JP2010233505A - Reagent kit for detecting nucleic acid amplification, having excellent preservation stability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent for maintaining the performance of a reagent for detecting the nucleic acid amplification, usable for genetic screening in clinical diagnosis. <P>SOLUTION: The preservation stability is improved by constituting the reagent for detecting the nucleic acid amplification, usable for the reaction for detecting the nucleic acid amplification by PCR of two or more liquid compositions, and formulating (a) a reagent composition containing at least a fluorescent labeled probe, a magnesium ion and a buffer, and having a pH of ≥6.5 but <8.5, and (b) a reagent composition containing at least a DNA polymerase and a buffer, in the different liquid compositions. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method and a kit for maintaining the performance of a reagent used in a nucleic acid amplification detection reaction by PCR.

Nucleic acid amplification detection reactions by PCR require many different reagents. Major reagents include nucleic acid amplification enzymes such as DNA polymerase, nucleoside triphosphates, oligonucleotide primers that are complementary to the target nucleic acid, magnesium ions, and other buffers. Furthermore, dye-labeled oligonucleotide probes for detecting amplification products and DNA-binding dyes such as SYBR Green I (registered trademark) are also necessary reagents for nucleic acid amplification detection reactions.
Some of these reagents are not stable depending on ambient temperature, pressure and humidity conditions. Therefore, conventionally, unstable reagents are put in separate containers in advance and stored at -20 ° C or lower, and when performing a nucleic acid amplification detection reaction, an operator removes each stored reagent and optimizes it. The reaction solution was carefully prepared by various methods. Preparation of such a reaction solution was not a special operation for researchers specializing in genetic engineering and molecular biology.

However, due to recent advances in nucleic acid amplification detection technology, genetic testing has been applied to various fields such as infectious diseases because it is more sensitive than conventional testing methods. In these fields, for example, when a laboratory technician uses it for clinical diagnosis, the reagent is not necessarily an expert who is used to handling genes and is not a person with advanced expertise. There are many.
In such a case, a reagent form that can be handled easily without complicated operations and that can be easily dispensed by an automated apparatus is desired. Furthermore, in the case of a reagent used for inspection, a reagent stored in a liquid form is preferred rather than thawing and using a cryopreserved reagent because it saves labor and time for thawing.

  In order to supply reagents that meet these requirements, the first thing that can be considered is to make it easier to use by mixing all the reagents necessary for the nucleic acid amplification detection reaction. It is known that the reagent performance is extremely reduced. In addition, even in the case of cryopreservation at −20 ° C., the inclusion of oligonucleotide primers, which are substrates for initiation of DNA polymerase, may reduce reagent performance due to increased non-specific reactions.

  One of the methods used as an amplification detection reagent for performing real-time PCR is the TaqMan (registered trademark) method. TaqMan® probes consist of DNA oligonucleotides labeled with donor and acceptor molecules. This probe is designed to bind to a specific region of one strand of the PCR amplification product. After the PCR primer anneals to this strand, the Taq enzyme extends the DNA by 5 'to 3' DNA polymerase activity. The TaqMan® probe is phosphorylated at the 3 'end to prevent initiation of Taq extension. When the TaqMan® probe hybridizes to the target nucleic acid, the growing Taq DNA polymerase hydrolyzes the probe from 5 ', releasing the donor from the acceptor as a basis for detection. The signal in this case is cumulative, and the concentration of free donor and acceptor molecules increases with each cycle of the amplification reaction (Non-Patent Document 1).

As another method used as an amplification detection reagent for performing real-time PCR, Patent Document 1 has C (cytosine) at the 3 ′ end, and C (cytosine) and G (guanine) are hydrogen-bonded. A Q-Probe method is disclosed in which PCR is performed using a primer labeled with a fluorescent dye that sometimes loses fluorescence. Thereby, it is supposed that the result of PCR can be simply confirmed by confirming the change of the fluorescence.
Usually, it is considered that the fluorescent dye may be deteriorated, and a method of detecting by adding a standard substance and correcting the obtained fluorescence value as a standard is used.

JP 2001-286300 A

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 7276-7280, August, 1991.

The TaqMan method and the Q-Probe method are technologies for detecting a nucleic acid using a fluorescently labeled probe, and are homogeneous detection technologies that can be detected at the same time that a DNA polymerase functions to amplify the nucleic acid. Since both detect fluorescence, maintaining the amount of fluorescence emitted by the fluorescent probe is a very important issue. In addition, since amplification and detection are performed simultaneously, there are many reagent components that constitute the reaction solution, and it is not easy to prepare a reaction solution with good storage stability. In the conventional reagent storage stabilization method, there has been no report on a reagent storage stabilization method including a fluorescent probe.

Furthermore, in homogeneous detection, maintaining the activity of an enzyme that amplifies the target nucleic acid is also an important issue. Until now, no useful method has been reported for stabilizing the enzyme while preserving the fluorescence of the fluorescent probe as much as possible and storing it in a state where it can be conveniently used.

An object of the present invention is to provide a reagent for maintaining the performance of a nucleic acid amplification detection reagent used for genetic testing in clinical diagnosis.

