that continuous extension reactions occur at least twice (for two bases).

The following shows examples of typical polymerase extension reaction and amplification reaction of the target site of the target nucleic acid fragment accompanied by the polymerase extension reaction. The simplest case is to perform the polymerase extension reaction in the 5 ′ → 3 ′ direction only once using the target nucleic acid fragment as a template. 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 initial target nucleic acid fragment. That is, it is a method 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 utilizing the polymerase extension reaction. For the amplification of the target nucleic acid, various methods that have been developed and invented so far can be used. The most popular and popular method for amplifying a target nucleic acid is the PCR (polymerase chain reaction) method. In the PCR method, denaturation (the step of denaturing a nucleic acid fragment from double-stranded to single-stranded) is controlled by periodically controlling the temperature rise and fall of the reaction solution → annealing (a primer is added to the nucleic acid fragment denatured to single-stranded). This is a method of amplifying a target portion of a target nucleic acid fragment by repeating the periodic steps of (hybridization step) → polymerase (Taq DNA polymerase) extension reaction → denature. Finally, the target site of the target nucleic acid fragment can be amplified up to 1,000,000 times the initial amount. Therefore, the amount of accumulated pyrophosphate generated in the polymerase extension reaction in the amplification process of the PCR method increases, and the detection becomes easy.

The cycling assay method using exonuclease described in JP-A-5-130870 is also one of the methods for amplifying a target site of a target nucleic acid fragment utilizing a polymerase extension reaction. This method is a method in which 5 '→ 3' exonuclease is acted together with a polymerase extension reaction starting from a primer specifically hybridized to a target site of a target nucleic acid fragment to decompose the primer from the reverse direction. A new primer hybridizes instead of the decomposed primer, and the extension reaction by the DNA polymerase proceeds again. The extension reaction by the polymerase and the decomposition reaction by the exonuclease which removes the previously extended chain are sequentially and periodically repeated. Here, the elongation reaction by the polymerase and the decomposition reaction by the exonuclease can be carried out under isothermal conditions. Also in this cycling assay method, the accumulated amount of pyrophosphate generated in the repeated polymerase extension reaction increases, and the detection becomes easy.

The LAMP method is a recently developed method for amplifying a target site of a target nucleic acid fragment. This method comprises at least four types of primers that complementarily recognize at least six specific sites of a target nucleic acid fragment,
By using a strand displacement type Bst DNA polymerase which has no nuclease activity in the 5 ′ → 3 ′ direction and which catalyzes the elongation reaction while releasing the double-stranded DNA on the template as single-stranded DNA, isothermal conditions can be obtained. Is a method of amplifying the target site of the target nucleic acid fragment as a special structure.
The amplification efficiency of this LAMP method is high, and the accumulated amount of pyrophosphate generated in the polymerase extension reaction is also very large, which facilitates detection.

When the target nucleic acid fragment is an RNA fragment, it is possible to carry out an extension reaction using reverse transcriptase having reverse transcription activity and using the RNA chain as a template. In addition, reverse transcriptase and T
The RT-PCR method in which an aq DNA polymerase is used in combination and an RT (reverse transcription) reaction is followed by a PCR reaction can be used. The presence of the RNA fragment of the target nucleic acid fragment can be detected by detecting pyrophosphate produced in this RT reaction or RT-PCR reaction. This method is effective in detecting the presence of RNA viruses.

(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, pyrophosphoric acid (PP
a) to adenosine triphosphate (AT) by sulfurylase
P) and adenosine triphosphate acts on luciferin by luciferase to detect luminescence. Therefore, in order to detect pyrophosphoric acid (PPi) by this method, a device capable of measuring luminescence is required.

[0034]

[Chemical 1]

A method for detecting pyrophosphoric acid suitable for the present invention is represented by Formula 2
Alternatively, the method shown in Expression 3 is used. The method shown in Formula 2 or Formula 3 is produced by converting pyrophosphate (PPi) into inorganic phosphate (Pi) by pyrophosphatase and reacting the inorganic phosphate (Pi) with xanthosine or inosine by purine nucleoside phosphorylase (PNP). Xanthine or hypoxanthine to xanthine oxidase (XOD)
Oxidize to produce uric acid, and hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process is used to produce peroxidase (PO
The color-developing agent (dye precursor) is caused to develop a color by D) and the color is compared. Since the results can be detected colorimetrically by the method shown in Formula 2 or Formula 3, pyrophosphoric acid (PPi) can be detected visually or by using a simple colorimetric measuring device.

[0036]

[Chemical 2]

[0037]

[Chemical 3]

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,
As EC1.11.1.7), commercially available products can be used. The color former (that is, the dye precursor) may be any one that produces a dye with hydrogen peroxide and peroxidase (POD), and for example, a composition that produces a dye by oxidation of a leuco dye (eg, US Pat. No. 4,089). , 74
7 and the like, triarylimidazole leuco dyes, JP-A-59-193352 (EP 01222641).
A) diarylimidazoleuko dyes described in A) and the like); compositions containing compounds that form dyes by coupling with other compounds when oxidized (for example, 4-aminoantipyrines and phenols or naphthols), etc. Can be used.

