WO2020175406A1 - Gene amplification method and gene amplification kit - Google Patents

Abstract

[Problem] To provide a gene amplification method, a gene amplification kit and others, in each of which gene amplification actually using a DNA polymerase under acidic conditions is employed and a method using pyridylboronic acid useful for the detection of pyrophosphoric acid under acidic conditions or the like can be utilized. [Solution] A gene amplification method comprising a step (I) of amplifying a target gene by a DNA polymerase reaction in an acidic reaction solution having a pH value of 4.2 to 6.9; a gene detection method comprising the step (I) and a step (II) of detecting the target gene on the basis of an amplification product or a reaction by-product produced in the step (I); and a gene amplification kit for carrying out the methods.

Description

\¥0 2020/175406 1 卩(: 17 2020/007232 Clarification

Title of invention: Gene amplification method and gene amplification kit

Technical field

The present invention relates to a gene amplification method, a gene amplification kit, and the like.

Background technology

[0002] Gene amplification technology is widely used in the medical field for pathogen testing, cancer diagnosis, etc., and in the life science field for food poisoning testing, water quality testing, genetically engineered crop testing, etc. .. As for gene amplification, a method is known in which the target gene is used as a template and allowed to act with an oral polymerase, and the generated target gene is detected by electrophoresis and staining. In addition, as a simple method for confirming gene amplification, a method for determining the amplification of a target gene by measuring pyrophosphate which is a reaction by-product is used.

As a method for measuring pyrophosphoric acid, which is a reaction by-product, there is a method of detecting with a fluorescent reagent, which has been put to practical use as a detection system such as real-time ◯. However, it is known that the method is expensive because it uses a fluorescent reagent and is easily affected by the fluorescent substance. As another method for measuring pyrophosphate, there is a method using an integrated transistor. This method is a method of evaluating gene amplification by capturing and detecting protons generated from pyrophosphoric acid, which is a by-product of the target gene amplification reaction, with a 1 ^to 1 sensor, and is also put to practical use as a detection system for an oral sequencer. ing. However, in this method, since it is necessary to capture the protons of the gene amplification reaction, it is necessary to suppress the buffering capacity in the reaction solution as much as possible. Therefore, when the enzyme reaction is repeated, the protons produced by the gene amplification reaction accumulate. As a result, it is known that the reaction solution tends to be acidic and deviates from the optimal levels 1 ^to 1 of the enzyme, resulting in a decrease in enzyme activity and a decrease in gene amplification (Non-Patent Document 1).

[0004] Therefore, as a method for solving these problems, there is a method using pyridylboronic acid that specifically recognizes pyrophosphate. This method does not require the use of a fluorescent reagent and is inexpensive, and pyrophosphoric acid and pyridylporonic acid can be produced under acidic conditions. \¥0 2020/175406 2 卩 (: 170? 2020 /007232

Since it works, even if the protons produced by the gene amplification reaction accumulate, it can be detected without any problem. However, the range of 1 ^to 1 that pyridylboronic acid can bind to pyrophosphate is 5 to 7, and the general gene amplification reaction is carried out on the alkaline side. It is known that depurination is likely to occur (Non-patent document 2), and in a gene where depurination has occurred, an oral polymerase cannot act, and therefore gene amplification does not occur. Therefore, this method has not been utilized because there is still no effective method for gene amplification under acidic conditions.

[0005] In addition, many studies have been conducted on gene amplification, and most of these methods are reactions on the alkaline side. Among them, there is a description about gene amplification when 1 ^to 1 is 7 or less. There are known documents (Patent Documents 1, 2, 3). However, since the known document does not aim to carry out gene amplification under acidic conditions, it does not show a specific composition or conditions for reacting an oral polymerase under acidic conditions. There is no mention of any examples of gene amplification.

[0006] Further, gene amplification has been studied using the 0.8 polymerase having an optimal 1 ^to 1 of 6 under acidic conditions (Non-Patent Document 3). However, in this method, the optimal 1 ^to 1 for gene amplification was 8.2, and gene amplification was not observed in 1 ^to 17.4. The optimal 1 ^to 1 and the optimal 1 ^to 1 of gene amplification of the enzyme is different, if it is 0 eight polymerase having the optimum! To acidic conditions, it does not mean it is gene amplification under acidic conditions. As described above, a gene amplification method using an oral polymerase under an acidic condition can be utilized so that a method using pyridylporonic acid, which is useful for detecting pyrophosphate under an acidic condition, can be utilized. It is a situation that has not yet occurred.

Prior art documents

Patent literature

[0007] Patent Document 1: Patent No. 4 6 9 9 4 1 5 Specification

Patent Document 2: Japanese Patent No. 5 0 0 7 4 4 0 Patent Document 3: JP 201 6 _ 52 1 1 20 JP

Non-patent literature

[0008] Non-Patent Document 1: Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research Research Results Report Issue No. 2670201 3 2nd Edition June 26, 2017

Non-patent document 2: Basic master Molecular biology P 1 5 — 1 6 Publication year 20 006 1 February Ohmsha

Non-Patent Document 3: E n z yme a n d M i c r o b i a l t e c h n o I o g y, 5 1, 334-34 1, 201 2

Summary of the invention

Problems to be Solved by the Invention

[0009] The present invention solves various problems in the prior art relating to gene amplification described above, and provides a method for selectively and conveniently amplifying a gene to be measured using a DNA polymerase under acidic conditions. The purpose is to provide.

Means for solving the problem

[0010] As a result of detailed examination of the method for amplifying a gene to be measured using DN A polymerase, the inventors unexpectedly can significantly perform a gene amplification reaction under acidic conditions. It was found that there are gene amplification reaction conditions such as the type of buffer solution and various salt concentrations, and the present invention was completed.

[0011] The present invention relates to the following aspects [1] to [8].

[1] A gene amplification method including a step (a) of amplifying a target gene by a DNA polymerase reaction in an acidic reaction solution of pH 4.2 to 6.9.

[2] The gene amplification method according to aspect [1], characterized in that the acidic reaction solution used in step (I) is prepared using a buffer solution having a pH of 4.0 to 6.9.

[3] The gene amplification method according to aspect [2], wherein the acidic reaction solution used in the step () includes 5 mM to 125 mM of potassium chloride.

[4] The DN A polymerase used in the step (丨) is derived from a T hermus genus, T her mo coccus genus, or Baci II us genus bacteriophage. \¥0 2020/175406 4 卩 (: 170? 2020 /007232

The gene amplification method according to aspect [1], which is a conventional O polymerase.

[5] The acidic reaction liquid used in step (I) contains a saccharide,

The gene amplification method described in [1].

[6] Aspect (1) to [5] Aspect (1), and a step of detecting the target gene based on the amplification product or reaction by-product obtained in the step () (II), including gene detection methods.

[7] In the step (II), the amount of the reaction byproduct generated in the step (丨) is measured, and the amount of the target gene is determined based on the measured amount of the reaction byproduct. Gene detection method.