As a result of intensive studies in view of the above problems, the present inventors have divided a reagent necessary for a nucleic acid amplification detection reaction into two containers and optimally combined main components, thereby stably storing a fluorescently labeled probe. As a result, the present invention has been completed. That is, the present invention has the following configuration.

[Claim 1]
A method for improving the storage stability of a nucleic acid amplification detection reagent used in a nucleic acid amplification detection reaction by PCR, having the following characteristics (1) to (2).
(1) The reagent is composed of two or more liquid compositions, and when the reagent is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) In this reagent, a DNA polymerase and a fluorescently labeled probe are blended in separate liquid compositions.
[Section 2]
Item 2. A method for improving the storage stability of a nucleic acid amplification detection reagent according to Item 1, wherein the reagent composition containing a fluorescently labeled probe contains magnesium ions.
[Section 3]
Item 3. A method for improving the storage stability of a nucleic acid amplification detection reagent according to Item 2, wherein the magnesium ion concentration is 2 mM to 40 mM.
[Claim 4]
The fluorescent dye of the fluorescently labeled probe is any one selected from the group consisting of fluorescein or a derivative thereof, TAMRA, Cy3, Cy5, Cy5.5, rhodamine or a derivative thereof, ROX, Hex, JOE, BODIPYs, Alexas, BHQs Item 4. A method for improving the storage stability of the nucleic acid amplification detection reagent according to any one of Items 1 to 3.
[Section 5]
Item 5. The method for improving the storage stability of a nucleic acid amplification detection reagent according to any one of Items 1 to 4, wherein the fluorescently labeled probe is labeled with a fluorescent dye that is quenched by hybridization to a target nucleic acid.
[Claim 6]
Item 6. The method for improving the storage stability of the nucleic acid amplification detection reagent according to any one of Items 1 to 5, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof.
[Claim 7]
Item 7. A method for improving the storage stability of a nucleic acid amplification detection reagent according to any one of Items 1 to 6, further comprising an oligonucleotide primer in the reagent composition containing a fluorescently labeled probe.
[Section 8]
A kit used for a nucleic acid amplification detection reaction by PCR having the following characteristics (1) to (2).
(1) The kit is composed of two or more liquid compositions, and when the kit is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) The kit includes (a) a reagent composition containing at least a fluorescently labeled probe, magnesium ions and a buffer, and having a pH of 6.5 or more and less than 8.5, and (b) at least a DNA polymerase and a buffer. The reagent composition containing the agent is blended in another liquid composition.
[Claim 9]
Item 9. The nucleic acid amplification detection reagent kit according to Item 8, wherein the magnesium ion concentration in (a) is 2 mM to 40 mM.
[Section 10]
The fluorescent dye of the fluorescently labeled probe is any one selected from the group consisting of fluorescein or a derivative thereof, TAMRA, Cy3, Cy5, Cy5.5, rhodamine or a derivative thereof, ROX, Hex, JOE, BODIPYs, Alexas, BHQs Item 10. The nucleic acid amplification detection reagent kit according to Item 8 or 9, wherein
[Section 11]
Item 11. The nucleic acid amplification detection reagent kit according to any one of Items 8 to 10, wherein the fluorescently labeled probe is labeled with a fluorescent dye that is quenched by hybridization to a target nucleic acid.
[Claim 12]
Item 12. The nucleic acid amplification detection reagent kit according to any one of Items 8 to 11, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof.
[Claim 13]
Item 13. The reagent kit for detecting detection of nucleic acid amplification according to any one of Items 8 to 12, wherein the reagent composition (a) comprises an oligonucleotide primer.

According to the present invention, a nucleic acid amplification detection reaction can be performed simply and rapidly in clinical diagnosis. Specifically, since it is stored without freezing, it is possible to provide an amplification detection reaction solution without repeating freezing and thawing and to provide a reagent that can maintain reagent performance for a long period of time.

It is a figure which shows the fluorescence change analysis result of the quenching probe in a temperature change. It is a figure which shows the detection peak showing the fluorescence variation | change_quantity of the quenching probe in a temperature change. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of A, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saves at 35 degreeC with the reagent form of B, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of C, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of D, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of E, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of F, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of G, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saved at 35 degreeC with the reagent form of H, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saves at 8 degreeC with the reagent form of C, and a measured value. It is a figure which shows transition of the dissociation fluorescence value when it preserve | saves at 8 degreeC with the reagent form of H, and a measured value. It is a figure which shows transition of the fluorescence value of a fluorescence labeled probe when preserve | saving at pH 6.5 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescence labeled probe when preserve | saved at pH 7.0 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescent label probe when preserve | saving at pH7.5 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescence labeled probe when preserve | saved at pH 8.0 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescent label probe when preserve | saving at pH8.5 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescence labeled probe when preserve | saving at pH9.0 and 35 degreeC. It is a figure which shows transition of the fluorescence value of a fluorescence labeled probe when preserve | saved at pH9.5 and 35 degreeC.

Method for Improving Storage Stability of Nucleic Acid Amplification Detection Reagent One embodiment of the present invention is the storage of a nucleic acid amplification detection reagent used in a nucleic acid amplification detection reaction by PCR having the following characteristics (1) to (2): This is a method for improving stability.
(1) The reagent is composed of two or more liquid compositions, and when the reagent is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) In this reagent, a DNA polymerase and a fluorescently labeled probe are blended in separate liquid compositions.