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

For quantitative analysis using such a dry analysis element, a fixed amount of a liquid sample is spotted on the surface of the spreading layer.
The liquid sample spread on the spreading layer reaches the reagent layer, where it reacts with the reagent and develops color. After spotting, the dry analytical element is kept at a constant temperature for a suitable time (incubation) to allow the color development reaction to proceed sufficiently, and then, 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 light is measured to obtain the reflection optical density, and quantitative analysis is performed based on the calibration curve obtained in advance.

Since the dry analytical element is stored and stored in a dry state until detection, it is not necessary to prepare the reagent at the time of use, and in general, the stability of the reagent is higher in the dry state. It is more convenient and quicker than the so-called wet method, which requires preparing a reagent solution before use. Further, it is also excellent as an inspection method capable of rapidly performing highly accurate inspection with a small amount of liquid sample.

(G) Pyrophosphoric acid quantitative dry analytical element: The pyrophosphoric acid quantitative dry analytical element that can be used in the present invention may have the same layer structure as various known dry analytical elements. The dry analytical element includes a support, a developing layer, a detection layer, a light shielding layer, an adhesive layer, in addition to the reagent for performing the reaction of the formula 2 or the formula 3 in the above item (E) (detection of pyrophosphoric acid (PPi)). It may be a multi-layer including a layer, a water absorbing layer, an undercoat layer and other layers. An example of such a dry analytical element is disclosed in Japanese Patent Laid-Open No. 49-53888 (corresponding US Pat. No. 3,99).
2,158), JP-A-51-40191 (corresponding US Pat. No. 4,042,335), and JP-A-55-164.
356 (corresponding U.S. Pat. No. 4,292,272) and JP-A-64-1959 (corresponding EPC published patent 016).
6365A).

The dry analytical element in the case of using the light-transmitting water-impermeable support may have the following practical constitution.
However, the content of the present invention is not limited to this. (1) One having a reagent layer on a support. (2) A support having a detection layer and a reagent layer in this order. (3) A support having a detection layer, a light reflection layer, and a reagent layer in this order. (4) A substrate having a second reagent layer, a light reflection layer, and a first reagent layer in this order on a support. (5) Detection layer, second reagent layer, light reflection layer, first layer on support
Those having reagent layers in this order.

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

A water absorption layer may be provided between the support and the reagent layer or the detection layer. A filter layer may be provided between the layers. A development layer may be provided on the reagent layer,
An adhesive layer may be provided between them.

The support may be any of light-impermeable (opaque), light-semi-transmissive (semi-transparent) and light-transmissive (transparent). Generally, it is light-transmissive and water-impermeable. Permeable supports are preferred. Polyethylene terephthalate and polystyrene are preferred materials for the light-transmitting water-impermeable support. In order to firmly bond 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. Examples of the fibrous material include filter paper, non-woven fabric,
Woven fabrics (eg plain weave fabrics), knitted fabrics (eg tricot knitted fabrics), glass fiber filter paper and the like can be used. As the non-fibrous material, a membrane filter made of cellulose acetate or the like described in JP-A-49-53888 and the like, JP-A-49-53888 and JP-A-5-1988.
5-90859 (corresponding US Pat. No. 4,258,00
1) JP-A-58-70163 (corresponding US Pat. No. 4,
486, 537) and the like, a continuous void-containing granular structure layer composed of inorganic or organic fine particles may be used.
JP 61-4959 A (Corresponding European Publication EP 01
66365A), JP-A-62-116258, JP-A-62-138756 (corresponding European publication EP 0).
226465A), JP-A-62-138757 (corresponding European publication EP 0226465A), JP-A-62.
-138758 gazette (corresponding European publication EP 02264
Also suitable are laminates of a plurality of partially bonded porous layers such as those described in 65A).

The porous layer may be a spreading layer having a so-called metering action, which spreads the liquid in an area approximately proportional to the amount of the liquid supplied. Of these, woven fabric, knitted fabric and the like are preferable as the spreading layer. Textile fabrics and the like may be subjected to glow discharge treatment as described in JP-A-57-66359. In the spreading layer, in order to control the spreading area, the spreading speed, etc., JP-A-60-222770 (corresponding: EP 0162301A), JP-A-63-21.
9397 gazette (corresponding West German patent publication DE 371791
3A), JP-A-63-112999 (correspondence: DE
3179913A), and a hydrophilic polymer or a surfactant as described in JP-A-62-182652 (corresponding to DE 3717913A) may be contained.

For example, after preliminarily impregnating or coating the reagent of the present invention on a paper, cloth, a porous membrane made of a polymer or the like, a special water-permeable layer, such as a detection layer, provided on the support, Kaisho 5
It is also a useful method to bond them by a method such as the one disclosed in JP-A-5-1645356.

The thickness of the reagent layer thus produced is not particularly limited, but when it is provided as a coating layer, it is 1 μm to 5 μm.
A range of about 0 μm, preferably 2 μm to 30 μm is suitable. In the case of a method other than coating, such as laminating by laminating, the thickness can greatly change in the range of several tens μm to several hundreds μm.