[8] The gene detection method according to Aspect [7], wherein the reaction by-product is pyrophosphate.

[9] The gene according to the aspect [8], wherein the amount of the reaction pyrophosphate generated in the step (丨) is measured by measuring the potential change due to the reaction with pyridylboronic acid in the step (丨丨) Detection method.

[10] The gene detection method according to the aspect [8], wherein the amount of pyrrolic acid generated in the step () is measured by the absorbance method in the step ().

[11] A kit for gene amplification for carrying out the gene amplification method according to any one of aspects [1] to [5], which comprises an oral polymerase, a buffer, and a reaction reagent. , The gene amplification kit.

[12] A gene detection device or system for carrying out the gene detection method according to any one of aspects [6] to [10], comprising the gene amplification kit according to aspect [11]. The gene detection device or system, including: Effect of the invention

[0012] In the prior art, it was impossible to selectively amplify the gene to be measured under acidic conditions to detect the gene, but in the gene amplification method according to the present invention, , Depurination of gene as seen in the prior art is not observed, and the gene to be measured is significantly amplified by the action of 0.8 polymerase even under acidic conditions. You can Furthermore, subsequently, it becomes possible to selectively or simply detect or measure the gene by using the acidic reaction solution used in the amplification reaction.

Brief description of the drawings

[0013] [Fig. 1] Fig. 1 is a diagram showing the influence of a buffer solution in a gene amplification reaction solution on a DNA polymerase derived from the genus Thermocococcus.

[Fig. 2] Fig. 2 is a diagram showing the influence of a buffer solution in a gene amplification reaction solution on a D N A polymerase derived from the genus Thermu s.

[Fig. 3] Fig. 3 is a diagram showing the influence of a buffer solution and a potassium chloride concentration in a gene amplification reaction under acidic conditions in a D N A polymerase derived from the genus Thermocococcus.

FIG. 4 is a graph showing the influence of magnesium concentration on the gene amplification reaction.

[FIG. 5] A graph showing the influence of potassium chloride concentration on the gene amplification reaction.

[Fig. 6] Fig. 6 is a view showing an effect of adding trehalose in a gene amplification reaction.

FIG. 7 is a diagram showing a comparison of the gene amplification reaction under acidic conditions in the composition of the gene amplification reaction described in the present invention and a known document.

[Fig. 8] Fig. 8 is a diagram showing the influence of a buffer solution in a gene amplification reaction solution on a DNA polymerase derived from Bac i l u u s s u b t i I i s bacteriophage. [Fig. 9] Fig. 9 is a diagram showing the effect of potassium chloride concentration in a gene amplification reaction solution on a DNA polymerase derived from Bac i I u s s u b t i I i s bacteriophage.

MODE FOR CARRYING OUT THE INVENTION

[0014] The present invention relates firstly to a gene amplification method. Steps of the gene amplification method of the present invention (

In I), pH 4.2 to 6.9, for example, in the case of DNA polymerases derived from the genera T hermus and T hermococcus, preferably pH 5.7 to 6.9 or DNs derived from Bacil I lussubtilis bacteriophage. In the case of A polymerase, the gene (DNA) to be measured is preferably amplified in the DN A polymerase reaction in an (acidic) reaction solution having a pH of 5.0 to 6.5. The pH of the above acidic reaction solution is \¥02020/175406 6 卩 (: 17 2020 /007232

I 干〇 I 〇 13リ 3属などの微生物由来や巳 8〇 I I I リ 3 3リ匕 1： 丨 丨 丨 3バクテリオファージなどのウィルス由来の〇 八ポリ メラーゼが好ましい。 また、 組換え型口 八ポリメラーゼでも良く、 合成し た 0 八ポリメラーゼでも良い。 可溶性酵素が好ましいが、 不溶性酵素に界 面活性剤を組み合わせても良く、 可溶化タンパクとの融合又は膜結合部分の 削除等により不溶性酵素を可溶化させた酵素でも良い。 口 八ポリメラーゼ の公知のアミノ酸配列を利用でき、 組換え型の口 八ポリメラーゼとしては 、 公知の口 八ポリメラーゼと 60 %、 65 %、 70 %、 75 %、 80 %、Since it fluctuates somewhat as the width reaction progresses, it is determined by I·! at the start of the gene amplification reaction. The eight polymerase used in the method of the present invention may be any of eight polymerases derived from prokaryotes, eukaryotes and viruses. For example, 680 1 I 1 3 gene, D 1 1 16 16 111 3 3 00000li 3 genus, Ding 1"16" 11100 000li 3 genus,

It is preferable to use the 08 polymerase derived from a microorganism such as genus I 〇 I 〇 13 li 3 or a virus such as 8 Ⅲ III 3 3 底 1: 丨 丨 丨 3 bacteriophage. Also, a recombinant type eight polymerase may be used, or a synthesized eighteen polymerase may be used. Soluble enzymes are preferable, but surfactants may be combined with insoluble enzymes, or enzymes in which insoluble enzymes are solubilized by fusion with solubilized proteins or deletion of membrane-bound portions may be used. The known amino acid sequence of the oral polymerase can be used.Recombinant oral polymerase includes 60%, 65%, 70%, 75%, 80% of the known oral polymerase.

A protein having an amino acid sequence having 85%, 90% or 95% or higher identity and having an eighty-eight polymerase activity may be used. In the gene amplification method of the present invention, by repeating amplification of the gene to be measured in an 0.8 polymerase reaction in an acidic reaction solution within a specific range of!! It becomes possible to increase the reaction product (reaction by-product) and then measure the reaction by-product under such acidic!! conditions. Therefore, it is preferable to use 0.8 polymerase having a significant gene amplification activity in the range of 1 ^to 1 of such a reaction solution. As described above, the optimality of the orchard polymerase itself!! does not necessarily match the optimality of gene amplification activity!!.

[0015] The method for preparing the oral polymerase used in the gene amplification method of the present invention may be any method known to those skilled in the art, for example, water is added to an object containing the oral polymerase, a grinder, an ultrasonic wave. After crushing with a crusher etc., the crushed crushed product is centrifuged to remove the solid matter by filtration, etc., and there is also a method of purifying or isolating the extract by column chromatography, etc. It is possible to use a polymerase such as Hashiguchi polymerase.

[0016] Concentration of oral polymerase in the acidic reaction solution used in the gene amplification reaction Can be appropriately determined by those skilled in the art according to the type of sample, the estimated gene concentration in the sample, and various reaction conditions such as reaction time and temperature. For example, B aci I lus genus, T hermus genus, P yrococcus genus, T her mo coccus genus, Su I f 〇 I obus genus-derived microorganisms or B aci I lussubti I is bacteriophage in the gene amplification reaction solution, etc. The concentration of the virus-derived DN A polymerase can be 0.5 M9/mL or more, more preferably 1 g/mL or more, and further preferably 3 g/mL or more. In any case, the method of the present invention has the advantage that it is not necessary to add an excessive amount of DN A polymerase to the expected gene in the sample, since DN A polymerase can be used repeatedly. Therefore, the upper limit of the DNA polymerase concentration can be appropriately set by those skilled in the art in consideration of economical efficiency.