Reagents for performing nucleic acid amplification reactions by PCR are often stored frozen at −20 ° C. or lower when used for normal research. When used, the required amount is taken after thawing at room temperature, and then stored again by freezing. When frozen and stored, it is necessary to perform such room temperature thawing at the time of use, which is not suitable for a rapid and simple clinical test. The present invention presupposes a reagent that can be used without freezing and thawing so that it can be used quickly and conveniently. 0 ° C. to 40 ° C. is a temperature at which the nucleic acid amplification reagent can be used without freezing and thawing, and is a temperature at which the reagent does not freeze.

Nucleic acid amplification reactions by PCR require many different reagents. The main reagent components include amplification enzymes such as DNA polymerase, nucleoside triphosphates (dNTPs) that serve as reaction substrates, oligonucleotide primers complementary to the target substance to initiate the reaction, and enzyme activation Magnesium ions and other buffers to be included. In addition, if detection is required after real-time PCR or amplification, a dye-labeled oligonucleotide probe, a DNA binding dye such as SYBR Green I ™, and an internal control nucleic acid are included.

Among these reagent components, mixing with specific components and storing them will reduce the activity of the amplification enzyme, hydrolyze the oligonucleotide primers and oligonucleotide probes, and fluorescence labeled with the oligonucleotide probes. There are combinations in which dyes and DNA-binding dyes deteriorate.
For example, it is generally known that the activity of DNA polymerase decreases when magnesium ions and DNA polymerase are mixed and stored. It is also known that even if DNA polymerase and an oligonucleotide primer serving as a reaction initiator are mixed and stored, a non-specific product is produced, and a specific reaction cannot be performed. It is also known that when a divalent metal ion and an oligonucleotide are mixed, DNase existing in a trace amount in the solution is activated and the oligonucleotide is easily hydrolyzed.

The term “stability” as used herein means, in a broad sense, that the amplification performance of the amplification reaction reagent is maintained, or that the fluorescence of the fluorescent dye of the detection reagent is maintained. Specifically, the activity of the DNA polymerase is maintained. That is, the sequence is maintained without hydrolysis of the oligonucleotide primer, and the fluorescent dye of the fluorescently labeled oligonucleotide probe is not separated from the probe, and the fluorescence amount of the fluorescent dye is maintained.

In general, the performance of amplification reagents largely depends on the activity of DNA polymerase and the performance of oligonucleotide primers. As for the storage stabilization method for maintaining the performance of the amplification reagent, how to stably store these two types of reagent components is decisive.
Storage of DNA polymerase with reduced activity leads to stabilization. For that purpose, a method for suppressing the activity of the DNA polymerase may be applied, and such a method is not particularly limited, but a method using an anti-DNA polymerase antibody, a method using an aptamer, and a method using BSA are used in combination. It is preferable. Furthermore, a method of storing at as low a temperature as possible and a method of freeze-drying DNA polymerase can be used in combination.
Oligonucleotide primers cause significant performance degradation due to hydrolysis. In particular, when the 3 ′ end is shortened by hydrolysis, the double-stranded binding ability is greatly changed, and sequence-specific binding may not be possible. Since this hydrolysis is caused by the action of DNase, it is necessary for stabilization of the oligonucleotide primer to prevent DNase contamination or to suppress the activity of DNase.

In order to stably store the amplification reagent, it is only necessary to maintain the activity of the DNA polymerase and the performance of the oligonucleotide primer. However, when the main components of these amplification reagents are divided into two containers, it is difficult to achieve compatibility because magnesium ions that activate DNA polymerase are simultaneously activators of DNase. In order to optimize the balance between the two, it is preferable to prepare and store a solution in which magnesium ions and oligonucleotide primers are mixed and DNase is removed as much as possible.

In general, the performance of the detection reagent depends on the amount of fluorescence possessed by a fluorescently labeled oligonucleotide probe (hereinafter abbreviated as probe) and the hybridization ability depending on the sequence of the probe. As for the storage stabilization method for maintaining the performance of the detection reagent, how to preserve the probe while maintaining the two kinds of capabilities of the probe is decisive.
The amount of fluorescence depends on, for example, the pH of the buffer solution to be stored. Keeping the pH in a suitable state is one way to stabilize it. Depending on the type of the fluorescent dye, there is a substance that significantly reduces the amount of fluorescence inherently contained by including a metal chelating agent such as EDTA in the storage solution.
As for the probe sequence, it is hydrolyzed in the same manner as the oligonucleotide primer, thereby causing a significant decrease in hybridization ability. Since this hydrolysis is caused by the action of DNase, it is necessary for the stabilization of the probe to prevent DNase contamination or to suppress DNase activity.

In order to stably store the amplification detection reagent, it is optimal if the amplification performance and detection performance of the reagent can be compatible. When the main component of the amplification reagent is divided into two containers, it is difficult to achieve compatibility because magnesium ions that activate DNA polymerase are simultaneously activators of DNase. In order to optimize the balance between the two, it is preferable to prepare and store a solution in which magnesium ions and oligonucleotide primers are mixed and DNase is removed as much as possible. When a detection reagent is added here in a homogeneous amplification detection reaction or the like, it is preferable to include the probe in a container containing oligonucleotide primers.