When the reagent layer is composed of a water-permeable layer composed of a hydrophilic polymer binder, examples of hydrophilic polymers that can be used include the following. Gelatin and derivatives thereof (eg phthalated gelatin), cellulose derivatives (eg hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymers and methacrylamide copolymers (eg acrylamide or methacrylamide with various vinylic monimers). Polymer), polyhydroxyethyl methacrylate,
Examples thereof include polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, and copolymers of acrylic acid and various vinylic monomers.

The reagent layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21677 (corresponding US Pat. No. 3,992,158) and JP-A-55-164356 (corresponding US Pat. No. 4,292,272). ), JP-A-54-
No. 101398 (corresponding US Pat. No. 4,132,52
8) It is provided by applying the aqueous solution or dispersion containing the reagent composition of the present invention and the hydrophilic polymer on another layer such as a support or a detection layer according to the method described in the description such as 8) and drying. You can 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 4 g / m ² to about 3
It is in the range of 0 g / m ² .

In addition to the reagent composition of formula 2 or formula 3, the reagent layer contains an enzyme activator for the purpose of improving various properties such as coating properties, diffusibility of diffusible compounds, reactivity, and storability. , Coenzymes, surfactants, pH buffer compositions, fine powders, antioxidants, and 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” edited by the Chemical Society of Japan.
Basics "(Maruzen Co., Ltd., issued in 1966) 1312-132
0 pages, R. M. C. Edited by Dawson et al, “Da
ta for Biochemical Research
h ”2nd edition (Oxford at the Claren
don Press, published in 1969) 476-508
Page, "Biochemistry," 5,467-477.
Page (1966), "Analytical Bioc
Chemistry ”104, pp. 300-310 (198).
There is a pH buffer system described in (year 0). Specific examples of the pH buffer system include borate-containing buffer; citric acid or citrate-containing buffer; glycine-containing buffer; bicine (Bi
Cine) containing buffer; HEPES containing buffer; M
Good buffers such as buffers containing ES are available. A buffer containing a phosphate cannot be used in the dry analysis element for detecting pyrophosphate.

The dry analytical element for determining pyrophosphoric acid which can be used in the present invention can be prepared by a known method described in the above-mentioned various patent specifications. The dry analytical element for the determination of pyrophosphoric acid is cut into a small piece such as a square having a side of about 5 mm to about 30 mm or a circle of about the same size.
No. 283331 (corresponding US Pat. No. 4,169,75
1), Japanese Utility Model Laid-Open No. 56-142454 (corresponding U.S. Pat. No. 4,387,990), Japanese Patent Laid-Open No. 57-63452, Japanese Utility Model Laid-Open No. 58-32350, Japanese Patent Publication No. 58-50.
No. 1144 (Corresponding International Public: WO083 / 0039
It is preferable to use it as a chemical analysis slide by putting it in the slide frame described in 1) and the like from the viewpoint of manufacturing, packaging, transportation, storage, measurement operation and the like. Depending on the purpose of use, long tapes can be stored in a cassette or magazine, or small pieces can be stored in a container with an opening, or small pieces can be attached or stored on an opening card, or cut pieces can be used. It can be used as it is.

The dry analytical element for quantifying pyrophosphoric acid which can be used in the present invention is capable of quantitatively detecting pyrophosphoric acid as a test substance in a liquid sample by the same operation as that described in the above-mentioned patent specifications. it can. 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. About 20 ℃ to about 45
At a constant temperature in the range of 30 ° C, preferably about 30 ° C to about 40 ° C
Incubate for 1-10 minutes at a constant temperature within the range. The amount of pyrophosphoric acid in the sample can be determined by measuring the color development or discoloration in the analytical element from the light-transmissive support side and using the calibration curve prepared in advance by the principle of the colorimetric measurement method. Quantitative analysis can be performed with high accuracy by keeping the amount of the liquid sample spotted, the incubation time and the temperature constant.

The measuring operation is carried out in JP-A-60-125543, JP-A-60-220862, and JP-A-61-2.
The chemical analyzer described in Japanese Patent No. 943667, Japanese Patent Laid-Open No. 58-161867 (corresponding U.S. Pat. No. 4,424,191), etc. enables highly accurate quantitative analysis with extremely easy operation. Depending on the purpose and required accuracy, the degree of color development may be visually determined to perform semi-quantitative measurement.

In the pyrophosphoric acid quantitative dry analytical element which can be used in the present invention, since it is stored and stored in a dry state until the analysis is performed, it is not necessary to prepare the reagent at the time of use, and in general, it is a dry state. Since the reagent has a higher stability, it is more convenient and quicker than the so-called wet method in which a reagent solution has to be prepared before use. Further, it is also excellent as an inspection method capable of rapidly performing highly accurate inspection with a small amount of liquid sample.

The dry analytical element for quantifying inorganic phosphorus that can be used in the second embodiment of the present invention can be prepared by removing pyrophosphatase from the reagent layer in the above-mentioned pyrophosphoric acid quantitative dry analytical element. In addition, JP-A-7
It is also possible to use the dry analysis element described in JP-A-197. The dry analytical element for quantitative determination of inorganic phosphorus has the same layer structure, manufacturing method, and use method as the aforesaid dry analytical element for quantitative determination of pyrophosphoric acid, except that the reagent layer does not contain pyrophosphatase.