[0017] The target gene (DNA) in the gene amplification method of the present invention can be obtained by any method known to those skilled in the art. For example, it can be appropriately prepared from samples such as blood, fresh food, dietary foods and beverages according to the purpose of the test. Any method known to those skilled in the art can be used as a method for preparing DN A from each sample. For example, the sample is dissolved by sodium dodecyl sulfate, enzyme, bead disruption, etc. After binding to a silica membrane etc., the DNA prepared by eluting the DNA with an eluent can be used as a DNA sample for gene amplification. Further, it may be DNA c DNA depending on the type of amplification reaction.

[0018] The acidic reaction solution having a specific pH range used in the gene amplification method of the present invention has, for example, pH 4.0 to 6.9, preferably pH 4.0 to 6.9, depending on the type of DNA polymerase and reaction conditions. It can be appropriately prepared by those skilled in the art using a buffer having an appropriate pH range of 1 ^to 15.0 ^to 6.5, more preferably pH 5.5 ^to 6.5. The buffer solution may be any buffer agent known to those skilled in the art, preferably N a H ₂ P0 ₄ -N a ₂ HP 〇 ₄ , N a H ₂ P 〇 ₄ -N a 〇 H, N a H. ₂ Prepare using a phosphate buffer such as P 0 ₄ -K 0 H Prepare using a phosphate buffer or a citrate buffer such as citric acid-sodium citrate or citric acid 1 N a 0 H Such as citrate buffer Can be used. Regarding the (final) concentration of the buffer in the acidic reaction solution, for example, when the DNA polymerase derived from the genus T hermo coccus is reacted in a reaction solution prepared using a buffer solution of pH 5.5, The concentration of phosphate therein is preferably 0.1 to 200 mM, more preferably 0.5 to 100 mM, and further preferably 5 to 5 OmM. The upper limit of the concentration of such a buffering agent can be appropriately set by those skilled in the art in consideration of economical efficiency and the like. The acidic reaction solution in the above pH range can be prepared by using about 1 to 25% by volume of a buffer agent having a concentration of 1 to 10 times the final volume of the acidic reaction solution. It

[0019] The acidic reaction solution used in the gene amplification method of the present invention preferably contains potassium chloride. The concentration of potassium chloride in the reaction solution can be appropriately determined by those skilled in the art depending on the type of DNA polymerase and the reaction conditions. It is known that, in the conventional general gene amplification reaction in which the reaction is carried out on the alkaline side pH, the gene amplification reaction is inhibited when the potassium chloride concentration becomes 75 mM or more. In the gene amplification reaction described below, it is preferable to add potassium chloride so that it becomes excessive as the pH of the reaction solution used becomes more acidic. For example, when the DNA polymerase derived from the genus T hermus is reacted in an acidic reaction solution prepared by using a pH 6.0 buffer, the concentration of potassium chloride in the reaction solution is preferably 5 to 115 mM. , And more preferably 30 to 115 mM. For example, when a DNA polymerase derived from the genus T hermo coccus is reacted in an acidic reaction solution prepared using a buffer solution of pH 5.5, the final concentration of potassium chloride in the reaction solution is 5 to 115 mM is good, and more preferably 30 to 115 mM. Furthermore, for example, when the DNA polymerase derived from Baci II ussubtilis bacteriophage is reacted in an acidic reaction solution prepared by using a pH 4.0 buffer, the final concentration of potassium chloride in the reaction solution is 5 to 125 mM is preferable, and 30 to 125 mM is more preferable. Considering the buffer concentration, potassium chloride concentration should be low when the buffer concentration is high and low when the buffer concentration is low. The potassium concentration should be high. Further, the upper limit of the potassium chloride concentration can be appropriately set by those skilled in the art in consideration of economical efficiency and the like.

[0020] The acidic reaction solution used in the gene amplification method of the present invention further contains divalent ions. As the divalent ion, magnesium, manganese, cobalt or the like can be used, but magnesium is preferably used. The concentration of magnesium in the reaction solution used in the reaction is appropriately determined. For example, when the DN A polymerase derived from the genus T herm X c O ecus is reacted with a buffer solution of pH 5.5, the concentration of magnesium in the reaction solution is preferably 1.5 to 9 mM, more preferably 2 to 6 mM. Good. For example, when a DNA polymerase derived from the genus T hermus is reacted in an acidic reaction solution prepared using a pH 6.0 buffer, the magnesium concentration in the reaction solution is preferably 2 to 6 mM, more preferably 2 mg. ~4 mM is recommended. For example, when a DN A polymerase derived from a virus such as B acillus subtilis bacteriophage is reacted in an acidic reaction solution prepared using a buffer of pH 4.0, the magnesium concentration in the reaction solution is 1 to 30 mM. Good, and more preferably 10 to 14 mM.

In the gene amplification method of the present invention, in order to amplify a gene, deoxyadenosine triphosphate (d ATP), deoxycytidine triphosphate (d CTP), deoxycytimidine triphosphate (d TTP) ), deoxyribonucleotide triphosphate (d NT P) mixed with 4 types of deoxyguanosine triphosphate (d GT P ). The concentration of dNTP in the reaction solution used in the present reaction can be appropriately determined by those skilled in the art according to various reaction conditions.For example, the concentration of dNTP is preferably 0.01 mM or more, more preferably It can be 0.1 mM or more, more preferably 0.2 mM or more.

[0022] Further, in the gene amplification method of the present invention, various kinds of primers are used to amplify a gene. The primer can be appropriately designed and prepared by those skilled in the art based on the specific nucleotide sequence of the gene to be amplified. The concentration of the primer in the reaction solution used for the reaction can be appropriately determined by those skilled in the art according to various reaction conditions. For example, the primer concentration is O. OI mM or more, \¥02020/175406 10 卩 (: 170? 2020 /007232

More preferably, it can be 0.101 IV! or higher, and even more preferably 〇.201 IV! or higher.

门を繰り返すことや、

[0023] The gene amplification reaction in the step () of the gene amplification method of the present invention is performed by any method known to those skilled in the art, for example, various polymerase chain reactions (○ [¾) and the like. The temperature and time in the cycle may be any temperature at which a gene amplification reaction occurs. For example, in the case of using 08 polymerases of 3 gen. 11 ㊀ ``01 li genus and 11 gen ⊀ `` 01 〇 〇 〇 〇 〇 Ⅲ, 98° 〇 1 036 〇 ® 55 ° 0 · 3 〇 36 〇 ® 72° 0 ·

Repeating the door,

、 I 门加熱、 30°〇まで冷却した後、 30°〇 1 6 「 ® 65 °0 1 0

I n処理することなどできる。 酸性条件下では、 高温ほど 遺伝子の脱プリン化が起こりやすいことから低温で遺伝子増幅を行うことが 好ましい。 门 ® 94. 〇 · 3 〇 36 〇 ® 62° 0 · 3 〇 36 〇 ® 72° 0 · 303 ø 〇 It is possible to make a 72 ^° 〇 1 0 1 door after repeating ø 〇. 630 1 1 I 3 When using a polymerase derived from Teriophage

, I After heating and cooling to 30° 〇, 30° 〇 1 6 “® 65 ° 0 1 0

I n can be processed. Under acidic conditions, gene depurination is more likely to occur at higher temperatures, so it is preferable to perform gene amplification at lower temperatures.