When used by a person who is not usually an expert, it is better to store them in a mixed state as much as possible for the sake of convenience. For example, it is preferable to store them in four parts, and it is more preferable to store them in three parts. . Saving in two is a more preferable storage method. Mixing and storing all the reagents necessary for the nucleic acid amplification reaction is not suitable for the present invention because the activity decreases as described above.

Fluorescent dye-labeled probe Generally, fluorescent dyes can be used that are labeled on oligonucleotides and used for the measurement and detection of nucleic acids. Oligonucleotides labeled with fluorescent dyes (hereinafter also referred to as fluorescently labeled probes). ) Is preferably used when the fluorescent dye labeled on the oligonucleotide favors detection when it is hybridized to the target nucleic acid. For example, fluorescein or derivatives thereof, such as fluorescein isothiocyanate (FITC) or derivatives thereof, such as Alexa, TAMRA, Cy3, Cy5, rhodamine 6G (R6G) or derivatives thereof , Tetramethylrhodamine (TMR), Texas Red (TEXAS red), BODIPY (trade name; manufactured by Molecular Probes, USA), ROX, Hex, JOE, BHQ Preferred are fluorescein, FITC, TAMRA, Cy3, Cy5, Te Threaded, and the like.

The fluorescent dye to be labeled on the fluorescently labeled probe may be any fluorescent substance that does not degrade or fluoresce during the nucleic acid amplification process, and that quenches upon hybridization. In particular, a fluorescent substance that causes quenching of fluorescence upon base pairing of guanine and cytosine at the end of the fluorescently labeled probe is preferable. Specifically, fluorescein or a derivative thereof (for example, fluorescein isothiocyanate (FITC)), BODIPY (registered trademark) series, rhodamine or a derivative thereof (for example, 5-carboxyrhodamine 6G (CR6G) or tetramethylrhodamine (TAMRA) )) And the like can be used, but the use of the BODIPY series and CR6G is particularly preferable. The method of binding the fluorescent dye to the oligonucleotide can be performed according to a usual method. By using quenching of fluorescent dyes, one type of fluorescent dye can be obtained without using dyes inserted into other double-stranded nucleic acid structures such as intercalators and without using two types of probes that cause the FRET phenomenon. A target nucleic acid sequence can be detected simply and specifically using a labeled probe. The labeling position of the fluorescent dye in the base sequence of the fluorescently labeled probe is not particularly limited, but is preferably labeled at the end.

Any desired labeling method known in the art can be used to attach the fluorescent dye to the oligonucleotide (Nature Biotechnology, Vol. 14, pp. 303-308, 1996; Applied and Environmental Microbiology, 63, 1143-1147, 1997; Nucleic acids Research, 24, 4532-4535, 1996). For example, when a fluorescent dye is bound to the 5 ′ end, first, for example, — (CH ₂ ) _n —SH is introduced as a spacer into the phosphate group at the 5 ′ end according to a conventional method. These introduced products may use commercially available products (Glen Research). In this case, n is an integer of 3 to 8, preferably 6. A labeled oligonucleotide can be synthesized by binding a fluorescent dye having SH group reactivity or a derivative thereof to this spacer. The oligonucleotide labeled with the phosphor group synthesized in this way can be purified by chromatography such as reverse phase to obtain a nucleic acid probe used in the present invention.

A fluorescent dye can also be bound to the 3 ′ end of the oligonucleotide. In this case, for example, — (CH ₂ ) _n —NH ₂ is introduced as a spacer into the OH group at the 3′-position C of ribose or deoxyribose. As these introduction bodies, products marketed in the same manner as described above may be used (Glen Research). In addition, a phosphate group is introduced, and, for example, — (CH ₂ ) _n —SH is introduced as a spacer into the OH group of the phosphate group. In these cases, n is an integer of 3-8, preferably 4-7. A labeled oligonucleotide can be synthesized by binding a phosphor group reactive to an amino group and an SH group or a derivative thereof to the spacer. The oligonucleotide labeled with the phosphor group synthesized as described above can be purified by chromatography such as reverse phase to obtain a nucleic acid probe used in the present invention.

It is also possible to introduce a fluorescent dye molecule into the strand of the nucleic acid probe (Analytical Biochemistry Vol. 225, pages 32-38 (1998)). In this case, it is convenient to use a thymine amidite (Glen Research) in which a commercially available fluorescent dye is already bound.

The pH when storing the fluorescently labeled probe is preferably 6.5 or more and less than 8.5, and more preferably pH 7.0 or more and less than 7.5.