(H) Kit: The analysis of the target nucleic acid of the present invention comprises 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 a dry analysis element for the determination of pyrophosphoric acid.

The form of the kit is such that at least a part of the target nucleic acid fragment has an opening capable of supplying a liquid containing a target nucleic acid fragment whose base sequence is known, at least one primer complementary to a part of the target nucleic acid fragment, and at least one kind. Deoxynucleoside triphosphate (dNTP), and at least one reaction cell part capable of holding at least one polymerase, a detection part capable of holding a dry analytical element for quantifying pyrophosphate, and the openings thereof, It may be a cartridge which is provided with a thin tube or a groove that connects the reaction cell section and the detection section and can move the liquid.

As such a cartridge, the cartridge described in US Pat. No. 5,919,711 and the like can be used. FIG. 2 shows an example of the cartridge type kit of the present invention. Kit 1
At 0, the sample solution containing the target nucleic acid can be supplied from the opening 31. The opening 31 is a thin tube 41.
Is connected to the reaction cell 32. Reaction cell 3
2 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. Furthermore, the reaction cell 32 is connected to the detection unit 33 by a thin tube 42. The dry analysis element 51 is held in advance in the detection unit 33. The sample solution in which the polymerase extension reaction has proceeded in the reaction cell 32 moves through the narrow tube 42 and is supplied onto the pyrophosphoric acid quantification dry analytical element 51 of the detection unit 33 to detect the pyrophosphate produced by the polymerase extension reaction. In the kit 10, the liquid can be moved between the opening 31 and the reaction cell 32 and between the reaction cell 32 and the detection unit 33 by using centrifugal force, electrophoresis, electroosmosis, or the like. Further, the reaction cell 32, the thin tubes 41 and 4
2. The detection unit 33 is preferably sealed by the base 21 and the lid 22.

When the cartridge type kit 10 as shown in FIG. 2 is used, as shown in FIG. 3, the temperature control parts 61 and 62 of the reaction cell 32 and the detection part 33 and the dry analysis for pyrophosphoric acid determination are carried out. Detection unit 7 capable of detecting color development or color change in element 51 by reflected light
It is desirable to use the devices comprising 1 and 72 together.

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

When the detection of pyrophosphoric acid produced by the polymerase extension reaction is carried out using a dry analytical element for quantitative determination of inorganic phosphorus after enzymatic conversion of pyrophosphoric acid into inorganic phosphoric acid, the first reaction cell is Preliminarily holding at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate (dNTP), and at least one polymerase, and carrying out a polymerase extension reaction in the first reaction cell. Then, the reaction solution in the first reaction cell is transferred to the second reaction cell, which is connected to the first reaction cell by a thin tube and in which pyrophosphatase is held in advance, and the polymerase in the first reaction cell is transferred. Pyrophosphoric acid generated in the extension reaction is converted into inorganic phosphoric acid in the second reaction cell, and then the reaction solution in the second reaction cell is mixed with the second reaction cell. Be linked by capillary into the cell is moved in advance to the detection unit quantifying inorganic phosphorus the dry analytical element is held, it is also possible to detect the inorganic phosphorus.

In addition, the "opening-
Tubule-reaction cell-capillary-detection section "are arranged in parallel,
Alternatively, a plurality of sets can be installed by arranging them in the radial direction of the 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 the target nucleic acid, a plurality of target nucleic acids can be detected simultaneously. It is possible to provide a possible kit.

The present invention will be described in detail below with reference to examples. However, the technical scope of the present invention is not limited by this embodiment.

[0067]

Examples Example 1: Detection of SRY gene-related site on the short arm of the Y chromosome using a dry analytical element for pyrophosphate determination (1) Preparation of a dry analytical element for pyrophosphate determination A gelatin subbing layer was provided. A colorless transparent polyethylene terephthalate (PET) smooth film sheet (support) having a thickness of 180 μm was coated with an aqueous solution of the composition (a) shown in Table 1 so as to have the following coverage, and dried to form a reagent layer. Provided.

[0068]

[Table 1]

An aqueous adhesive layer solution having the composition (b) shown in Table 2 below was applied onto the reagent layer so as to have the following coverage, and dried to form an adhesive layer.

[0070]

[Table 2]

Then, water was supplied to the entire surface of the adhesive layer at a rate of 30 g / m ² to swell the gelatin layer, and a broad woven fabric made of pure polyester was laminated on it by lightly applying pressure. Then, a porous spreading layer was provided.

Next, an aqueous solution having the composition (c) shown in Table 3 below was applied on the spread layer almost uniformly so as to have the following coverage, and dried to prepare a dry analytical element for quantifying pyrophosphoric acid. .

[0073]

[Table 3]

(2) Preparation of Target Nucleic Acid Fragment Sample Solution A commercially available nucleic acid extraction and purification kit (manufactured by QIAGEN, Inc.) was used for blood samples collected from one male and one female respectively.
A target nucleic acid fragment sample solution was prepared by recovering the genomic nucleic acid fragment extracted and purified using QIAamp DNA Blood Mini Kit) in 1 mL of purified distilled water.

(3) Preparation of primer The primer was synthesized as a set of oligonucleotide primers (primer 1, primer 2) having a base sequence designed to specifically recognize the SRY gene on the short arm of the Y chromosome. did.