[0024] Further, the amount of the reaction product can be further increased by including (coexisting) a saccharide in the acidic reaction solution used in the gene amplification method of the present invention. As saccharides, saccharose, trehalose, and other saccharides known to those skilled in the art can be used. The concentration of saccharides in the reaction solution used for the reaction can be appropriately determined by those skilled in the art according to various reaction conditions. For example,

リメラーゼを 1^~16.0の緩衝液を用いて調製し た酸性反応液中で反応させる場合、 該反応液中の糖類濃度は、 1 %以上がよ く、 より好ましくは 5%以上がよい。 例えば、

When Eight polymerase is reacted with a buffer solution of 1 ^to 15.5, the saccharide concentration in the reaction solution is preferably 10% or less, more preferably 5% or less. For example, Ding

When reacting limerase in an acidic reaction solution prepared using a buffer solution of 1 ^to 16.0, the saccharide concentration in the reaction solution is preferably 1% or more, more preferably 5% or more. For example,

III 3 When a virus-derived 08 polymerase such as a bacteriophage is reacted in an acidic reaction solution prepared using a buffer solution of 1 ^to 14.0, the saccharide concentration in the reaction solution is preferably 0.01% or more, It is more preferably 1% or more. \¥02020/175406 11 11 (: 170? 2020 /007232

[0025] Furthermore, by adding (coexisting with) bovine serum albumin (Miwa 38), a nonionic preparation, and an ammonium ion to the acidic reaction solution used in the gene amplification method of the present invention, the stability of the enzyme and the mouth can be improved. And the amount of reaction products can be increased. The concentration of Min 3 can be appropriately determined by those skilled in the art according to various reaction conditions. For example, in the case of reacting an oral polymerase derived from genus 116000001 genus 3 with a buffer solution of 1 ^to 15.5, the concentration of Min 3 in the reaction solution is preferably 0.001% or more, more preferably 0.005% or more, More preferably, it is 0.01% or more.As the nonionic preparation, nonionic preparations known to those skilled in the art such as -40 and Triton X-100 can be used. The concentration of the nonionic preparation can be appropriately determined by those skilled in the art according to various reaction conditions.For example, in the case of reacting an oral polymerase derived from 3 genus 0100000 with a buffer solution of 1 ^to 15.5, The concentration of the nonionic drug in the composition is preferably 0.0005% or more, more preferably 0.001% or more, further preferably 0.01% or more, and the ammonium ion is ammonium ion known to those skilled in the art such as ammonium sulfate. The concentration of ammonium ion in the reaction solution used for the reaction can be appropriately determined by those skilled in the art according to various reaction conditions. When the reaction is carried out with a buffer solution of 1 ^to 15.5, the ammonium ion concentration in the reaction solution is preferably 1 2001 IV! or less, more preferably 1 0001 IV! or less, still more preferably 80 IV! or less.

[0026] Each reaction component such as a reaction reagent/enzyme used in the step () of the method of the present invention is an arbitrary method known to those skilled in the art as long as it is an addition method in which a gene amplification reaction occurs under a predetermined acidic condition. It can be added to the reaction system by procedures, etc. For example, each component may be added in advance to the reaction solution at once before the start of the reaction, or a sample containing the oral polymerase or gene may be added last to react. Therefore, for example, as in the examples of the present specification, potassium chloride, divalent ions, saccharides, and the like can be contained in the O buffer solution in advance.

[0027] Secondly, the present invention relates to an eight-eight polymer in an acidic reaction solution of 1 ^to 14.2 to 6.9. \¥0 2020/175406 12 12 (: 170? 2020 /007232

— A method for detecting a gene, comprising a step (II) of detecting the target gene based on the amplification product or reaction by-product obtained in the step (丨) after the step (丨) of amplifying the target gene by a zease reaction. Pertain.

The detection of the target gene based on the amplification product or the reaction by-product can be performed qualitatively, semi-quantitatively, or quantitatively by using any method and means known to those skilled in the art.

For example, it is possible to detect qualitatively or semiquantitatively by electrophoresing an amplified gene, which is an amplification product, and then staining it. Alternatively, there is a method of detecting pyrophosphoric acid, which is a reaction by-product, with a fluorescent reagent.

Alternatively, the amount of each reaction byproduct such as pyrophosphate and hydrogen ions generated in the step () is measured, and the amount of the target gene is determined (quantitatively detected) based on the measured amount of the reaction byproduct. Can be done. In this method, it is possible to use a calibration curve based on a certain correlation between the amount of reaction by-products and the amount of measured gene. In particular, any method and means known to those skilled in the art can be used for measuring the amount of pyrophosphoric acid generated in the step () of the present invention. In particular, since the gene to be measured can be amplified under acidic conditions in the step () of the present invention, a method for measuring a potential change by pyridylporonic acid specifically detecting pyrophosphate, which is carried out under acidic conditions. Can be used effectively. Further, the pyrophosphate produced in the step () of the present invention is combined with hypoxanthine-guanine phosphoribosyl transferase, xanthine oxidase or xanthine dehydrogenase, or pyrophosphate is treated with inorganic pyrophosphatase to form two molecules of phosphorus. By measuring the acid and its phosphoric acid, pyrophosphoric acid can be measured by the absorbance method with a higher sensitivity, for example, a method combining luminol with inorganic pyrophosphatase, pyruvate oxidase and peroxidase. Further, in measuring the proton (hydrogen ion) generated from pyrophosphate in the gene amplification reaction, it is possible to use a glass electrode for detecting hydrogen ion or a measuring method for measuring the potential change by an ion-sensitive field effect transistor. it can. Pyrophosphate, hydrogen ion, etc. generated in the step () of the present invention are appropriately \¥02020/175406 13 卩 (: 170? 2020 /007232

It can be measured by separating it from the solution for gene amplification reaction. The method for separating pyrophosphoric acid, hydrogen ions and the like from the reaction solution is not particularly limited as long as it does not affect the measurement, but examples thereof include vapor chromatography separation and separation using a microfluidic device. To be

等） 並びに、 糖類等の各種添加剤等を含む、 遺伝 子増幅用キッ トを提供する。 当該キッ トは、 安定化剤又は緩衝剤等の当業者 に公知の他の任意成分を適宜含有させ、 前記酵素等試薬成分の安定性を高め ても良い。 測定に影響の無い成分であれば特に限定されないが、 例えば、 卵 白アルブミン、 糖アルコール類、 カルボキシル基含有化合物、 酸化防止剤、 界面活性剤等を例示できる。 [0028] Further, the present invention is for carrying out the above-mentioned method of the present invention, wherein each of the above-mentioned components necessary for amplifying the target gene, for example, 〇 polymerase, buffer, reaction reagent (primer and

Etc.), and a kit for gene amplification containing various additives such as sugars. The kit may appropriately contain other optional components known to those skilled in the art, such as a stabilizer or a buffer, to enhance the stability of the reagent component such as the enzyme. The component is not particularly limited as long as it does not affect the measurement, and examples thereof include ovalbumin, sugar alcohols, carboxyl group-containing compounds, antioxidants and surfactants.