DNA polymerase DNA polymerase may be used as long as it can be generally used for nucleic acid amplification reaction, and currently known DNA polymerase (single or combination) Taq DNA polymerase, EX-Taq, LA-Taq, Expand series , Platinum series, Tbr, Tfl, Tru, Tth, T1i, Tac, Tne, Tma, Tih, Tfi (PolI type enzyme), Pfu, Pfutubo, Pyrobest (registered trademark), Pwo, KOD, Bst, Sac, Sso , Poc, Pab, Mth, Pho, ES4, VENT, DEEPVENT (the above are α-type enzymes) and the like.
A natural DNA polymerase (mutant) in which one or several amino acid sequences are deleted, substituted or added by known means may be used. Alternatively, the above enzyme (natural type or mutant) may be further modified by means such as chemical modification. Alternatively, the above enzyme (natural type or mutant) may be further modified by means such as chemical modification.
For example, the most common Taq DNA polymerase and KOD DNA polymerase can be used. KOD DNA polymerase can be easily obtained from Toyobo (product code KOD-101, etc.).

It is further preferred that the DNA polymerase does not have 5 'exonuclease activity. In the present invention, the amount of fluorescence change from the labeled probe is sometimes used for detection of the target nucleic acid. Therefore, when the labeled probe and the second primer are hybridized to the extension product of the first primer in the high-speed PCR process, the labeled probe is used for a DNA polymerase having 5 ′ exonuclease activity, such as Taq DNA polymerase. With a non-specific signal increase. The DNA polymerase having no 5 'exonuclease activity is not particularly limited, but KOD DNA polymerase is preferred.

dNTPs
dNTPs is a mixture of dATP, dTTP, dCTP, and dGTP, and is a substrate substance for nucleic acid amplification reaction. It is preferable to mix four compounds in equal amounts. In an environment where freezing is not performed, dNTPs are preferably contained in a solution containing DNA polymerase. The concentration of dNTPs is not particularly limited, but is preferably 50 mM or more and 500 mM or less. Various of these are commercially available.

Magnesium ion Magnesium ion usually uses a compound in a salt state. Magnesium salts suitable for nucleic acid amplification reactions are preferred.
In the present invention, the magnesium salt shall mean any substance containing magnesium in such a form that magnesium ions are released into the aqueous solvent. Examples of the magnesium salt include, but are not limited to, magnesium chloride, magnesium hydroxide, magnesium carbonate, and magnesium sulfate. In particular, magnesium chloride and magnesium sulfate are preferable.

The concentration of magnesium ions is not particularly limited, but is preferably adjusted so as to be the concentration of the final reaction solution from 1 mM to 8 mM.
The final concentration of 1 mM to 8 mM indicates a suitable magnesium concentration used for the nucleic acid amplification reaction. When the reagent is stored, it is stored at a higher concentration to be finally mixed with another reagent mixture. For example, in the case of a nucleic acid amplification reaction system having a total volume of 50 μL and adding 12.5 μL of a DNA polymerase-containing mixture and 12.5 μL of a magnesium-containing mixture, the final solution volume is 4 times, so the magnesium ion concentration during storage is 4 mM To 32 mM is preferred. When mixing two reagents, the concentration ratio is suitably in the range of 2 to 10 times, preferably 2 to 8 times, more preferably 3 to 5 times. The magnesium ion concentration when considering such a concentration ratio is not particularly limited, but 2 mM to 40 mM is preferable. More preferably, it is 3 mM to 40 mM. When the concentration is less than 2 mM, for example, a 5-fold concentration results in a magnesium ion concentration of less than 0.4 mM, so that there is a possibility that PCR amplification will not occur without activation of the DNA polymerase. Moreover, since the density | concentration of the magnesium salt to melt | dissolve when it exceeds 40 mM is too high, preparation is difficult.

In addition, in the method of the present invention, a buffer solution, an amino acid or protein, a saccharide, a reducing agent polyhydric alcohol, and the like may be appropriately disposed in each liquid composition as necessary as long as the reaction is not affected.
Although it does not specifically limit as a buffer solution, Good buffer, such as Tris and Hepes, a phosphate buffer solution, etc. are used, Specifically, various buffers (pH 7.5-9 (pH 7.5-9 ( 25 ° C)). Tris buffer is preferable. A preferred buffered pH range is 7-9.
Amino acids or proteins may include sodium glutamate, albumin, skim milk, etc., sugars such as sucrose and maltose, reducing agents such as glutathione and mercaptoethanol, and polyhydric alcohols such as glycerol and sorbitol.
Further, a surfactant or the like may be included. The surfactant is preferably a nonionic surfactant, preferably exemplified by Triton X-100, Tween 20, Nonidet P40 and the like. The surfactant may be contained so as to be 0.0001 to 1%, preferably 0.001 to 0.1% during the reaction stage, that is, the polymerase reaction.

On the other hand, the concentration of the mixed solution containing DNA polymerase is preferably stored in a state where the concentration is as high as possible. However, in consideration of the ease of operation during mixing, the concentration ratio is in the range of 2 to 10 times, preferably 2 It is preferable to store at a magnification of 8 to 8 times, more preferably 3 to 5 times.

Kit Used for Nucleic Acid Amplification Detection Reaction One of the embodiments of the present invention is a kit used for a nucleic acid amplification detection reaction by PCR having the following characteristics (1) to (2).
(1) The kit is composed of two or more liquid compositions, and when the kit is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) The kit includes (a) a reagent composition containing at least a fluorescently labeled probe, magnesium ions and a buffer, and having a pH of 6.5 or more and less than 8.5, and (b) at least a DNA polymerase and a buffer. The reagent composition containing the agent is blended in another liquid composition.