<Base sequence of primer> Primer 1: 5'-GATCAGCAAGGAGCT
GGGATACACGTG-3 ′ (SEQ ID NO: 1) Primer 2: 5′-CTGTAGCTTCCCGTT
GCGGTG-3 '(SEQ ID NO: 2)

(4) Amplification of Target Nucleic Acid Fragment by Polymerase Extension Reaction A target nucleic acid fragment was amplified by PCR with the composition of the reaction solution shown below. PCR is [Denature:
It was carried out by repeating 30 cycles of 94 ° C. for 30 seconds, annealing: 65 ° C. for 30 seconds, and polymerase extension reaction: 72 ° C. for 1 minute.

<Composition of reaction liquid> Purified water 36.5 μL 10 x PCR buffer 5 μL 2.5 mM dNTP 4 μL Taq FP (Nippon Gene Co., Ltd.) 0.5 μL 20 μM primer 2 μL 30 ng / μL target nucleic acid fragment sample solution 2 μL

(5) Detection of Pyrophosphoric Acid Using Pyrophosphoric Acid Quantitative Analysis Element When a sample solution containing no target nucleic acid fragment was used in the amplification reaction of the target nucleic acid fragment by the polymerase extension reaction of (4) above (control) ), Using a target nucleic acid sample solution prepared from blood collected from a man (Sample M), and using a target nucleic acid sample solution prepared from blood collected from a woman (Sample F)
10 μL of the liquid after each reaction was spotted on the dry analytical element for quantifying pyrophosphoric acid prepared in (1) above, and the dry analytical element for quantifying pyrophosphoric acid was incubated at 37 ° C. for 5 minutes, and then the wavelength was 650 nm. At the reflection density (O
D _R ) was measured and found to be 0.287, 1.143, and 0.281 for the control, sample M, and sample F, respectively.

Example 1 shows that the SRY gene-related site on the short arm of the Y chromosome, which is unique to males, can be specifically detected. From the results of Example 1, it is possible to detect the presence of the target nucleic acid fragment by the method of the present invention in which pyrophosphoric acid produced by the progress of polymerase extension reaction is detected using a dry analytical element for quantifying pyrophosphoric acid. I know there is.

Example 2: Detection of single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase gene (ALDH2 gene) -related site using a dry analytical element for pyrophosphate determination (1) Preparation of target nucleic acid fragment sample solution The ALDH2 gene-related site differs in a specific single nucleotide species by sequencing
A target nucleic acid fragment was prepared in the same manner as in the method described in (2) of Example 1 based on a blood sample collected from one person who is known to be DH2 active type or ALDH2 inactive type. Sample liquids were prepared as a sample ALDH2 active type and a sample ALDH2 inactive type, respectively.

(2) Primer design The primer is a base designed as a primer specific to the ALDH2 active base sequence for a specific portion of the ALDH2 gene-related site on chromosome 12 that determines the activity of ALDH2. It was synthesized as a set of an oligonucleotide primer (primer 1) having a sequence and an oligonucleotide primer (primer 2) having a base sequence designed as a primer specific to the downstream base sequence of the specific site.

<Base sequence of primer> Primer 1: 5'-CAGGCATACACT GAA
GTGAAAACTG-3 ′ (SEQ ID NO: 3) (ALDH2 inactive form occurs when the underlined GAA base sequence becomes AAA) Primer 2: 5′-AGGTCCTGAACTTCC
AGCAG-3 '(SEQ ID NO: 4)

(3) Detection of Pyrophosphoric Acid Using Pyrophosphoric Acid Quantitative Analytical Element Preparation of the pyrophosphoric acid quantitative analytical element was carried out in the same manner as in Example 1 (1).
Amplification (PCR) of the target nucleic acid fragment by the polymerase extension reaction is performed in (4) of Example 1, and the reaction solution after the polymerase extension reaction is measured by the pyrophosphate analysis dry analysis element of Example 1. The reflection density (ORR) was measured in the same manner as in (5). As a result, the control, sample ALDH2 active type, and sample ALDH2 inactive type each showed 0.256,
It was 1.003 and 0.262.

Example 2 shows ALDH2 on chromosome 12.
It is shown that among the gene-related sites, the difference in the base sequence of the specific portion that determines the activity of ALDH2 can be specifically detected. From the results of Example 2, it is possible to detect the base sequence of the target nucleic acid fragment by the method of the present invention in which pyrophosphoric acid produced by the progress of polymerase extension reaction is detected using a dry analytical element for quantifying pyrophosphoric acid. It can be seen that it is.

Example 3 Detection of SRY Gene Related Site on Short Arm of Y Chromosome Using Inorganic Phosphorus Quantitative Dry Analyzing Element In preparation of the pyrophosphoric acid quantifying dry assay element shown in (1) of Example 1. Using a dry analytical element for quantitative determination of inorganic phosphorus prepared in the same manner except that pyrophosphatase was removed from the composition (a) of the reagent layer aqueous solution in Table 1, 100 μL of the reaction solution after the PCR reaction was treated with 10 units of pyrophosphatase. The measurement was performed in the same manner as above except that the treatment (pH 7.0, 37 ° C., 10 minutes) was performed, and the reflection density (OR _R ) was measured. For the control, sample M, and sample F, 0.268 and 1. 268 and 0.273
Met.