[0029] The present invention also provides a gene detection device or a gene detection system including the gene amplification kit for carrying out the gene detection method (measuring method). In addition to the kit for gene amplification, the gene detection device or the gene detection system according to the present invention, based on the amplification product or reaction by-product obtained in the step of amplifying the target gene in the method of the present invention (), Various means for carrying out the step (II) for detecting: Depending on the method, each of the means: Any reagents, devices, instruments, kits, etc. that are known in the technical field and are required for the method It can be included as appropriate.

Hereinafter, the present invention will be described specifically with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.

Example 1

(Preparation of polymerase derived from hyperthermophile)

由来の口 八ポリメ ラーゼ遺伝子 （非特許文献 3） を組み込んだプラスミ ド 巳丁 28匕 （+） で大腸菌 〇 361 1 3 2（0巳 3） 1_ _{7 3}3を形質転換し、 発現株として 用いた。 発現株について、 カナマイシンを終濃度 50 9/ 1_及びクロラム フエニコールを終濃度 34 M g/m L含む L B培地により 37°Cで 4時間培養 後、 終濃度〇. 2mMとなるように 丨 PTGを添加した。 さらに、 培養液を 25^°Cで終夜培養後、 集菌を行い、 得られた菌体を超音波破砕し、 無細胞抽 出液を調製した。 調製した無細胞抽出液について遠心分離を行い、 得られた 上清の一部を用いて電気泳動法により目的酵素の発現を確認した。 次いで残 りの上清をアフイニテイカラム （商品名 ： H i T r a p H e p a r i n H P、 GEヘルスケア製） により夾雑タンパクを除去し、 0. 1 5 m g/mL の T h e r mo c o c c u s属由来の D N Aポリメラーゼを得た。 なお、 T h e r m u s属由来の D N Aポリメラーゼはタカラバイオ製 （商品名 ： E x T a q HS） 、 B a c i I I u s s u b t i l i sバクテリオフアー ジ由来の D N Aポリメラーゼは関東化学製 （商品名 ： p h i 29 D N Aポ リメラーゼ） を用いた。 Ding 1" 16 "1 1 100000 Ri 3

Escherichia coli 〇 361 1 3 2 (0 _ 3) 1_ _{7 3} 3 was transformed with the plasmid (28) (+) containing plasmid derived from the lipophilic polymerase gene (Non-patent document 3) and used as an expression strain. I was there. For expression strains, kanamycin was added to a final concentration of 50 9/1_ and chloram. After culturing at 37°C for 4 hours in LB medium containing phenicol at a final concentration of 34 M g/mL, PTG was added to a final concentration of 0.2 mM. Furthermore, after culturing the culture solution at 25 ^° C overnight, the cells were collected, and the obtained cells were ultrasonically disrupted to prepare a cell-free extract. The prepared cell-free extract was centrifuged, and a part of the obtained supernatant was used to confirm the expression of the target enzyme by electrophoresis. Then, the remaining supernatant was subjected to an affinity column (trade name: Hi Trap Heparin HP, manufactured by GE Healthcare) to remove contaminating proteins, and 0.15 mg/mL of T her mo coccus-derived A DNA polymerase was obtained. The DNA polymerase derived from the genus T hermus is manufactured by Takara Bio (trade name: Ex Taq HS), and the DNA polymerase derived from Baci II ussubtilis bacterium is manufactured by Kanto Kagaku (trade name: phi 29 DNA polymerase). Using.

[0032] The base sequence of the polymerase DNA derived from the genus Therm oc cccus is as follows.

[D NA Polymerase from the genus T her mo c o c c u s]

ATGGGGATCCTGGACGCAGACTACATTACGGAAGATGGCAAGCCGGTCATTCGTGTGTTCAAGAAAGAA AAGGGCGAATTCAAAATCAATTATGATCGTGACTTTGAACCGTATATTTACGCTCTGCTGAAAGATGAC AGCGCGATCGAAGATATTAAAAAGATCACCGCTGAACGTCACGGTACCACGGTCCGTGTGACGCGCGCC GAACGTGTTAAAAAGAAATTTCTGGGTCGCCCGGTTGAAGTCTGGAAACTGTATTTCACCCATCCGCAG GATGTGCCGGCTATTCGTGACAAAATCCGCGAACACCCGGCGGTGGTTGATATTTATGAATACGACATC CCGTTTGCAAAGCGTTATCTGATTGATAAAGGCCTGATCCCGATGGAGGGTAACGAAGAACTGCGCATG CTGGCGTTTGACATTGAAACCCTGTACCATGAAGGCGAAGAATTCGGCGAAGGTCCGATTCTGATGATC AGCTATGCGGATGAAGAAGGTGCCCGTGTGATTACCTGGAAAAATATCGACCTGCCGTATGTTGAAAGT GTCTCCACGGAAAAAGAAATGATTAAGCGCTTTCTGAAAGTGATCCAGGAAAAAGATCCGGACGTTCTG ATTACCTATAACGGCGATAATTTTGACTTCGCGTACCTGAAGAAACGTTCAGAAACGCTGGGTGTTAAG TTCATTCTGGGCCGCGATGGTTCGGAACCGAAAATCCAACGTATGGGCGACCGCTTTGCCGTGGAAGTT AAAGGTCGCATCCACTTCGATCTGTACCCGGTGATTCGTCGCACCATCAACCTGCCGACCTATACGCTG GAAACGGTGTACGAAGCCATTTTTGGCCAGCCGAAAGAAAAGGTTTATGCAGAAGAAATCGCACAAGCT \¥02020/175406 15 15 (: 170? 2020 /007232

実施例 2

Example 2

[0033] (Effect of Buffer in Gene Amplification Reaction Solution under Acidic Conditions in 08 Polymerase from Genus 6)

As the final concentration at the time of polymerase chain reaction (○[¾)), the one having the composition shown in Table 1 was used as a 〇[buffer]. 〇 [¾ in each buffer solution, 2.5 1\/ 1 for 4 !_, 〇.1% for 3/8 for 2.5 !-, 〇.01% 7 V \ I 〇 n X — 1 0 \¥02020/175406 16 卩 (: 170? 2020 /007232

実施例 1 で取得した丁 6 「 0100000 リ 3属由来の口 八ポリメラーゼを 1 !_添加し、 50 !_の 〇 増幅反応液を調製した。 〇 の反応温度と時間は、 表 2に示す 条件で実施した。 0 to 5 1-, 1 0 1\/1 Forward primer (1): 〇 080 〇 〇 〇 〇 〇 〇 〇 〇 口 ody 〇 0010, and reverse primer (1 ): 〇 Dynamic 〇 〇 〇 〇 〇 〇 888-chome 080-chome-chome 〇-chome 880-chome 2!_, 6.