Although the preparation method of each liquid composition in the kit of this invention is not specifically limited, The above-mentioned method can be illustrated.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not particularly limited to the following examples.

(Sample collection)
Genes were extracted from stomach biopsy material using QIAamp (registered trademark) DNA micro Kit (manufactured by Qiagen). The extracted DNA was stored at -20 ° C.

(Preparation of template plasmid)
PCR was performed using 1 μL of the extracted DNA solution as a template. The primer pair used here was a primer capable of full-length amplification of the H. pylori 23S rRNA gene. All were designed from the known sequence of the 23S rRNA gene (GenBank accession number U27270) of H. pylori.

  PCR was performed using GeneAmp (registered trademark) 9700 (manufactured by Applied Biosystems) as a thermal cycler, with a total volume of 26 μL. 1 μL of the DNA solution and a reagent mixture (KOD-plus 0.5 U, 10 × PCR buffer 2.5 μL, 200 μM dNTPs, each 5 μM primer) were mixed. After initial denaturation of the target DNA at 94 ° C. for 2 minutes, a cycle of 94 ° C./15 seconds denaturation step, 60 ° C./30 seconds annealing step, and 68 ° C./60 seconds extension step was repeated 35 times. The obtained nucleic acid fragment was ligated to pUC18 which was cleaved with a blunt end with a restriction enzyme, and a transformant was obtained using competent cell JM109. The transformant in which the target nucleic acid fragment was inserted was subjected to liquid culture and a plasmid was extracted.

  The nucleic acid sequence of the obtained plasmid was analyzed by an autosequencer to obtain a plasmid having the wild-type base sequence of 23S rRNA gene of H. pylori. Hereinafter, the wild type is indicated as WT.

(Synthesis of primer and labeled probe)
Primers and fluorescently labeled probes used for detection were obtained by consigning synthesis to a nucleic acid synthesis contractor.

The base sequences of the fluorescently labeled probe and primer used are shown.
The labeled probe is a 16-base sequence shown in SEQ ID NO: 1, the primer F is a sequence shown in 25 bases of SEQ ID NO: 2, and the primer R is a sequence shown in 26 bases of SEQ ID NO: 3.

(Detection of wild-type H. pylori)
Table 1 shows the reaction composition prepared by preparing each reagent as needed in a total amount of 10 μL. For nucleic acid amplification, a PCR method using KOD-plus- manufactured by Toyobo was used. For detection, the QP probe method (Fluorescent quenching-based quantitative detection of specific DNA / RNA using a BODIPY nucleoprobe. 29, No. 6, e34). Optimization of the reaction composition based on the composition in Table 1 can be easily performed by those skilled in the art. The amount of plasmid DNA was prepared so that about 1000 copies were contained in a total volume of 10 μL. Table 2 shows the reaction conditions and detection conditions during PCR amplification. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for the measurement of amplification and fluorescence. The measurement mode was 530 nm. Hereinafter, unless otherwise specified, all measurements are made with a light cycler (registered trademark). In the melting curve analysis, an increase in the amount of fluorescence when the labeled probe dissociates from the target nucleic acid is detected.
A sample containing WT (wild type) and NC (negative control: water is used as a template) are amplified and detected under the above conditions, and the analysis results of FIGS. 1 and 2 are obtained by analysis using the light cycler software. It was. The vertical axis in FIG. 1 indicates the fluorescence intensity, and the horizontal axis indicates the temperature (° C.). The vertical axis in FIG. 2 indicates the amount of change in fluorescence as the value of the inverse sign of the first derivative of the fluorescence intensity (−dF / dt), and the horizontal axis indicates the temperature (° C.).
FIG. 1 shows the change in fluorescence of the labeled probe in the melting curve analysis. In WT, as the temperature is raised, the labeled probe dissociates from the target nucleic acid, and the fluorescence value at 75 ° C. is the fluorescence value when the labeled probe completely dissociates from the target nucleic acid. The dissociated fluorescence value indicates the fluorescence value at 40 ° C. of NC (when water is used as a template), and can be used as a reference for the fluorescence amount of the fluorescently labeled probe.

  As a result of the analysis, FIG. 1 shows the fluorescence change of the labeled probe in the melting curve analysis. The fluorescence value at 75 ° C. is the fluorescence value when the labeled probe is completely dissociated from the target nucleic acid. The dissociation fluorescence value indicates a fluorescence value at 40 ° C. when water is used as a template, and can be used as a reference for the fluorescence amount of the fluorescently labeled probe. FIG. 2 shows a detection peak indicating the dissociation between the target nucleic acid and the fluorescently labeled probe in the melting curve analysis. The detection peak indicates the presence of the target nucleic acid that hybridizes with the fluorescently labeled probe.