From Example 3, pyrophosphoric acid produced by the progress of the polymerase extension reaction, which is the second embodiment of the present invention, was converted into inorganic phosphoric acid by pyrophosphatase, and then the dry analytical element for quantifying inorganic phosphorus was used. It can be seen that the presence of the target nucleic acid fragment can be specifically detected even by the method of detection.

Example 4: Detection of Pseudomonas aeruginosa in human whole blood using a dry analytical element for the determination of pyrophosphate (experiment based on a model of examination of Pseudomonas aeruginosa sepsis) (1) Pseudomonas aeruginosa was added Preparation of human whole blood LB medium (Luria-Bertani medium)
Pseudomonas Sy cultured overnight in
lingae), a solution of which concentration was changed by dilution with PBS was added to human whole blood sampled with EDTA to give 0, 5 × 10 ⁵ , 1 mL / mL, respectively.
Six levels of human whole blood containing 5 × 10 ⁶ , 2.5 × 10 ⁶ , 5 × 10 ⁷ , and 1 × 10 ⁸ cells were prepared. here,
The number of bacterial cells is a value estimated using a spectrophotometer.

(2) Preparation of Dry Analytical Element for Quantifying Pyrophosphoric Acid A colorless transparent polyethylene terephthalate (PET) smooth film sheet (support) having a thickness of 180 μm and provided with a gelatin subbing layer is shown in Table 4 above. An aqueous solution having the composition (a) as described was applied so as to have the following coverage, and dried to form a reagent layer.

[0090]

[Table 4]

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

[0092]

[Table 5]

Then, water was supplied to the entire surface of the adhesive layer at a rate of 30 g / m ² to swell the gelatin layer, and a woven fabric cloth made of pure polyester was gently and evenly pressed on the gelatin layer. Laminated to provide a porous spreading layer.

Next, an aqueous solution of the composition (c) shown in Table 6 below was applied onto the spread layer almost uniformly so as to obtain the following coverage, dried, and cut into 13 mm × 14 mm, and the plastic mount was mounted. By putting it in the material, a dry analytical element for quantifying pyrophosphoric acid was prepared.

[0095]

[Table 6]

(3) Extraction and purification of nucleic acid from human whole blood 6 levels of human whole blood prepared by adding Pseudomonas aeruginosa prepared in (1) above were used as samples, and commercially available nucleic acid was extracted from each of them. Purification kit (QIAamp D, manufactured by QIAGEN)
The nucleic acid sample solution containing the target nucleic acid fragment was prepared by recovering the genomic nucleic acid fragment extracted and purified using NA Blood Mini Kit) in 1 mL of purified distilled water.

(4) PCR amplification Using the nucleic acid sample solution containing the target nucleic acid fragment obtained by extracting and purifying from the 6-level human whole blood sample in the above (3), PCR amplification was performed under the following conditions. I went.

<Primer> Pseudomonas aeruginosa genomic nucleic acid (Ice Nucleation Protein (I
The following primer set with the sequence (nak) N-terminal) was used. Primer (upper); 5'-GCGATGCTGTAATGACTCTCGACAAGC-3 '(SEQ ID NO: 5) Primer (lower); 5'-GGTCTGCAAATTCTGCGGCGTCGCTC-3' (SEQ ID NO: 6)

With the composition of the reaction solution shown below, [Modification: 94
PCR amplification was carried out by repeating 30 cycles of 1 minute at [deg.] C., annealing: 1 minute at 55 [deg.] C., extension reaction of polymerase: 72 minutes at 1 minute.

[0100] <Composition of reaction liquid> 10 × PCR buffer-5 μL 2.5 mM dNTP 4 μL 20 μM primer (upper) 1 μL 20 μM Primer (lower) 1 μL Pyrobest 0.25 μL 5 μL of nucleic acid sample solution obtained in (3) Purified water 33.75 μL

(5) Detection Using Pyrophosphoric Acid Quantitative Analytical Element The solution after the PCR amplification reaction in the above (4) was directly applied onto the dry analytical element for pyrophosphoric acid quantitative determination prepared in the above (2), 20 μL each. Then, after incubating the dry analysis element for the determination of pyrophosphoric acid at 37 ° C. for 5 minutes, the wavelength of 650
The time course of the reflection optical density (OD _R ) measured from the support side in nm is shown in FIG. 4, the reflection optical density (OD _R ) after 5 minutes is shown in FIG. 5, and the green pus in human whole blood is shown. The relationship between the number of bacteria and the reflection optical density (OD _R ) after 5 minutes is shown in FIG.

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

Example 5 Detection of Single Nucleotide Polymorphisms (SNPs) at Aldehyde Dehydrogenase Gene (ALDH2 Gene) Related Sites Using a Dry Analytical Element for Pyrophosphate Determination (The portion corresponding to the single nucleotide polymorphism was designated as primer 3) 'Example of setting near the end) (1) Preparation of nucleic acid sample solution containing target nucleic acid fragment Because one specific base type of the ALDH2 gene-related site differs in advance by sequencing the base sequence, AL
A commercially available nucleic acid extraction and purification kit (manufactured by QIAGEN, from each of blood samples collected from one person known to be DH2 active type or ALDH2 inactive type)
The nucleic acid sample solution containing the target nucleic acid fragment was prepared by recovering the genomic nucleic acid fragment extracted and purified using QIAamp DNA Blood Mini Kit) in 1 mL of purified distilled water.