1 !_ was added to the polymerase 6 derived from the genus 01000003, which was obtained in Example 1, to prepare 50 !_ amplification reaction solution. The reaction temperature and time of ◯ are the conditions shown in Table 2. It was carried out in.

[0034]

table 1

* Composition of each buffered product;

Miscellaneous ³ \¥02020/175406 18 18 (: 170? 2020 /007232

? 1 ^to 1 ₂ 〇 ₄ - 3_Rei_1 ^to 1 ¹ to 15.5 and 6.0, as well as, 5_Rei_11 \ / 1 ₃ 1 ^- 1 ₂ 〇?

₄ -? At ₃₂ 1 ^to 1 〇 ₄ 1 ^- 16. Samples with 0 (Practical Product for 5-7), amplify the desired gene was confirmed.

Example 4

[0036] (Effect of buffer in gene amplification reaction solution under the acidic conditions on T he "Li3 genera-derived eight polymerase)

〇 As the final concentration during the reaction, the ones with the compositions shown in Table 3 and Table 4 were used as buffer solution. To each 〇 buffer, 2. 5 1 \ / 1-chome a 4 1 -.!, 1 0 1 \ / 1 forward primer (1) and reverse primer (1) each 〇 4 _, 6.4门₉ / !_ of Suu Hachi 1 !_, º _X 7 aq 1 ^to 13 Polymer

-0.25%, and trehalose was added so that the final concentration was 10% (only the products 8, 9, and 10 were added), and 50 times of the amplification reaction solution was prepared. The reaction temperature and time of ○ were carried out under the conditions shown in Table 5.

[0037]

Table 3

*Each! The p-scale buffer is composed of substances with

〔 ^:0038 \\0 2020/175 406 20 20201007232

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Size ₁ 〇

View

Example 5

[0039] ○ After amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the products 8 to 16 prepared in Example 4, and as shown in FIG. Amplification of the target gene was confirmed in the sample (Product 8) using N a H ₂ P 〇 ₄ -N a ₂ HP 〇 ₄ (pH 6.0), but the sample using citrate buffer was used. No amplification was observed in any of the above. In addition, in the sample (Product 11) using ₂ mM Na H ₂ P0 ₄ -K0H (pH 6.0), amplification of the target gene was confirmed, but Na H ₂ P 〇 ₄ -K 〇 No amplification was observed in the samples using H (pH 5.0 or 5.5). Furthermore, no amplification was observed in the samples using the McKilvein buffer (pH 5.0, 5.5, or 6.0).

Example 6

[0040] (Effect of Buffer and Potassium Chloride Concentration in Gene Amplification Reaction Solution under Acidic Conditions in Thermocococcus-Derived DNA Polymerase)

As the final concentration during the PC R reaction, the composition shown in Table 6 was used as the PC R buffer solution. Add 2.5 mM d NTP to 4 !_, 0.1% BSA to 2.5 !_, 0.01% Triton X—100 to 5 !_, 10^M in each PC R buffer. Forward primer (1) and reverse primer (2): GCATTGCCCGT CAGGCTAATTCTGAA was 2 !_, 6. ng/^L of sou DNA was 2.5 !_, and DNA derived from the genus T her mo coccus obtained in Example 1 1 ML of polymerase was added to prepare 50 ML of PC R amplification reaction solution. The PCR reaction temperature and time were set as shown in Table 7.

[0041]

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Operation

«^ ¾ * Easy 3 ¾ ¾ ¾-^ · ® ¾ *! _!? 52_Rei ₂ 6 Ri 4 _£,,

Art ₉

Example 7

[0042] [After amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the products 17 and 22 prepared in Example 6, and as shown in FIG. In the sample using MN a H ₂ P ○ _4- NaOH (pH 5.5), amplification of the target gene was observed in the samples containing 5 mM KC I and 30 mM KC I (Products 17 and 18). Although confirmed, no amplification was observed in the sample supplemented with 50 mM KC I. In addition, in the sample using 100 mM Na H ₂ P 〇 ₄ -N a 0 H (pH 5.5), amplification was not observed in any of the samples to which 5, 30, and 50 mM KCl were added. ..

Example 8

(Effect of magnesium concentration on gene amplification reaction)

As the final concentration during the PC R reaction, the composition shown in Table 8 was used as the PC R buffer solution. For each PCR buffer, 2.5 mM d NTP is 41_, 0.1% BSA is 2.5 !_, 0.01% Triton X—100 is 5!_, 10^M Forward Apply two primer (1) and two reverse primer (2),

1 L of 6.4 ng/L of Sue DNA and 1 L of D N A polymerase derived from the genus Thermococcus obtained in Example 1 were added to prepare a 50 !_ PCR amplification reaction solution. The PCR reaction temperature and time were set as shown in Table 7.

Example 9

[0044] After PCR amplification, for the products 23 to 27 prepared in Example 8, amplification of the target gene was confirmed by agarose gel electrophoresis, and as shown in Fig. 4, the final concentration during PCR reaction was 2, 4 Amplification of the gene of interest was confirmed in samples using PCR buffer containing 6 mM Mg C ₂ (Products 24 to 26). In addition, pyrophosphoric acid in the solution after the PCR amplification reaction was measured using a pyrophosphoric acid measurement kit (trade name: PP i Light Inorganic Pyrophosphate Assay, manufactured by L〇NZA). Therefore, the amount of pyrophosphate was highest when the PCR buffer containing 2 mM MgC ₂ was used as the final concentration in the PCR reaction. The amount of pyrophosphate was shown as a relative value, with the highest value being 100%.

[0045] \¥0 2020/175406 24 卩 (: 17 2020 /007232

Maki ₆

ToshiKasumikumo I 5 righteousness 5蠢 _{V · § 9 V (, -} ° ¾4 '

₈

Example 10

[0046] (Effect of potassium chloride concentration on gene amplification reaction)

〇 [The final concentration at the time of the reaction has the composition shown in Table 10 It was For each PC buffer, 2.5 mM d NT P 41_, 0.1% BSA 2.5 !_, 0.01% Triton X—100 5^L, 10^M Forward Apply two primer (1) and two reverse primer (2),

1 L of 6.4 ng/L of Sue DNA and 1 L of D N A polymerase derived from the genus Thermococcus obtained in Example 1 were added to prepare a 50 !_ PCR amplification reaction solution. The PCR reaction temperature and time were set as shown in Table 7.