Examination of amplification detection reagent performance at 35 ° C storage (reagent storage)
The reagent composition of the amplification detection reagent was fixed to the component amounts shown in Table 1. The main components of the amplification detection reagent were classified into five groups: DNA polymerase, dNTPs, magnesium ion, primer set, and fluorescently labeled probe, and each component was stored in two reagent mixtures. As shown in Table 3, each component was combined to prepare three reagent mixture sets. As the probe and primer, the labeled probe, primer F and primer R described in Example 1 were used. Each reagent mixed solution was put in a 1.5 mL screwed tube and arranged in a light-shielding rack. As for the storage method, in the case of 35 ° C. storage, it was stored in INCUBATOR MIR-153 manufactured by Sanyo Electric, and the set temperature was set to 35 ° C. In the case of storage at 8 ° C., the product was stored in INCUBATOR MIR-553 manufactured by Sanyo Electric Co., Ltd., and the set temperature was set at 8 ° C. During use, the two reagent mixtures were mixed before use.

(Reagent measurement)
A reaction solution 10 μL was prepared by mixing 2.5 μL of the DNA polymerase mixed solution, 2.5 μL of the primer mixed solution, and 5 μL of the plasmid DNA solution. The template amount was prepared so that about 1000 copies were contained in 5 μL, and the others were carried out according to the method of Example 1. The reagent stored at 35 ° C. was measured every week and was measured for up to 10 weeks.

(Data analysis)
Using the analysis software attached to LightCycler (registered trademark), the dissociated fluorescence value was calculated from FIG. 1, and the measured value was calculated from the height of the detection peak shown in FIG. . As the dissociation fluorescence value, the fluorescence value at 40 ° C. of the melting curve analysis is used as it is, and shows the fluorescence value that is the basis of the fluorescence-labeled probe. The confirmation method of amplification detection reagent performance can be compared by the height of the measured value. As the measured value, a value 10 times the absolute value of the height of the detected peak found in the graph obtained by differentiating the fluorescence value of the melting curve analysis is used.

(result)
The analysis results of the storage stability of the 35 ° C. storage reagent are shown in FIGS. As shown in FIGS. 3 to 6, when the combination of reagent components was changed from A to D, the dissociated fluorescence value could be detected even in the measurement result at the 13th week. It can be said that the storage of amplification detection reagents by these combinations is an excellent storage method because the fluorescently labeled probe showed fluorescence even at the 13th week. A composition common to A to D is that the enzyme KOD DNA polymerase and the fluorescently labeled probe are stored as separate mixed solutions. Further, the fact that the storage solution property of the fluorescently labeled probe is pH 7.4 is one factor that enables the fluorescently labeled probe to be stably stored.

  Further, when comparing A or B with C or D, it was possible to maintain the fluorescence value until the 13th week, but for A or B, the measured value was zero at the 10th week. A or B is considered to be caused by a decrease in the activity of the enzyme because KOD DNA polymerase, which is an enzyme, and magnesium ions are mixed and stored. From this result, in addition to stabilizing the fluorescence of the fluorescently labeled probe by storing the KOD DNA polymerase and the fluorescently labeled probe separately, the enzyme activity can be obtained by mixing magnesium ions in the reagent mixture containing the fluorescently labeled probe. It was found that storing in a combination of C or D that can maintain the above is preferable as a method for storing the amplification detection reagent.

Comparative Example 1

(Method)
Reagent storage, reagent measurement, and data analysis were performed according to Example 2. As shown in Table 4, among the main components of the amplification detection reagents classified into five, four types of reagent mixture sets in which the enzyme and the fluorescently labeled probe were mixed and stored were prepared. The sample was stored at 35 ° C. as in Example 2.

(result)
7 to 10 show the results of storage at 35 ° C. In the four reagent mixture sets from E to H, the dissociation fluorescence value at 13 weeks was lower than the detection sensitivity of the light cycler, and the fluorescence value was 0. In addition, F, G, and H have a faster decrease in enzyme activity than the dissociated fluorescence value, and the amplification detection performance as an amplification detection reagent is lost. On the other hand, although the enzyme activity was maintained in E, the fluorescence detection performance was lost in the 13th week because the fluorescence value was below the detection sensitivity of the light cycler due to the deterioration of the fluorescently labeled probe. From this result, it was found that when KOD and a fluorescently labeled probe were mixed and stored, deterioration of fluorescence of the fluorescently labeled probe was promoted.

Examination of amplification detection reagent performance by storage at 8 ° C. Reagent storage, reagent measurement, and data analysis were performed according to Example 2. The reagent mixing set of C and the reagent mixing set of H in Table 2 were used. The reagent stored at 8 ° C. was measured every month for up to 16 months.

(result)
The analysis results of the storage stability of the 8 ° C. storage reagent are shown in FIG. 11 or FIG. From FIG. 11, when the combination of the reagent components is C, the dissociation fluorescence value exceeds 1.2 even in the measurement result at the 16th month. Also, from FIG. 12, when the combination of the reagent components is set to H, the dissociation fluorescence value is 1.2 or less as a result of measurement at the 16th month. The combination of H indicates that the fluorescence of the fluorescently labeled probe is reduced in the performance of the amplification detection reagent. Similarly to the result of storage at 35 ° C., it was found that the amplification detection reagent stored by the combination of C did not significantly decrease the fluorescence value even when stored for 16 months by refrigeration. Further, it was found that even when stored at C for 16 months, the measured value serving as an index of the performance of the amplification detection reagent was maintained.