(2) Preparation of dry analytical element for quantifying pyrophosphoric acid By the method described in Example 4, a dry analytical element for quantifying pyrophosphoric acid was prepared.

(3) PCR Amplification Using the nucleic acid sample solution containing the target nucleic acid fragment obtained by extracting and purifying from the ALDH2 active or ALDH2 inactive human whole blood sample in the above (1) as it is. ,
PCR amplification was performed under the following conditions.

<Primer> The primer determines a common primer (upper) in the ALDH2 gene-related site on chromosome 12 and the activity of ALDH2 1.
Set the part corresponding to the nucleotide polymorphism near the 3'end (lower-1
And lower-2) (underlined portions of the primer base sequences), corresponding to ALDH2 active type and inactive type, two types of primer (lower-1) and primer
A set of (lower-2) was used.

[0107] Primer (upper): 5'-AACGAAGCCCAGCAAATGA- 3 '( SEQ ID NO: 7) primers (lower-1): 5'- GGGCTGCAGGCATACACA G A-3' ( SEQ ID NO: 8) or, primer - (upper): 5 '-AACGAAGCCCAGCAAATGA-3' (SEQ ID NO: 9) Primer (lower-2): 5'-GGGCTGCAGGCATACACA A A-3 '(SEQ ID NO: 10)

With the composition of the reaction solution shown below, [denaturation: 94
PCR amplification was carried out by repeating 35 cycles of 20 ° C. for 20 seconds, annealing for 60 ° C. for 30 seconds, and polymerase extension reaction for 72 ° C. for 1 minute 30 seconds.

[0109] <Composition of reaction liquid> 10 × PCR buffer-5 μL 2.5 mM dNTP 5 μL 5 μM primer (upper) 2 μL 5 μM Primer (lower-1 or -2) 2 μL Taq 0.5 μL Nucleic acid fragment sample solution obtained in (1) 0.5 μL Purified water 35 μL

(4) Detection Using Pyrophosphoric Acid Quantitative Analytical Element The solution after the PCR amplification reaction in the above (3) was directly applied onto the dry analytical element for pyrophosphoric acid quantifying prepared in (2) above in an amount of 20 μL. Then, after incubating the dry analytical element for the determination of pyrophosphoric acid at 37 ° C. for 5 minutes, the wavelength of 650
The time change of the reflection optical density (OD _R ) obtained by measuring from the support side in nm is shown in FIG. 7, and the reflection optical density (OD _R ) after 5 minutes is shown in FIG.

From the results of Example 5, the common primer:
PCR was performed using a primer set with two types of primers corresponding to ALDH2 active type and inactive type, in which a portion corresponding to a single nucleotide polymorphism that determines the activity of ALDH2 was set near the 3'end, and Using the solution after the PCR amplification reaction as it is,
Using a dry analysis element for the determination of pyrophosphoric acid, the reflection optical density (O
D _R ), and its reflection optical density (OD _R )
ALDH of the sample depending on the relationship between the size and the two types of primers corresponding to the ALDH2 active and inactive types used.
2 active form, that is, the aldehyde dehydrogenase gene (A
It can be seen that single nucleotide polymorphisms (SNPs) in the LDH2 gene) -related site can be detected.

[0112]

INDUSTRIAL APPLICABILITY According to the present invention, a target nucleic acid fragment having a specific nucleotide sequence, which is effective for clinical examination of infectious diseases caused by viruses, bacteria, etc., and examination of genetic diseases due to individual genetic characteristics, etc. Simple and rapid methods and kits for analysis are provided.

[0113]

[Sequence list]

SEQUENCE LISTING <110> Fuji Photo Film Co. Ltd., <120> A method for analyzing a target nucleic acid
fragment and a kit for analyzing a target nucleic
acid fragment <130> A21324A <160> 10

<210> 1 <211> 27 <212> DNA <213> Artificial DNA <400> 1 gatcagcaag cagctgggat acacgtg 27

<210> 2 <211> 21 <212> DNA <213> Artificial DNA <400> 2 ctgtagcttc ccgttgcggt g 21

<210> 3 <211> 25 <212> DNA <213> Artificial DNA <400> 3 caggcataca ctgaagtgaa aactg 25

<210> 4 <211> 20 <212> DNA <213> Artificial DNA <400> 4 aggtcctgaa cttccagcag 20

<210> 5 <211> 27 <212> DNA <213> Artificial DNA <400> 5 gcgatgctgt aatgactctc gacaagc 27

<210> 6 <211> 25 <212> DNA <213> Artificial DNA <400> 6 ggtctgcaaa ttctgcggcg tcgtc 25

<210> 7 <211> 19 <212> DNA <213> Artificial DNA <400> 7 aacgaagccc agcaaatga 19

<210> 8 <211> 20 <212> DNA <213> Artificial DNA <400> 8 gggctgcagg catacacaga 20

<210> 9 <211> 19 <212> DNA <213> Artificial DNA <400> 9 aacgaagccc agcaaatga 19

<210> 10 <211> 20 <212> DNA <213> Artificial DNA <400> 10 gggctgcagg catacacaaa 20

[Brief description of drawings]

FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention.