Example 11

[0047] After PCR amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the products 28 to 33 prepared in Example 10, and as shown in Fig. 5, at the final concentration during the PCR reaction, The amplification of the target gene was confirmed in the samples using the PCR buffer containing 30, 50, 75, or 100 mM KCI (Products 30 to 33). The pyrophosphoric acid in the solution after the PCR amplification reaction was measured using a pyrophosphoric acid measurement kit (trade name: P Pi Light Inorganic Pyrophosphate Asassay, manufactured by L〇NZA). The maximum amount of pyrophosphate was highest when using a PCR buffer containing 75 or 100 mM KC I at the final concentration in the PCR reaction. The amount of pyrophosphoric acid was shown as a relative value, with the highest value being 100%.

[0048]

\¥0 2020/175 406 26 卩 (: 17 2020 /007232

Example 12

[0049] (Effect of addition of trehalose on gene amplification reaction under acidic conditions)

〇 [The final concentration at the time of reaction should be one with the composition shown in Table 12 as 〇 buffer solution. did. For each PCR buffer, add 2.5 mM dNTP 4 !_, 10^M forward primer (1) and reverse primer (1) at 4 ML each, 6.4 n 9/1 _. S_0_8_11_ and Ex Taq HS polymerase were added to 0.25 ML to prepare a 5 OML gene amplification reaction solution. The PCR reaction temperature and time were set as shown in Table 5.

Example 13

[0050] After PCR amplification, the samples 34 to 37 prepared in Example 12 were treated with agaro.

-When amplification of the target gene was confirmed by electrophoresis, amplification of the target gene was confirmed in all samples as shown in Fig. 6. In addition, pyrophosphoric acid in the solution after the reaction was measured using a pyrophosphoric acid measurement kit (trade name: PP i Light Inorganic Pyrophosphate Assay, manufactured by L〇NZA), and as shown in Table 13, trehalose The amount of pyrophosphate generated in the gene amplification reaction increased with the addition of the. The amount of pyrophosphate was shown as a relative value, with the highest value being 100%.

[0051]

\\0 2020/175 406 28 20201007232

52

¾: «X ¾ ¾! ¾ ¾ ¾ ¾ · ¾ 9-«? ¾ Ⅱ _/ ¾ 〇 _< 1 _{¾ |} 4 _< .' _| ,

Art _{2 I}

[0052] [Comparative Example 1]

(Comparison of the gene amplification reaction under acidic conditions in the present invention and the gene amplification reaction composition described in the known literature) \¥02020/175406 29 卩 (: 170? 2020 /007232

Commercially available 〇 eight polymerase!? (Trade name: Michichi Ding 39 ^1-13, Takara Bio Co., Ltd.) in the attachment buffer on the basis of the composition as claimed in only ₂ IV ₃ 1 ^~ 1 2 〇 ₄ _ 3 ₂ 1 change in ^~ 1? 〇 ₄ ⁽¹ to 16.0), as the final concentration at the time 〇 reaction, having composition of comparative product 1 shown in Table 1 4 was 0 buffer. 0 Buffer solution 5!_

の反応温度と時間は、 表 5に示す条 件で実施した。 , 2.5 11\/1 for 4 1_, 1 0 1\/1 for forward primer (1) and reverse primer (1) for each 0.4 !_, and 6.4 ng/ML for sou 8 1_ the Michichi 7 aq 1 ^~ 13 polymerase 〇. 25 1_ was added, to prepare a gene amplification reaction solution 50 1_.

The reaction temperature and time were determined according to the conditions shown in Table 5.

Example 14

[0053] 〇 For the final concentration at the time of reaction, use the composition of the product 38 of Table 14

緩衝液に、 2. 5 1\/1 1\1丁？を 4 ! -、 1 0 1\/1 フォワードプライマー（ 1）及びリバースプライマー（ 1）を各 0.4 し、 6 . 4 n g/MLのスー〇 八を 1 !_、 º X 7 a q 1^~13ポリメラーゼを 〇. 25 し添加し、 5〇 しの遺伝子増幅反応液を調製した。 〇［¾の反 応温度と時間は、 表 5に示す条件で実施した。 また、 表 1 4の実施品 39の 組成のものを 〇 緩衝液とし、

緩衝液に、 2. 5 1\/1 1\1丁？を 4 !_、 〇. 1 % 巳3八を 2. 5 !_、 〇. 01 %、 7 V \ I〇 n X- 1 00を 5 ! -、 1 0 !\/1 フォワードプライマー（ 1）及びリバースプライ マー（2）を各 2 !_、 6. 4门 9/ !_のスーロ 八を 1 !_、 丁 1"16 「 111It was used as a buffer solution.

In the buffer solution, 2.5 1\/1 1\1 pc? 4 !-, 1 0 1\/1 0.4 forward primer (1) and reverse primer (1) 0.4 each, and add 6.4 ng/ML sou 1 to 1 !_, º X 7 aq 1 ^to 13 polymerase Was added to prepare 50 gene amplification reaction solutions. ◯ [The reaction temperature and time were measured under the conditions shown in Table 5. The composition of the product 39 in Table 14 was used as the 〇 buffer solution.

In the buffer solution, 2.5 1\/1 1\1 pc? 4 !_, 〇 .1% 巳 3 8 to 2.5 !_, 〇 .01%, 7 V \ I 〇 n X-100 5 !-, 1 0 !\/1 Forward primer (1) And reverse primer (2) for each 2 !_ and 6.4 for 9/ !_ Suuro 8 1 !_, Ding 1" 16 "111

の反応温度と時間は、 表 7に示す 条件で実施した。 A total of 50 !_ gene amplification reaction liquids were prepared by adding 1 !_ of a polymerase derived from 3 genus. Also,

The reaction temperature and time were as shown in Table 7.

[0054] \ ¥ 0 2020/175406 30卩(: 17 2020/007 232 Subesun _I

Example 15

[0055] After amplification, Comparative Product 1 and Working Product 3 prepared in Comparative Example 1 and Example 16

Amplification of the target gene was confirmed by agarose electrophoresis for 8 and 39. Around this time, as shown in Fig. 7, gene amplification was not recognized in the conventional gene amplification composition, but gene amplification was recognized under acidic conditions by changing the type of buffer solution and potassium chloride concentration.

Example 16

(Effect of Buffer in Gene Amplification Reaction Solution under Acidic Conditions on Bacillus subtilis Bacteriophage-Derived DNA Polymerase) As a final concentration during PCR reaction, PCR buffer with the composition shown in Table 15 was used. It was a liquid. ○ [¾ buffer solution 21_, 0.5 ja g/jaL·p UC 119 11_, 100 M-13 F primer: 2 L of CAGTCGTCATGCATTGCCTGCTC, 5 !_ of sterilized water, 10 ML A template mixture solution was prepared. The template mixture was heated at 95°C for 1 minute and then cooled to 30°C at 0.1°C/sec. After cooling, 〇 [¾ buffer solution 21_, 25 mM d NTP 0.8 1_, 100 mM DTT 11_, 100 U/mL pyrophosphatase 0.2 L, 50^9/mL B acillus subtilis bacterio 2 mL of phage-derived DNA polymerase and 4 mL of sterilized water were added, and 10 mL of the reaction mixture was added to the template mixture, and the PCR reaction was performed at 30 ^° C for 16 hours.

[0057]

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94 ^ !*:: _/ ^ _{..-¾ ¾} ^ · "- ^ * "" _{0-6 :}

Example 17

[0058] 〇 [After amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the products 40 to 44 prepared in Example 16 and as shown in FIG. M citric acid-sodium citrate pH 4.0, 35 mM citric acid sodium citrate pH 4.5, 35 mM N a H ₂ P 〇 ₄ -N a 〇 H pH 5.0 and 35 mM Na Amplification of the target gene was confirmed in the sample using H ₂ P 〇 ₄ -N a ₂ HP 〇 ₄ pH 6.0 (implemented products 41 to 44), and the gene was detected near room temperature under acidic conditions. It was confirmed that the gene can be amplified in acidic condition under the condition of pH 4 by performing amplification.

Example 18

(Effect of Potassium Chloride Concentration in Gene Amplification Reaction Solution under Acidic Conditions on B N A Polymerase from Bac i l u s s u b t i l i s Bacteriophage)

As the final concentration during PCR reaction, PCR buffer solution having the composition shown in Table 16 was used. ○ [¾ buffer solution 21_, 0.5 ja g/jaL·p UC 119 11_, 100 M- 13 F primer: CAGTCGTCATGCATTGCCTGCTC 2 L, sterilized water 5!_ added, 10 ML A template mixture solution was prepared. The template mixture was heated at 95 °C for 1 minute and then cooled to 30 °C at 0.1 °C/sec. After cooling, 〇[¾ buffer solution 2 !_, 25 mM d NTP 0.8 !_, 100 mM DTT 11 1_, 100 U/mL pyrophosphatase 0.2 L, 50^9/m LB acillussubtilis 2 mL of bacteriophage-derived DNA polymerase and 4 mL of sterilized water were added, and 10 mL of the reaction mixture was added to the template mixture, and a PCR reaction was carried out at 30 ^° C for 16 hours.

[0060]

\¥0 2020/175406 35 卩 (: 170? 2020 /007232

8 The final concentration at the time of the reaction was 75, 115, or 125 1\/1 <(including 3 〇 〇 [¾ Samples using buffer solution (Products 45 to 47) Amplification of the gene was confirmed.

[0062] From the above results, in the gene amplification reaction in the method of the present invention, it was possible to selectively and easily amplify the gene to be measured under acidic conditions by using the 88 polymerase. As shown in Examples 2 to 7, gene amplification under acidic conditions is changed in each type of octamerase by setting the kind of buffer solution and the concentration of the buffer solution and potassium chloride in an appropriate range. I found out. Further, as shown in Examples 8 to 11 and Examples 18 to 19, the magnesium and potassium chloride concentrations in the reaction solution are set to appropriate ranges, and the results are shown in Examples 12 to 13 As described above, it was found that the addition of trehalose to the reaction solution also changed the gene amplification under acidic conditions. Further, as shown in Comparative Example 1 and Examples 14 to 15, under conventional gene amplification conditions, gene amplification did not occur under acidic conditions, but in the method of the present invention, the type of buffer solution and It was found that gene amplification can be achieved even under acidic conditions by setting the potassium chloride concentration within an appropriate range. Furthermore, as shown in Examples 16 to 17, it was found that gene amplification can be performed even under acidic conditions of !4 units when gene amplification is performed near room temperature.

Industrial availability

[0063] Under conventional gene amplification conditions, no specific composition or conditions for reacting an oral polymerase under acidic conditions have been shown. On the other hand, in the gene amplification method according to the present invention, the reaction composition and reaction conditions of an appropriate buffer solution, various salts, additives, etc. for amplifying the gene to be measured under acidic conditions are found, It was possible to amplify the gene of interest under acidic conditions. As a result, in the gene amplification method according to the present invention, the reaction proceeds without problems even if the protons produced in the gene amplification reaction accumulate. As a result, immediately use the acidic reaction solution used in the gene amplification method of the present invention in a gene detection method using pyridylboronic acid that specifically recognizes pyrophosphoric acid, which is useful as an excellent gene detection technique. Is possible \¥0 2020/175 406 36 卩 (: 17 2020 /007232

.. Furthermore, the method of the present invention does not require the use of fluorescent reagents and is inexpensive.

Claims

\¥0 2020/175 406 37 卩(: 17 2020/007232 Claims [Claim 1] A gene amplification method, comprising a step (a) of amplifying a target gene by an 0.8 polymerase reaction in an acidic reaction solution of 1 ^to 14.2 to 6.9. [Claim 2] The method for gene amplification according to claim 1, wherein the acidic reaction solution used in the step () is prepared using the buffer solution of 1 ^to 14.0 to 6.9. [Claim 3] The gene amplification method according to claim 2, wherein the acidic reaction solution used in the step () comprises 5 IV! to 125 V IV! potassium chloride. [Claim 4] The oral polymerase used in the step (丨) is as follows. The gene amplification according to claim 1, characterized in that it is an oral polymerase derived from a bacterium belonging to the genus 3 genus or 6 3 0 1 II 3 genus bacteriophage. Law. [Claim 5] The gene amplification method according to claim 1, wherein the acidic reaction solution used in the step () includes a saccharide. [Claim 6] The step (I) according to any one of claims 1 to 5, and the step (I) ) The method for detecting a gene, which comprises the step (II) of detecting the target gene based on the amplification product or the reaction by-product obtained in (4). [Claim 7] In the step (丨丨), the amount of the reaction byproduct generated in the step (丨) is measured, and the amount of the target gene is determined based on the measured amount of the reaction byproduct. And the gene detection method. [Claim 8] The gene detection method according to claim 7, wherein the reaction by-product is pyrophosphate. [Claim 9] The amount of pyrophosphoric acid produced in the step (丨) is measured by measuring a potential change due to a reaction with pyridylboronic acid in the step (丨丨). Gene detection method. [Claim 10] The gene detection method according to claim 8, wherein the amount of pyrophosphate produced in the step () is measured by an absorbance method in the step (). [Claim 11] A kit for gene amplification for carrying out the gene amplification method according to any one of claims 1 to 5, comprising an oral polymerase, a buffer, and a reaction reagent. The gene amplification kit. \¥0 2020/175406 38 卩 (: 170? 2020 /007232 [Claim 12] A gene detection device or system for carrying out the gene detection method according to any one of claims 6 to 10, comprising the gene amplification kit according to claim 11. The gene detection device or system including.