(Reagent storage)
The time-dependent change of the fluorescence-labeled probe in the pH change when stored at 35 ° C. was examined. A solution (10 mM Tris-H ₂ SO ₄ pH 6.5 to 9.5) for storing a fluorescently labeled probe was prepared. Further, each storage solution was added to 1 μM of the fluorescently labeled probe shown in SEQ ID NO: 1 to obtain a fluorescently labeled probe solution. Each fluorescently labeled probe solution was placed in a light-shielding rack by putting 500 μL in a 1.5 mL screwed tube. The preservation | save method was preserve | saved for SANYO Electric INCUBATOR MIR-153, and preset temperature was set to 35 degreeC.

(Reagent measurement)
A solution was prepared by diluting each fluorescently labeled probe solution 5-fold with 1 × KOD-plus-PCR buffer. 20 μL was filled into a light cycler (registered trademark) dedicated glass capillary, and the measurement temperature was set to 30 ° C. in Instrument's Real Time Fluorometer mode of the light cycler (registered trademark), and fluorescence measurement at 530 nm was performed. Each fluorescently labeled probe solution stored at 35 ° C. was measured every week for up to 6 weeks.

(Data analysis and results)
Fluorescence measurement results in a light cycler (registered trademark) were compiled for each pH to create a graph. The results are shown in FIGS. When compared with the difference in fluorescence values between the 0th week and the 6th week, it was 2.0 or more when stored in a solution of pH 8.5 or higher, and was less than 2.0 when stored at a pH of less than 8.5. It was. Furthermore, at pH 7.0 and pH 7.5, the difference in fluorescence value between the 0th week and the 6th week was less than 1.5, confirming that the deterioration of the fluorescent dye was further suppressed.

  According to the present invention, the reagent preparation can be carried out more easily and quickly by storing the nucleic acid amplification detection reagent, which has been stored for each component by cryopreservation, in a reagent mixture that can be stored in a refrigerator. Since the preparation is simple, genetic testing using nucleic acid amplification detection can be easily performed even in a clinical diagnosis place that does not have specialized knowledge in nucleic acid amplification technology, and it is expected to greatly contribute to the industry.

Claims (13)

A method for improving the storage stability of a nucleic acid amplification detection reagent used in a nucleic acid amplification detection reaction by PCR, having the following characteristics (1) to (2).
(1) The reagent is composed of two or more liquid compositions, and when the reagent is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) In this reagent, a DNA polymerase and a fluorescently labeled probe are blended in separate liquid compositions. The method for improving the storage stability of the nucleic acid amplification detection reagent according to claim 1, wherein the reagent composition containing a fluorescently labeled probe contains magnesium ions. The method for improving the storage stability of the nucleic acid amplification detection reagent according to claim 2, wherein the magnesium ion concentration is 2 mM to 40 mM. The fluorescent dye of the fluorescently labeled probe is any one selected from the group consisting of fluorescein or a derivative thereof, TAMRA, Cy3, Cy5, Cy5.5, rhodamine or a derivative thereof, ROX, Hex, JOE, BODIPYs, Alexas, BHQs The method for improving the storage stability of the nucleic acid amplification detection reagent according to any one of claims 1 to 3. The method for improving the storage stability of a nucleic acid amplification detection reagent according to any one of claims 1 to 4, wherein the fluorescently labeled probe is labeled with a fluorescent dye that is quenched by hybridization to a target nucleic acid. The method for improving the storage stability of the nucleic acid amplification detection reagent according to any one of claims 1 to 5, wherein the DNA polymerase is KOD DNA polymerase (from Thermococcus kodakaraensis KOD1) or a variant thereof. The method for improving the storage stability of the nucleic acid amplification detection reagent according to claim 1, further comprising an oligonucleotide primer in the reagent composition containing a fluorescently labeled probe. A kit used for a nucleic acid amplification detection reaction by PCR having the following characteristics (1) to (2).
(1) The kit is composed of two or more liquid compositions, and when the kit is used, a reaction solution is prepared by mixing all of the two or more liquid compositions.
(2) The kit includes (a) a reagent composition containing at least a fluorescently labeled probe, magnesium ions and a buffer, and having a pH of 6.5 or more and less than 8.5, and (b) at least a DNA polymerase and a buffer. The reagent composition containing the agent is blended in another liquid composition. The nucleic acid amplification detection reagent kit according to claim 8, wherein the magnesium ion concentration in (a) is 2 mM to 40 mM. The fluorescent dye of the fluorescently labeled probe is any one selected from the group consisting of fluorescein or a derivative thereof, TAMRA, Cy3, Cy5, Cy5.5, rhodamine or a derivative thereof, ROX, Hex, JOE, BODIPYs, Alexas, BHQs The nucleic acid amplification detection reagent kit according to claim 8 or 9, wherein The nucleic acid amplification detection reagent kit according to any one of claims 8 to 10, wherein the fluorescently labeled probe is labeled with a fluorescent dye that is quenched by hybridization to a target nucleic acid. The nucleic acid amplification detection reagent kit according to any one of claims 8 to 11, wherein the DNA polymerase is KOD DNA polymerase (derived from Thermococcus kodakaraensis KOD1) or a variant thereof. The nucleic acid amplification detection reagent kit according to any one of claims 8 to 12, wherein the reagent composition (a) comprises an oligonucleotide primer.

Images