FIG. 2 is a perspective view showing an example of a kit in the form of a cartridge of the present invention.

FIG. 3 is a perspective view showing a system configuration when a kit in the form of a cartridge of the present invention is used.

FIG. 4 shows the relationship between the number of Pseudomonas aeruginosa in human whole blood and the change with time of the reflected optical density (OD _R ).

FIG. 5 shows the relationship between the number of Pseudomonas aeruginosa in human whole blood and the reflection optical density (OD _R ) after 5 minutes.

FIG. 6 shows the relationship between the number of Pseudomonas aeruginosa in human whole blood and the reflection optical density (OD _R ) after 5 minutes.

FIG. 7 shows the relationship between the active / inactive form of ALDH-2 of a sample and the change with time of the reflection optical density (OD _R ).

FIG. 8 is a sample AHDH-2 type / primer.
The relationship between the species and the reflection optical density (OD _R ) after 5 minutes is shown.

[Explanation of symbols]

10 ... Kit 21 ... Base 22 ... Lid 31 ... Opening 32 ... Reaction cell 33 ... Detector 41 ... Capillary 51 ... Dry analytical element 61 ... Temperature control section 62 ... Temperature control section 71 ... Detection unit 72 ... Detection unit 81 ... Primer 82 ... Deoxynucleoside triphosphate (dNTP) 83 ... Polymerase

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 C12N 15/00 A (72) Inventor Yoshihide Iwaki 3-11-46 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. (72) Inventor Yumiko Takeshita 3-11-46 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. F-term (reference) 4B024 AA11 CA01 GA18 HA11 HA19 4B029 AA07 AA21 BB16 CC03 CC11 FA13 4B063 QA01 QA18 QQ03 QQ42 QQ89 QR03 QR07 QR08 QR13 QR32 QR38 QR41 QR55 QR57 QR58 QR62 QR82 QS25 QS34 QS39 QX01

Claims (12)

[Claims] 1. A target nucleic acid fragment whose base sequence is at least partially known, at least one primer complementary to a part of the target nucleic acid fragment, at least one deoxynucleoside triphosphate, and at least one polymerase. Reacting and detecting the presence or absence of progress of a polymerase extension reaction starting from the 3 ′ end of the primer using the target nucleic acid fragment as a template,
In the method for analyzing the target nucleic acid fragment, the presence or absence of the progress of the polymerase extension reaction is detected by detecting pyrophosphate produced along with the polymerase extension reaction. 2. The method for analyzing a target nucleic acid fragment according to claim 1, wherein pyrophosphoric acid is detected by using a colorimetric method. 3. The method for analyzing a target nucleic acid fragment according to claim 1, wherein pyrophosphoric acid is detected using a dry analytical element. 4. The dry analytical element for pyrophosphate determination, comprising a reagent layer containing xanthosine or inosine, pyrophosphatase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a color former. Item 4. A method for analyzing a target nucleic acid fragment according to Item 3. 5. The polymerase is DNA polymerase I, Klenow fragment of DNA polymerase I, Bst D.
The method for analyzing a target nucleic acid fragment according to any one of claims 1 to 4, wherein the method is selected from the group consisting of NA polymerase and reverse transcriptase (reverse transcriptase). 6. The method for detecting pyrophosphoric acid, which comprises enzymatically converting pyrophosphoric acid into inorganic phosphoric acid, and then providing a reagent layer containing xanthosine or inosine, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a coloring agent. The method for analyzing a target nucleic acid according to claim 1, wherein the method is performed by using a dry analysis element for use. 7. An enzyme for converting pyrophosphate into inorganic phosphate,
The method for analyzing a target nucleic acid fragment according to claim 6, which is pyrophosphatase. 8. The polymerase is DNA polymerase I, Klenow fragment of DNA polymerase I, Bst D.
The method for analyzing a target nucleic acid fragment according to claim 6 or 7, wherein the method is selected from the group consisting of NA polymerase and reverse transcriptase (reverse transcriptase). 9. The analysis of a target nucleic acid fragment according to claim 1, wherein the analysis of the target nucleic acid fragment is detection of the presence or amount of the target nucleic acid fragment or detection of the base sequence of the target nucleic acid fragment. Method. 10. The method for analyzing a target nucleic acid fragment according to claim 9, 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. 11. A target comprising at least one primer complementary to a part of a target nucleic acid fragment to be analyzed, at least one deoxynucleoside triphosphate, at least one polymerase, and each element of a dry analytical element for quantifying pyrophosphate. Nucleic acid fragment analysis kit. 12. At least one primer complementary to a part of a target nucleic acid fragment to be analyzed, at least one deoxynucleoside triphosphate, at least one polymerase, pyrophosphatase, and each element of a dry analytical element for quantifying inorganic phosphorus. A kit for analyzing a target nucleic acid fragment, which comprises: