WO2012043183A1 - Method for synthesizing target nucleic acid in feces - Google Patents

Method for synthesizing target nucleic acid in feces Download PDF

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WO2012043183A1
WO2012043183A1 PCT/JP2011/070473 JP2011070473W WO2012043183A1 WO 2012043183 A1 WO2012043183 A1 WO 2012043183A1 JP 2011070473 W JP2011070473 W JP 2011070473W WO 2012043183 A1 WO2012043183 A1 WO 2012043183A1
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nucleic acid
stool
target nucleic
synthesizing
reaction
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French (fr)
Japanese (ja)
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長岡　智紀
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オリンパス株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a method for highly accurately synthesizing a target nucleic acid to be analyzed in feces using a nucleic acid synthesis reaction.
  • Patent Document 1 and Non-Patent Document 1 include a method for examining colon cancer by detecting non-apoptotic DNA frequently observed in nucleic acids derived from cancer cells, in particular, Alu repeat region and alphoid repeat region, A method for examining colorectal cancer based on the difference in fragment length of cancer-related genes such as p53 has been disclosed.
  • nucleic acid such as nucleic acid derived from cancer cells in stool
  • feces contain a large amount of digest residue and bacteria
  • nucleic acids are very easily degraded.
  • the analysis accuracy is impaired by introducing foreign substances in the stool into the nucleic acid collected from the stool. For this reason, in order to obtain a more reliable nucleic acid analysis result, a method for recovering highly purified nucleic acid from feces while preventing decomposition or the like has been developed.
  • Patent Document 2 discloses a method of stabilizing the stool structure by cooling the stool to a temperature below the gel freezing point, separating cells from the stool in this state, and analyzing the DNA extracted therefrom. ing.
  • Non-Patent Document 2 discloses that after removing contaminants such as proteins from a stool sample, RNA is extracted using phenol and chaotropic salt, and the extracted RNA is further extracted.
  • a method of recovering by adsorbing on a silica-containing solid support is disclosed.
  • Patent Document 3 discloses a method for preparing a stool sample for analyzing an oncogene in stool. This is a method in which a stool sample is homogenized at a solvent ratio of at least 5 with respect to stool mass 1, and then DNA derived from mammalian cells is collected including bacterial DNA.
  • the collected stool is homogenized in the presence of an RNase inhibitor, RNA is directly extracted from the prepared suspension, and Cox-2 (cyclooxygenase-2), which is an oncogene.
  • a method for detecting a transcript of a gene is disclosed.
  • feces contain substances having an inhibitory action on nucleic acid synthesis reactions such as PCR (Polymerase Chain Reaction) such as bile acids and salts thereof (for example, see Non-Patent Document 3).
  • PCR Polymerase Chain Reaction
  • bile acids and salts thereof for example, see Non-Patent Document 3.
  • the average amount of feces excreted by an adult is about 200 to 400 g / day, but there is a report that 200 to 650 mg / day of bile acid is excreted in feces of healthy people. That is, when converted to 1 g of stool, a healthy person contains about 0.5 mg to 3.25 mg, and a patient contains 10 times as much bile acid.
  • JP 2005-514073 A Japanese National Patent Publication No. 11-511982 Special Table 2002-539765 Japanese Patent No. 4134047 WO2010 / 024251A1
  • iron chloride is known to inhibit nucleic acid synthesis reactions such as PCR. Further, iron chloride is metabolized by bacteria in feces and the like, and there is a possibility that reactions such as PCR may be inhibited by irons other than iron chloride.
  • the present invention is a method of synthesizing a target nucleic acid to be analyzed in stool using a nucleic acid synthesis reaction, and an object thereof is to improve reaction efficiency and obtain a highly reliable result.
  • the present inventor reacted in the presence of an iron chelator when the nucleic acid extracted / recovered from stool is used as a template for a nucleic acid synthesis reaction such as reverse transcription reaction or PCR. As a result, it was found that the reaction efficiency can be improved, and the present invention was completed.
  • the present invention has the following configuration.
  • (1) A method for synthesizing a target nucleic acid in stool, in which a nucleic acid synthesis reaction using a nucleic acid collected from stool as a template in a reaction solution containing an iron chelator, (2) The method for synthesizing a target nucleic acid in feces according to (1), wherein the iron chelator is tetrasodium 3-hydroxy-2,2′-iminodisuccinate, (3) The method for synthesizing a target nucleic acid in stool according to (1) or (2), wherein the reaction solution further contains EDTA, (4) The method for synthesizing a target nucleic acid in stool according to any one of (1) to (3), wherein the nucleic acid synthesis reaction is a reverse transcription reaction or a nucleic acid amplification reaction using a polymerase, (5) The nucleic acid collected from the stool is a nucleic acid collected from the extraction solution after the nucleic acid extraction solution is added to and mixed with the stool
  • Example 1 it is the graph which showed the relative value of the expression level (copy number) of Cox-2 gene in RNA collect
  • the nucleic acid synthesis reaction means a reaction for synthesizing a nucleic acid using a reverse transcription reaction by a reverse transcriptase (reverse transcriptase) or a base chain extension reaction by a polymerase or ligase.
  • a reverse transcriptase reverse transcriptase
  • a base chain extension reaction by a polymerase or ligase examples include PCR (polymerase chain reaction), real-time PCR, and SDA (Standard Displacement Amplification).
  • Examples of the base chain extension reaction by ligase include LCR (ligase chain reaction).
  • the method for synthesizing a target nucleic acid in stool of the present invention (hereinafter sometimes referred to as the synthesis method of the present invention) is a method of synthesizing a target nucleic acid in stool, using a nucleic acid collected from stool as a template
  • the nucleic acid synthesis reaction is performed in a reaction solution containing an iron chelator.
  • an inhibitory substance means a substance that acts in an inhibitory manner on an enzymatic reaction using a nucleic acid as a substrate.
  • the enzyme reaction is not particularly limited as long as it is an enzyme reaction using a nucleic acid as a substrate, and also includes a nucleic acid synthesis reaction such as reverse transcription reaction and PCR.
  • the reaction efficiency of the nucleic acid synthesis reaction can be improved by adding an iron chelating agent to the reaction solution.
  • the reason why such an effect is obtained is not clear, but it is presumed that the reaction efficiency can be increased as a result of reducing the influence of the inhibitor remaining in the nucleic acid collected from the stool.
  • the type of inhibitor whose inhibitory action is reduced by the iron chelator is not clear, but is thought to be iron ions or their inclusions.
  • the iron chelating agent used in the synthesis method of the present invention is not particularly limited as long as it is a compound capable of chelating iron ions, but is preferably an iron chelating agent having high specificity for iron ions.
  • an iron chelating agent with high affinity for other ions such as magnesium ion, it may chelate even the ions required for the enzyme activity used in the nucleic acid synthesis reaction. Pay attention to the amount.
  • iron chelating agent used in the synthesis method of the present invention include 3-hydroxy-2,2′-iminodisuccinic acid tetrasodium (HIDS), EDTA (ethylenediaminetetraacetic acid), deferoxamine, deferiprone, deferasirox. Etc.
  • HIDS is preferably used because of its high specificity to iron ions.
  • the iron chelating agent added to the reaction solution may be only one type, or two or more types may be used in combination.
  • a higher reaction efficiency improvement effect can be achieved by using HIDS and EDTA together in appropriate amounts.
  • the concentration of the iron chelating agent added to the reaction solution in the synthesis method of the present invention is not particularly limited as long as the concentration is sufficient to obtain the reaction efficiency improvement effect of the nucleic acid synthesis reaction. It can be determined as appropriate in consideration of the type of nucleic acid, the amount of nucleic acid added to the reaction solution, the method for recovering nucleic acid from stool, and the like.
  • the HIDS concentration in the reaction solution is preferably 5 mM or more, more preferably 5 mM to 1 M, and even more preferably 10 mM to 1 M.
  • the EDTA concentration in the reaction solution is preferably 10 mM to 0.1 M.
  • the HIDS content in the reaction solution is preferably 5 mM or more and the EDTA content is preferably 1 to 10 mM.
  • the nucleic acid used as a template for the nucleic acid synthesis reaction may be a nucleic acid recovered from stool, and may be a nucleic acid recovered from stool by any known method.
  • it may be a nucleic acid obtained by separating and recovering a cell containing a target nucleic acid from stool in advance, as in Patent Document 2 and the like, and extracted and purified from the recovered cell.
  • It may be a nucleic acid recovered directly from stool without going through a separation step. Since the effect of improving the reaction efficiency of the nucleic acid synthesis reaction of the present invention can be exhibited more remarkably, in the present invention, it is preferable to use a nucleic acid recovered directly from stool or a solid component thereof.
  • Examples of the method for directly recovering nucleic acid from stool or its solid component include a method of adding a nucleic acid extraction solution directly to stool and mixing it, and then recovering the nucleic acid eluted in the extraction solution.
  • the method involves nucleic acids of all biological species contained in the stool, mainly nucleic acids derived from animals excreting the stool, and bacteria such as intestinal resident bacteria without performing the separation operation of cells and contaminants.
  • the nucleic acid derived from the stool is simultaneously extracted and recovered.
  • the nucleic acid contained in the stool includes, in addition to animal-derived nucleic acids and bacteria-derived nucleic acids, food-derived nucleic acids ingested by the animals.
  • the extraction solution may be any solution that can denature proteins in solid components and elute nucleic acids into the extraction solution from cells such as mammalian cells and intestinal resident bacteria in the solid content.
  • the solution is not particularly limited, and any solution used in the technical field may be used.
  • a solution in which a compound usually used as a protein denaturant such as a chaotropic salt, an organic solvent, or a surfactant is added as an active ingredient to an appropriate solvent can be used as the extraction solution.
  • These active ingredients may be a combination of two or more.
  • the surfactant that can be an active ingredient of the extraction solution is preferably a nonionic surfactant.
  • the nonionic surfactant include Tween 80, CHAPS (3- [3-Colamidopropyldimethylammonio] -1-propanesulfonate), Triton X-100, Tween 20, and the like.
  • the concentration of the chaotropic salt and the surfactant is not particularly limited as long as the nucleic acid can be eluted from the solid component, and the mixing ratio of the stool amount (solid component amount) and the extraction solution, or the recovered amount is recovered. It can be determined appropriately in consideration of the nucleic acid synthesis method.
  • Phenol is preferable as the organic solvent that can be an active ingredient of the extraction solution.
  • Phenol may be neutral or acidic. When acidic phenol is used, RNA can be selectively extracted into the aqueous layer rather than DNA.
  • a solvent for adding these active ingredients to prepare an extraction solution for example, a phosphate buffer or a Tris buffer can be used.
  • a drug in which DNase is inactivated by high-pressure steam sterilization or the like is preferable, and a drug containing a protease such as proteinase K is more preferable.
  • a citrate buffer or the like can be used as the elution agent.
  • RNA is a substance that is very easily decomposed, so RNase such as guanidine thiocyanate or guanidine hydrochloride is used. It is preferable to use a buffer containing an inhibitor.
  • the solid component Before adding the extraction solution, other solutions such as an appropriate storage solution are added to the collected stool, and if the suspension is in the form of a suspension, the solid component is recovered from the suspension. You may add the solution for extraction to the collect
  • the recovery of the solid component from the suspension can be performed by a known solid-liquid separation process such as a centrifugal separation process or a filter filtration process.
  • the recovered solid component may be washed with an appropriate buffer and then the extraction solution may be added.
  • any conventionally known solution may be used, but a solution containing a nucleic acid stabilizer is preferable.
  • the nucleic acid stabilizer include water-soluble organic solvents, protease inhibitors, polycations, and high-concentration salts.
  • Recovery of the nucleic acid eluted in the extraction solution can be performed by a known method such as ethanol precipitation or cesium chloride ultracentrifugation. Further, after the nucleic acid eluted in the extraction solution is adsorbed on the inorganic support, the nucleic acid can be recovered by eluting the adsorbed nucleic acid from the inorganic support using a certain volume of solvent. .
  • the inorganic support for adsorbing nucleic acid a known inorganic support capable of adsorbing nucleic acid can be used.
  • the shape of the inorganic support is not particularly limited, and may be in the form of particles or a film.
  • the inorganic support examples include silica-containing particles (beads) such as silica gel, siliceous oxide, glass, and diatomaceous earth, and porous membranes such as nylon, polycarbonate, polyacrylate, and nitrocellulose.
  • Solvents for eluting adsorbed nucleic acids from inorganic supports are usually used to elute nucleic acids from these known inorganic supports in consideration of the type of nucleic acid to be recovered, the subsequent nucleic acid analysis method, and the like.
  • a solvent can be appropriately used.
  • the elution solvent is particularly preferably purified water.
  • the inorganic support on which the nucleic acid has been adsorbed is preferably washed with an appropriate washing buffer before the nucleic acid is eluted.
  • the denatured protein Before recovering the nucleic acid, the denatured protein may be removed from the extraction solution from which the nucleic acid has been eluted.
  • the quality of the recovered nucleic acid can be improved by removing the protein that has been denatured in advance before recovering the nucleic acid.
  • the protein can be removed from the extraction solution by a known method.
  • the denatured protein can be removed by precipitating the denatured protein by centrifugation and collecting only the supernatant.
  • centrifugation is performed, and the denatured protein is precipitated and only the supernatant is recovered. Protein can be removed.
  • nucleic acid recovery from feces can also be performed using a commercially available kit such as a nucleic acid extraction kit.
  • a nucleic acid synthesis reaction is performed in a reaction solution containing an iron chelator.
  • the nucleic acid synthesis reaction can be performed by a conventional method except that an iron chelator is added to the reaction solution.
  • the target nucleic acid in the present invention is a nucleic acid to be synthesized, and is particularly limited as long as it has a nucleotide sequence that has been clarified to the extent that it can be analyzed by a method used for analysis of normal nucleic acids such as PCR. It is not something.
  • the target nucleic acid in the present invention is preferably a nucleic acid derived from a mammalian cell, and more preferably an RNA derived from a mammalian cell (for example, mRNA or the like).
  • RNA expression level can also be detected.
  • mRNA expression analysis K-ras gene mutation analysis
  • DNA methylation analysis DNA methylation analysis
  • analyzes can be performed by methods known in the art.
  • a commercially available analysis kit such as a K-ras gene mutation analysis kit or a methylation detection kit may also be used.
  • nucleic acid recovered from feces since it is a nucleic acid recovered from feces, it is preferable to use a nucleic acid derived from gastrointestinal cells such as the large intestine, small intestine, stomach, etc. as a target nucleic acid, and a nucleic acid derived from a large intestine exfoliated cell as a target nucleic acid. Is more preferable.
  • a nucleic acid derived from a marker gene for neoplastic transformation (including cancer) or a marker gene for inflammatory gastrointestinal diseases is preferably used as a target nucleic acid
  • a nucleic acid derived from a marker gene for colon cancer is preferably used as a target nucleic acid.
  • the “gene-derived nucleic acid” means an expression product such as genomic DNA or mRNA of the gene.
  • the marker showing neoplastic conversion include known cancer markers such as Cox-2 gene, carcinoembryonic antigen (CEA), sialyl Tn antigen (STN), APC gene, p53 gene, K-ras gene, etc. The presence or absence of mutations.
  • markers that indicate inflammatory digestive tract diseases include Cox-2 gene-derived nucleic acids.
  • the Cox-2 gene-derived nucleic acid is also used as a marker indicating neoplastic transformation.
  • the reaction efficiency can be increased, and as a result, a stable reaction can be performed.
  • the synthesis method of the present invention it is possible to obtain a highly accurate and highly reliable result for the target nucleic acid in the nucleic acid recovered from stool containing a large amount of contaminants.
  • highly reliable results of gene expression analysis of stool-derived RNA can be obtained. The result thus obtained is preferably used for clinical examination.
  • a nucleic acid derived from a marker gene for colorectal cancer such as Cox-2 gene in feces can be synthesized with high accuracy.
  • the synthetic product thus obtained can be used for colorectal cancer detection. That is, information for clinical examination can be provided by using the synthesis method of the present invention.
  • the synthesis method of the present invention can be carried out more easily by making an iron chelating agent into a kit with a reagent used in a nucleic acid synthesis reaction.
  • a nucleic acid synthesis kit preferably includes HIDS or HIDS and EDTA as an iron chelator.
  • the reagent used for the nucleic acid synthesis (nucleic acid amplification) reaction include an enzyme (nucleic acid synthase), a buffer, a primer, and the like.
  • a product obtained by adding an iron chelator to a commercially available kit for reverse transcription reaction or PCR can be used as the nucleic acid synthesis kit of the present invention.
  • the kit for nucleic acid synthesis of the present invention may be provided with reagents used for recovering nucleic acid from stool.
  • reagents used for recovering nucleic acid from stool.
  • examples of such reagents include a storage solution added to feces, a nucleic acid extraction solution, an inorganic support, a solvent for elution from the inorganic support, and the like.
  • what added the iron chelating agent to the commercially available nucleic acid extraction kit can also be used as the nucleic acid synthesis kit of the present invention. In the present invention, it is preferable to dissolve the iron chelating agent in advance in the solvent for elution from the inorganic support, because the labor of mixing can be saved.
  • MKN45 cells used were cultured by a conventional method.
  • Example 1 Synthesis of nucleic acid derived from Cox-2 gene in RNA extracted from stool derived from pseudo-colon cancer patient
  • derived from Cox-2 gene in RNA extracted from stool sample derived from pseudo-colorectal cancer patient Nucleic acids were synthesized and detected.
  • a stool sample derived from a pseudo-colorectal cancer patient (a stool pseudo-sample collected from a colorectal cancer patient) contains MKN45 cells, a cultured cell line that highly expresses the Cox-2 gene, in the same manner as colorectal cancer cells. A mixture was used.
  • the obtained stool samples derived from pseudo colorectal cancer patients were dispensed into three 1.5 mL tubes of 100 ⁇ L each. To these three 1.5 mL tubes, 1 mL each of RLT buffer containing guanidine salt was added and stirred with Vortex. Centrifugation was performed after stirring, 300 ⁇ L of the supernatant was collected, and an equal amount of 70% ethanol was added and mixed. The obtained mixed solution is passed through an RNA recovery column of RNeasy midi kit (manufactured by Qiagen), and the RNA recovery column is subjected to washing operation and RNA elution operation according to the attached protocol, whereby 50 ⁇ L of total RNA is obtained. It was collected as a solution. The total RNA solution collected in three tubes was mixed to form one total RNA solution, the RNA concentration of the solution was measured, and the total RNA amount collected was quantified.
  • a reverse transcription reaction was performed in the presence of HIDS, EDTA, or BSA to synthesize cDNA.
  • a reverse transcription reaction was performed under the condition where none of HIDS, EDTA, and BSA was added.
  • Table 1 shows the concentrations of HIDS, EDTA, and BSA in the reaction solution of each sample.
  • a reaction solution for the reverse transcription reaction was prepared so that 1 ⁇ g of RNA was added to each reaction solution.
  • M-MLV (TaKaRa) was used as a reverse transcriptase.
  • Real-time PCR was performed using the obtained cDNA as a template, and the expression product (mRNA) of the Cox-2 gene was detected.
  • Cox-2 primer probe MIX (Catalog No: Hs00153133_m1) manufactured by Applied Biosystems was used. Specifically, 1 ⁇ L of each cDNA was dispensed into a 0.2 mL 96-well PCR plate. Thereafter, 8 ⁇ L of ultrapure water and 10 ⁇ L of nucleic acid amplification reagent “TaqMan GeneExpression Master Mix” (Applied Biosystems) were added to each well, and 1 ⁇ L of Cox-2 primer probe MIX (Applied Biosystems) was further added.
  • PCR reaction solution was prepared.
  • the PCR plate was placed in an ABI real-time PCR apparatus, treated at 95 ° C. for 10 minutes, and then subjected to 40 cycles of thermal cycling at 95 ° C. for 1 minute, 56.5 ° C. for 1 minute, and 72 ° C. for 1 minute. Further, PCR was performed while measuring the fluorescence intensity over time by treating at 72 ° C. for 7 minutes.
  • the measurement result of the fluorescence intensity was analyzed, and the expression level of the Cox-2 gene in each sample was examined.
  • the relative value of the expression level of other samples was calculated with the expression level of the control without addition of HIDS and the like (sample 17 in Table 1) being 1.
  • the calculation results are shown in the graph of FIG.
  • EDTA also has an effect of chelating trivalent iron ions (Fe 3+ ).
  • a target nucleic acid in stool can be synthesized and detected with high accuracy using a nucleic acid synthesis reaction, and thus can be used particularly in the field of clinical examinations.

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Abstract

The objective of the present invention is to provide a method that synthesizes a target nucleic acid in feces using a nucleic acid synthesis reaction, improves reaction efficiency, and obtains a highly reliable result. The method for synthesizing a target nucleic acid in feces is characterized by performing the nucleic acid synthesis reaction, which uses a nucleic acid collected from feces as a template, in a reaction liquid containing an iron chelator. The iron chelator can be tetrasodium 3-hydroxy-2,2'-iminodisuccinate. The present invention further provides a method for providing information for inspecting for colon cancer using the method for synthesizing a target nucleic acid in feces. The present invention further provides a kit that is for nucleic acid synthesis, has the iron chelator, and is used in the method for synthesizing a target nucleic acid in feces.

Description

糞便中の標的核酸の合成方法Method for synthesizing target nucleic acid in feces
　本発明は、糞便中の解析対象である標的核酸を、核酸合成反応を用いて高精度に合成するための方法に関する。 The present invention relates to a method for highly accurately synthesizing a target nucleic acid to be analyzed in feces using a nucleic acid synthesis reaction.
　近年、糞便中の癌遺伝子を増幅し、解析することによって大腸癌を検出する方法が開示されている。例えば、特許文献１及び非特許文献１には、癌細胞由来の核酸に多く観察される非アポトーシス性ＤＮＡを検出することによって大腸癌を検査する方法、特に、Ａｌｕ反復領域・アルフォイド反復領域や、ｐ５３等の癌関連遺伝子の断片長の差異に基づいて大腸癌を検査する方法が開示されている。 Recently, a method for detecting colorectal cancer by amplifying and analyzing an oncogene in feces has been disclosed. For example, Patent Document 1 and Non-Patent Document 1 include a method for examining colon cancer by detecting non-apoptotic DNA frequently observed in nucleic acids derived from cancer cells, in particular, Alu repeat region and alphoid repeat region, A method for examining colorectal cancer based on the difference in fragment length of cancer-related genes such as p53 has been disclosed.
　このように、糞便中の癌細胞由来の核酸等の核酸を解析するためには、糞便から高品質の核酸を回収することが重要である。例えば、糞便中には、消化残留物やバクテリアが大量に含まれているため、核酸は非常に分解されやすいという問題がある。また、糞便から回収された核酸に、糞便中の夾雑物が持ち込まれることにより、解析精度が損なわれるという問題もある。このため、より信頼性の高い核酸解析結果を得るために、糞便から、分解等を防止しつつ精製度の高い核酸を回収するための方法の開発がなされている。 Thus, in order to analyze nucleic acid such as nucleic acid derived from cancer cells in stool, it is important to collect high-quality nucleic acid from stool. For example, since feces contain a large amount of digest residue and bacteria, there is a problem that nucleic acids are very easily degraded. In addition, there is a problem in that the analysis accuracy is impaired by introducing foreign substances in the stool into the nucleic acid collected from the stool. For this reason, in order to obtain a more reliable nucleic acid analysis result, a method for recovering highly purified nucleic acid from feces while preventing decomposition or the like has been developed.
　例えば特許文献２には、糞便をゲル氷点未満の温度まで冷却することによって便の構造を安定化させ、この状態の糞便からの細胞を分離し、そこから抽出したＤＮＡを解析する方法が開示されている。その他、糞便試料からＲＮＡを回収する方法として、非特許文献２には、糞便試料からタンパク質等の夾雑物を除去した後、フェノールとカオトロピック塩を用いてＲＮＡを抽出し、抽出されたＲＮＡをさらにシリカ含有固形支持体に吸着させることにより回収する方法が開示されている。 For example, Patent Document 2 discloses a method of stabilizing the stool structure by cooling the stool to a temperature below the gel freezing point, separating cells from the stool in this state, and analyzing the DNA extracted therefrom. ing. In addition, as a method for recovering RNA from a stool sample, Non-Patent Document 2 discloses that after removing contaminants such as proteins from a stool sample, RNA is extracted using phenol and chaotropic salt, and the extracted RNA is further extracted. A method of recovering by adsorbing on a silica-containing solid support is disclosed.
　また、糞便から細胞を分離回収せずに、糞便から直接核酸を回収する方法もある。例えば特許文献３には、糞便中の癌遺伝子を解析するための、糞便サンプルの調整方法が開示されている。これは、糞便サンプルを、糞便質量１に対して、少なくとも５の溶媒比でホモジナイズした後、哺乳細胞由来のＤＮＡを、細菌のＤＮＡを含めて回収する方法である。また、特許文献４には、採取された糞便をＲＮＡ分解酵素阻害剤の存在下で均質化し、調製された懸濁物から直接ＲＮＡを抽出し、癌遺伝子であるＣｏｘ－２（ｃｙｃｌｏｏｘｙｇｅｎａｓｅ－２）遺伝子の転写産物を検出する方法が開示されている。 There is also a method of directly recovering nucleic acid from stool without separating and collecting cells from stool. For example, Patent Document 3 discloses a method for preparing a stool sample for analyzing an oncogene in stool. This is a method in which a stool sample is homogenized at a solvent ratio of at least 5 with respect to stool mass 1, and then DNA derived from mammalian cells is collected including bacterial DNA. In Patent Document 4, the collected stool is homogenized in the presence of an RNase inhibitor, RNA is directly extracted from the prepared suspension, and Cox-2 (cyclooxygenase-2), which is an oncogene. A method for detecting a transcript of a gene is disclosed.
　一方で、糞便中には、胆汁酸やその塩等の、ＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ）等の核酸合成反応に対して阻害作用を有する物質が含まれている（例えば、非特許文献３参照）。例えば、大人の平均的な排泄糞便量は、約２００～４００ｇ／日とされるが、健常人ではその糞便中に２００～６５０ｍｇ／日の胆汁酸が排泄されるという報告がある。すなわち、便１ｇあたりに換算した場合、健常人で約０．５ｍｇ～３．２５ｍｇ、患者でその１０倍の胆汁酸が含まれることになる。一方で、胆汁酸塩によるＰＣＲの阻害効果は、５０μｇ／ｍＬ程度の濃度で生じるとの報告もある。したがって、糞便から核酸を抽出し、それをＰＣＲ等で増幅する場合は、増幅効率を向上させるために、胆汁酸塩等の核酸合成反応阻害物質による影響を低減させることが好ましい。阻害物質による影響を考慮しなければ、例えば、鋳型として多量の糞便由来ＲＮＡを反応液中に持ち込んで逆転写反応を行った場合には、反応阻害が起こり、反応産物が得られないという問題が生じてしまう。 On the other hand, feces contain substances having an inhibitory action on nucleic acid synthesis reactions such as PCR (Polymerase Chain Reaction) such as bile acids and salts thereof (for example, see Non-Patent Document 3). For example, the average amount of feces excreted by an adult is about 200 to 400 g / day, but there is a report that 200 to 650 mg / day of bile acid is excreted in feces of healthy people. That is, when converted to 1 g of stool, a healthy person contains about 0.5 mg to 3.25 mg, and a patient contains 10 times as much bile acid. On the other hand, there is a report that the inhibitory effect of PCR by bile salts occurs at a concentration of about 50 μg / mL. Therefore, when nucleic acid is extracted from stool and amplified by PCR or the like, it is preferable to reduce the influence of a nucleic acid synthesis reaction inhibitor such as bile salt in order to improve amplification efficiency. If the effect of the inhibitor is not taken into account, for example, when a large amount of stool-derived RNA is brought into the reaction solution as a template and a reverse transcription reaction is performed, reaction inhibition occurs and a reaction product cannot be obtained. It will occur.
特表２００５－５１４０７３号公報JP 2005-514073 A 特表平１１－５１１９８２号公報Japanese National Patent Publication No. 11-511982 特表２００２－５３９７６５号公報Special Table 2002-539765 特許第４１３４０４７号公報Japanese Patent No. 4134047 WO２０１０／０２４２５１A１WO2010 / 024251A1
　特に近年では、生活習慣からくる慢性的な貧血症状のため、鉄分を含む経口薬剤や健康食品を摂取する人が多く、こうした人々の便から核酸を回収し、その便中の遺伝子を解析するために逆転写反応や核酸合成反応を行う場合、便中に排泄された過剰量の鉄分が抽出された核酸中に残存し、反応阻害を引き起こす可能性がある。例えば塩化鉄等は、ＰＣＲ等の核酸合成反応を阻害することが知られている。また、糞便中の細菌等によって、塩化鉄が代謝され、塩化鉄以外の態様の鉄によっても、ＰＣＲ等の反応を阻害し得る可能性がある。 Especially in recent years, because of chronic anemia due to lifestyle habits, many people take oral drugs containing iron and health foods, and in order to collect nucleic acids from these people's stool and analyze the genes in the stool In addition, when a reverse transcription reaction or a nucleic acid synthesis reaction is performed, an excessive amount of iron excreted in feces remains in the extracted nucleic acid, which may cause reaction inhibition. For example, iron chloride is known to inhibit nucleic acid synthesis reactions such as PCR. Further, iron chloride is metabolized by bacteria in feces and the like, and there is a possibility that reactions such as PCR may be inhibited by irons other than iron chloride.
　本発明は、糞便中の解析対象である標的核酸を、核酸合成反応を用いて合成する方法であって、反応効率を改善し、信頼性の高い結果を得る方法を目的とする。 The present invention is a method of synthesizing a target nucleic acid to be analyzed in stool using a nucleic acid synthesis reaction, and an object thereof is to improve reaction efficiency and obtain a highly reliable result.
　本発明者は、上記課題を解決すべく鋭意研究した結果、糞便から抽出・回収した核酸を、逆転写反応やＰＣＲ等の核酸合成反応の鋳型として用いる場合に、鉄キレート剤の存在下で反応を行うことにより、反応効率を改善し得ることを見出し、本発明を完成させた。 As a result of diligent research to solve the above-mentioned problems, the present inventor reacted in the presence of an iron chelator when the nucleic acid extracted / recovered from stool is used as a template for a nucleic acid synthesis reaction such as reverse transcription reaction or PCR. As a result, it was found that the reaction efficiency can be improved, and the present invention was completed.
　すなわち、本発明は、下記の構成をとる。
（１）　糞便から回収された核酸を鋳型とする核酸合成反応を、鉄キレート剤を含有する反応液中で行う、糞便中の標的核酸の合成方法、
（２）　前記鉄キレート剤が、３－ヒドロキシ－２，２’－イミノジコハク酸４ナトリウムである、前記（１）に記載の糞便中の標的核酸の合成方法、
（３）　前記反応液が、さらにＥＤＴＡを含む、前記（１）又は（２）に記載の糞便中の標的核酸の合成方法、
（４）　前記核酸合成反応が、逆転写反応、又はポリメラーゼを用いた核酸増幅反応である前記（１）～（３）のいずれか一つに記載の糞便中の標的核酸の合成方法、
（５）　前記糞便から回収された核酸が、動物より採取された糞便又はその固形成分に、核酸抽出用溶液を添加して混合した後、当該抽出用溶液から回収された核酸である、前記（１）～（４）のいずれか一つに記載の糞便中の標的核酸の合成方法、
（６）　前記標的核酸が、哺乳細胞由来の核酸である、前記（１）～（５）のいずれか一つに記載の糞便中の標的核酸の合成方法、
（７）　前記標的核酸が、新生物性転化を示すマーカー又は消化器系疾患を示すマーカーである、前記（６）に記載の糞便中の標的核酸の合成方法、
（８）　前記標的核酸が、Ｃｏｘ－２遺伝子由来核酸である、前記（７）に記載の糞便中の標的核酸の合成方法、
（９）　前記標的核酸を大腸癌のマーカー遺伝子由来の核酸として前記（１）～（８）のいずれか一つに記載の糞便中の標的核酸の合成方法を行い、
　当該合成方法の産物を検出する
ことを含む、大腸癌検査のための情報提供方法、
（１０）　糞便からの核酸抽出用溶液；
　核酸合成酵素及びバッファーを含む、核酸増幅試薬；及び
鉄キレート剤を有する、核酸合成用キット。 That is, the present invention has the following configuration.
(1) A method for synthesizing a target nucleic acid in stool, in which a nucleic acid synthesis reaction using a nucleic acid collected from stool as a template in a reaction solution containing an iron chelator,
(2) The method for synthesizing a target nucleic acid in feces according to (1), wherein the iron chelator is tetrasodium 3-hydroxy-2,2′-iminodisuccinate,
(3) The method for synthesizing a target nucleic acid in stool according to (1) or (2), wherein the reaction solution further contains EDTA,
(4) The method for synthesizing a target nucleic acid in stool according to any one of (1) to (3), wherein the nucleic acid synthesis reaction is a reverse transcription reaction or a nucleic acid amplification reaction using a polymerase,
(5) The nucleic acid collected from the stool is a nucleic acid collected from the extraction solution after the nucleic acid extraction solution is added to and mixed with the stool collected from the animal or its solid component ( 1) A method for synthesizing a target nucleic acid in feces according to any one of (4),
(6) The method for synthesizing a target nucleic acid in stool according to any one of (1) to (5), wherein the target nucleic acid is a nucleic acid derived from a mammalian cell,
(7) The method for synthesizing a target nucleic acid in stool according to (6), wherein the target nucleic acid is a marker indicating neoplastic conversion or a marker indicating digestive system disease,
(8) The method for synthesizing a target nucleic acid in stool according to (7), wherein the target nucleic acid is a Cox-2 gene-derived nucleic acid,
(9) The method for synthesizing a target nucleic acid in stool according to any one of (1) to (8), wherein the target nucleic acid is a nucleic acid derived from a marker gene for colorectal cancer,
A method for providing information for colorectal cancer testing, comprising detecting a product of the synthesis method;
(10) Nucleic acid extraction solution from feces;
A nucleic acid synthesis kit comprising a nucleic acid amplification reagent comprising a nucleic acid synthetase and a buffer; and an iron chelator.
　本発明の糞便中の標的核酸の合成方法により、夾雑物の多い糞便から回収された核酸を、核酸合成反応の鋳型として用いる場合であっても、従来になく信頼性の高い結果を得ることができる。 According to the method for synthesizing a target nucleic acid in stool of the present invention, even when a nucleic acid collected from stool containing a large amount of contaminants is used as a template for a nucleic acid synthesis reaction, a highly reliable result can be obtained. it can.
実施例１において、各サンプルから回収されたＲＮＡ中のＣｏｘ－２遺伝子の発現量（コピー数）の相対値を示したグラフである。In Example 1, it is the graph which showed the relative value of the expression level (copy number) of Cox-2 gene in RNA collect | recovered from each sample.
　本発明及び本願明細書において、核酸合成反応とは、逆転写酵素(リバーストランスクリプターゼ)による逆転写反応や、ポリメラーゼ又はリガーゼによる塩基鎖の伸長反応を利用して、核酸を合成する反応を意味する。ポリメラーゼによる塩基鎖伸長反応としては、ＰＣＲ（ｐｏｌｙｍｅｒａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）、リアルタイムＰＣＲ、ＳＤＡ（Ｓｔａｎｄａｒｄ　Ｄｉｓｐｌａｃｅｍｅｎｔ　Ａｍｐｌｉｆｉｃａｔｉｏｎ）等が挙げられる。リガーゼによる塩基鎖伸長反応としては、ＬＣＲ（ｌｉｇａｓｅ　ｃｈａｉｎ　ｒｅａｃｔｉｏｎ）等が挙げられる。 In the present invention and the present specification, the nucleic acid synthesis reaction means a reaction for synthesizing a nucleic acid using a reverse transcription reaction by a reverse transcriptase (reverse transcriptase) or a base chain extension reaction by a polymerase or ligase. To do. Examples of the base chain elongation reaction by polymerase include PCR (polymerase chain reaction), real-time PCR, and SDA (Standard Displacement Amplification). Examples of the base chain extension reaction by ligase include LCR (ligase chain reaction).
　本発明の糞便中の標的核酸の合成方法（以下、本発明の合成方法、ということがある）は、糞便中の標的核酸を合成する方法であって、糞便から回収された核酸を鋳型とする核酸合成反応を、鉄キレート剤を含有する反応液中で行うことを特徴とする。 The method for synthesizing a target nucleic acid in stool of the present invention (hereinafter sometimes referred to as the synthesis method of the present invention) is a method of synthesizing a target nucleic acid in stool, using a nucleic acid collected from stool as a template The nucleic acid synthesis reaction is performed in a reaction solution containing an iron chelator.
　本発明及び本願明細書において、阻害物質とは、核酸を基質とする酵素反応に対して阻害的に作用する物質を意味する。当該酵素反応としては、核酸を基質とする酵素反応であれば特に限定されるものではなく、逆転写反応やＰＣＲ等の核酸合成反応も含まれる。 In the present invention and the specification of the present application, an inhibitory substance means a substance that acts in an inhibitory manner on an enzymatic reaction using a nucleic acid as a substrate. The enzyme reaction is not particularly limited as long as it is an enzyme reaction using a nucleic acid as a substrate, and also includes a nucleic acid synthesis reaction such as reverse transcription reaction and PCR.
　本発明の合成方法では、反応液に鉄キレート剤を添加することにより、核酸合成反応の反応効率を改善することができる。このような効果が得られる理由は明らかではないが、糞便から回収された核酸中に残存する阻害物質による影響を低減させる結果、反応効率を高めることができる、と推察される。鉄キレート剤によって阻害作用が低減される阻害物質の種類は明らかではないが、鉄イオン又はその含有物であると考えられる。 In the synthesis method of the present invention, the reaction efficiency of the nucleic acid synthesis reaction can be improved by adding an iron chelating agent to the reaction solution. The reason why such an effect is obtained is not clear, but it is presumed that the reaction efficiency can be increased as a result of reducing the influence of the inhibitor remaining in the nucleic acid collected from the stool. The type of inhibitor whose inhibitory action is reduced by the iron chelator is not clear, but is thought to be iron ions or their inclusions.
　本発明の合成方法において用いられる鉄キレート剤としては、鉄イオンをキレートし得る化合物であれば、特に限定されるものではないが、鉄イオンに対する特異性の高い鉄キレート剤であることが好ましい。マグネシウムイオン等の他のイオンに対する親和性も高い鉄キレート剤を用いる場合には、核酸合成反応に用いる酵素活性に必要とされるイオンまでもキレートしてしまうおそれがあるため、反応液への添加量に注意を要する。 The iron chelating agent used in the synthesis method of the present invention is not particularly limited as long as it is a compound capable of chelating iron ions, but is preferably an iron chelating agent having high specificity for iron ions. When using an iron chelating agent with high affinity for other ions such as magnesium ion, it may chelate even the ions required for the enzyme activity used in the nucleic acid synthesis reaction. Pay attention to the amount.
　本発明の合成方法において用いられる鉄キレート剤としては、具体的には、３－ヒドロキシ－２，２’－イミノジコハク酸４ナトリウム（ＨＩＤＳ）、ＥＤＴＡ（エチレンジアミン四酢酸）、デフェロキサミン、デフェリプロン、デフェラシロクス等が挙げられる。本発明においては、鉄イオンへの特異性が高いことから、ＨＩＤＳを用いることが好ましい。 Specific examples of the iron chelating agent used in the synthesis method of the present invention include 3-hydroxy-2,2′-iminodisuccinic acid tetrasodium (HIDS), EDTA (ethylenediaminetetraacetic acid), deferoxamine, deferiprone, deferasirox. Etc. In the present invention, HIDS is preferably used because of its high specificity to iron ions.
　また、反応液に添加される鉄キレート剤は、１種類のみであってもよく、２種類以上を併用して用いてもよい。例えば、ＨＩＤＳとＥＤＴＡを適当量ずつ併用することによって、より高い反応効率改善効果を奏することができる。 Also, the iron chelating agent added to the reaction solution may be only one type, or two or more types may be used in combination. For example, a higher reaction efficiency improvement effect can be achieved by using HIDS and EDTA together in appropriate amounts.
　本発明の合成方法において反応液に添加される鉄キレート剤の濃度は、核酸合成反応の反応効率改善効果が得られるために十分な濃度であれば、特に限定されるものではなく、鉄キレート剤の種類や、反応液に添加する核酸量、糞便から核酸を回収する際の方法等を考慮して適宜決定することができる。例えば、鉄キレート剤としてＨＩＤＳを添加する場合には、反応液中のＨＩＤＳ濃度が５ｍＭ以上であることが好ましく、５ｍＭ～１Ｍであることがより好ましく、１０ｍＭ～１Ｍであることがさらに好ましい。鉄キレート剤としてＥＤＴＡを添加する場合には、反応液中のＥＤＴＡ濃度が１０ｍＭ～０．１Ｍであることが好ましい。また、ＨＩＤＳとＥＤＴＡを併用する場合には、反応液中のＨＩＤＳ含有量が５ｍＭ以上、ＥＤＴＡの含有量が１～１０ｍＭであることが好ましい。 The concentration of the iron chelating agent added to the reaction solution in the synthesis method of the present invention is not particularly limited as long as the concentration is sufficient to obtain the reaction efficiency improvement effect of the nucleic acid synthesis reaction. It can be determined as appropriate in consideration of the type of nucleic acid, the amount of nucleic acid added to the reaction solution, the method for recovering nucleic acid from stool, and the like. For example, when HIDS is added as an iron chelating agent, the HIDS concentration in the reaction solution is preferably 5 mM or more, more preferably 5 mM to 1 M, and even more preferably 10 mM to 1 M. When EDTA is added as an iron chelating agent, the EDTA concentration in the reaction solution is preferably 10 mM to 0.1 M. When HIDS and EDTA are used in combination, the HIDS content in the reaction solution is preferably 5 mM or more and the EDTA content is preferably 1 to 10 mM.
　本発明の合成方法において、核酸合成反応の鋳型として用いる核酸は、糞便から回収された核酸であればよく、公知のいずれの方法によって糞便から回収された核酸であってもよい。例えば、特許文献２等のように、糞便から標的核酸を含む細胞を予め分離回収し、この回収された細胞から抽出・精製された核酸であってもよく、特許文献４等のように、細胞分離工程を経ることなく、糞便から直接回収された核酸であってもよい。本発明の核酸合成反応の反応効率改善効果がより顕著に発揮することができるため、本発明においては、糞便又はその固形成分から直接回収された核酸を用いることが好ましい。 In the synthesis method of the present invention, the nucleic acid used as a template for the nucleic acid synthesis reaction may be a nucleic acid recovered from stool, and may be a nucleic acid recovered from stool by any known method. For example, it may be a nucleic acid obtained by separating and recovering a cell containing a target nucleic acid from stool in advance, as in Patent Document 2 and the like, and extracted and purified from the recovered cell. It may be a nucleic acid recovered directly from stool without going through a separation step. Since the effect of improving the reaction efficiency of the nucleic acid synthesis reaction of the present invention can be exhibited more remarkably, in the present invention, it is preferable to use a nucleic acid recovered directly from stool or a solid component thereof.
　糞便又はその固形成分から核酸を直接回収する方法としては、例えば、糞便に直接核酸抽出用溶液を添加して混合した後、当該抽出用溶液中に溶出された核酸を回収する方法が挙げられる。当該方法は、細胞や夾雑物等の分離操作を行うことなく、糞便に含まれている全ての生物種の核酸、主に当該糞便を排泄した動物由来の核酸と腸内常在菌等のバクテリア由来の核酸とを同時に糞便から抽出し回収する方法である。ここで、糞便に含まれている核酸としては、動物由来の核酸とバクテリア由来の核酸に加えて、当該動物が摂取した食物由来の核酸等が挙げられる。 Examples of the method for directly recovering nucleic acid from stool or its solid component include a method of adding a nucleic acid extraction solution directly to stool and mixing it, and then recovering the nucleic acid eluted in the extraction solution. The method involves nucleic acids of all biological species contained in the stool, mainly nucleic acids derived from animals excreting the stool, and bacteria such as intestinal resident bacteria without performing the separation operation of cells and contaminants. In this method, the nucleic acid derived from the stool is simultaneously extracted and recovered. Here, the nucleic acid contained in the stool includes, in addition to animal-derived nucleic acids and bacteria-derived nucleic acids, food-derived nucleic acids ingested by the animals.
　抽出用溶液としては、固形成分中のタンパク質を変性させ、この固形分中の哺乳細胞や腸内常在菌等の細胞から、核酸を抽出用溶液中に溶出させることが可能な溶液であれば、特に限定されるものではなく、当該技術分野において用いられているいずれの溶液を用いてもよい。例えば、カオトロピック塩、有機溶媒、界面活性剤等の、通常タンパク質の変性剤として用いられている化合物を有効成分として適当な溶媒に添加した溶液を、抽出用溶液として用いることができる。なお、これらの有効成分は２種類以上を組み合わせたものであってもよい。 The extraction solution may be any solution that can denature proteins in solid components and elute nucleic acids into the extraction solution from cells such as mammalian cells and intestinal resident bacteria in the solid content. The solution is not particularly limited, and any solution used in the technical field may be used. For example, a solution in which a compound usually used as a protein denaturant such as a chaotropic salt, an organic solvent, or a surfactant is added as an active ingredient to an appropriate solvent can be used as the extraction solution. These active ingredients may be a combination of two or more.
　抽出用溶液の有効成分となり得るカオトロピック塩としては、例えば、塩酸グアニジン、グアニジンイソチオシアネート、ヨウ化ナトリウム、過塩素酸ナトリウム、及びトリクロロ酢酸ナトリウム等がある。抽出用溶液の有効成分となり得る界面活性剤としては、非イオン性界面活性剤であることが好ましい。該非イオン性界面活性剤として、例えば、Ｔｗｅｅｎ８０、ＣＨＡＰＳ（３－［３－コラミドプロピルジメチルアンモニオ]－１－プロパンスルホネート）、Ｔｒｉｔｏｎ　Ｘ－１００、Ｔｗｅｅｎ２０等がある。カオトロピック塩や界面活性剤の濃度は、固形成分から核酸を溶出可能な濃度であれば、特に限定されるものではなく、糞便量（固形成分量）と抽出用溶液との混合比や、回収された核酸の合成方法等を考慮して、適宜決定することができる。 Examples of chaotropic salts that can be an active ingredient of the extraction solution include guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, sodium perchlorate, and sodium trichloroacetate. The surfactant that can be an active ingredient of the extraction solution is preferably a nonionic surfactant. Examples of the nonionic surfactant include Tween 80, CHAPS (3- [3-Colamidopropyldimethylammonio] -1-propanesulfonate), Triton X-100, Tween 20, and the like. The concentration of the chaotropic salt and the surfactant is not particularly limited as long as the nucleic acid can be eluted from the solid component, and the mixing ratio of the stool amount (solid component amount) and the extraction solution, or the recovered amount is recovered. It can be determined appropriately in consideration of the nucleic acid synthesis method.
　抽出用溶液の有効成分となり得る有機溶媒としては、フェノールであることが好ましい。フェノールは中性であってもよく、酸性であってもよい。酸性のフェノールを用いた場合には、ＤＮＡよりもＲＮＡを選択的に水層に抽出することができる。 有機 Phenol is preferable as the organic solvent that can be an active ingredient of the extraction solution. Phenol may be neutral or acidic. When acidic phenol is used, RNA can be selectively extracted into the aqueous layer rather than DNA.
　これらの有効成分を添加して抽出用溶液を調製する溶媒としては、例えば、リン酸バッファーやトリスバッファー等を用いることができる。高圧蒸気滅菌等により、ＤＮａｓｅを失活させた薬剤であることが好ましく、さらにプロテイナーゼＫ等のタンパク質分解酵素を含有させた薬剤であることがより好ましい。一方、ＲＮＡを回収する場合には、該溶出用薬剤として、例えば、クエン酸バッファー等を用いることができるが、ＲＮＡは非常に分解されやすい物質であるため、チオシアン酸グアニジンや塩酸グアニジン等のＲＮａｓｅ阻害剤を含有したバッファーを用いることが好ましい。 As a solvent for adding these active ingredients to prepare an extraction solution, for example, a phosphate buffer or a Tris buffer can be used. A drug in which DNase is inactivated by high-pressure steam sterilization or the like is preferable, and a drug containing a protease such as proteinase K is more preferable. On the other hand, when recovering RNA, for example, a citrate buffer or the like can be used as the elution agent. However, RNA is a substance that is very easily decomposed, so RNase such as guanidine thiocyanate or guanidine hydrochloride is used. It is preferable to use a buffer containing an inhibitor.
　抽出用溶液を添加する前に、採取された糞便にその他の溶液、例えば、適当な保存用溶液を添加し、懸濁液としていた場合には、当該懸濁液から固形成分を回収し、この回収した固形成分に抽出用溶液を添加してもよい。懸濁液からの固形成分の回収は、遠心分離処理やフィルター濾過等の処理等の公知の固液分離処理により行うことができる。なお、回収された固形成分を、適当なバッファーにより洗浄した後に、抽出用溶液を添加してもよい。 Before adding the extraction solution, other solutions such as an appropriate storage solution are added to the collected stool, and if the suspension is in the form of a suspension, the solid component is recovered from the suspension. You may add the solution for extraction to the collect | recovered solid component. The recovery of the solid component from the suspension can be performed by a known solid-liquid separation process such as a centrifugal separation process or a filter filtration process. The recovered solid component may be washed with an appropriate buffer and then the extraction solution may be added.
　採取された糞便と混合させる保存用溶液としては、従来公知のいずれの溶液を用いてもよいが、核酸安定化剤を含有する溶液であることが好ましい。核酸安定化剤としては、例えば、水溶性有機溶媒、プロテアーゼ阻害剤、ポリカチオン、高濃度の塩類等が挙げられる。中でも、核酸安定化効果に優れていることから、特許文献５に開示されているような水溶性有機溶媒を有効成分とする核酸安定化剤溶液であることが好ましい。 As the storage solution to be mixed with the collected stool, any conventionally known solution may be used, but a solution containing a nucleic acid stabilizer is preferable. Examples of the nucleic acid stabilizer include water-soluble organic solvents, protease inhibitors, polycations, and high-concentration salts. Especially, since it is excellent in the nucleic acid stabilization effect, it is preferable that it is the nucleic acid stabilizer solution which uses the water-soluble organic solvent as disclosed in patent document 5 as an active ingredient.
　抽出用溶液に溶出させた核酸の回収は、例えば、エタノール沈殿法や塩化セシウム超遠心法等の公知の手法で行うことができる。
　また、抽出用溶液に溶出させた核酸を無機支持体に吸着させた後、この吸着させた核酸を、一定容量の溶媒を用いて無機支持体から溶出させることにより、核酸を回収することができる。核酸を吸着させる無機支持体は、核酸を吸着することができる公知の無機支持体を用いることができる。また、該無機支持体の形状も特に限定されるものではなく、粒子状であってもよく、膜状であってもよい。該無機支持体として、例えば、シリカゲル、シリカ質オキシド、ガラス、珪藻土等のシリカ含有粒子（ビーズ）や、ナイロン、ポリカーボネート、ポリアクリレート、ニトロセルロース等の多孔質膜等がある。吸着させた核酸を無機支持体から溶出させる溶媒は、回収する核酸の種類やその後の核酸解析方法等を考慮して、これらの公知の無機支持体から核酸を溶出するために通常用いられている溶媒を適宜用いることができる。該溶出用溶媒として、特に精製水であることが好ましい。なお、核酸を吸着させた無機支持体は、核酸を溶出させる前に、適当な洗浄バッファーを用いて洗浄することが好ましい。 Recovery of the nucleic acid eluted in the extraction solution can be performed by a known method such as ethanol precipitation or cesium chloride ultracentrifugation.
Further, after the nucleic acid eluted in the extraction solution is adsorbed on the inorganic support, the nucleic acid can be recovered by eluting the adsorbed nucleic acid from the inorganic support using a certain volume of solvent. . As the inorganic support for adsorbing nucleic acid, a known inorganic support capable of adsorbing nucleic acid can be used. The shape of the inorganic support is not particularly limited, and may be in the form of particles or a film. Examples of the inorganic support include silica-containing particles (beads) such as silica gel, siliceous oxide, glass, and diatomaceous earth, and porous membranes such as nylon, polycarbonate, polyacrylate, and nitrocellulose. Solvents for eluting adsorbed nucleic acids from inorganic supports are usually used to elute nucleic acids from these known inorganic supports in consideration of the type of nucleic acid to be recovered, the subsequent nucleic acid analysis method, and the like. A solvent can be appropriately used. The elution solvent is particularly preferably purified water. The inorganic support on which the nucleic acid has been adsorbed is preferably washed with an appropriate washing buffer before the nucleic acid is eluted.
　核酸を回収する前に、核酸が溶出された抽出用溶液から、変性させたタンパク質を除去してもよい。核酸を回収する前に、予め変性させたタンパク質を除去することにより、回収される核酸の品質を向上させることができる。抽出用溶液からのタンパク質の除去は、公知の手法で行うことができる。例えば、遠心分離により、変性タンパク質を沈殿させて上清のみを回収することにより、変性タンパク質を除去することができる。また、クロロホルムを添加し、ボルテックス等により充分に攪拌混合させた後に遠心分離を行い、変性タンパク質を沈殿させて上清のみを回収することにより、単に遠心分離を行う場合よりも、より完全に変性タンパク質を除去することができる。 Before recovering the nucleic acid, the denatured protein may be removed from the extraction solution from which the nucleic acid has been eluted. The quality of the recovered nucleic acid can be improved by removing the protein that has been denatured in advance before recovering the nucleic acid. The protein can be removed from the extraction solution by a known method. For example, the denatured protein can be removed by precipitating the denatured protein by centrifugation and collecting only the supernatant. In addition, after adding chloroform and thoroughly stirring and mixing by vortexing, etc., centrifugation is performed, and the denatured protein is precipitated and only the supernatant is recovered. Protein can be removed.
　その他、糞便からの核酸の回収は、核酸抽出キット等の市販のキットを用いて行うこともできる。 In addition, nucleic acid recovery from feces can also be performed using a commercially available kit such as a nucleic acid extraction kit.
　回収された核酸を鋳型として、鉄キレート剤を含有する反応液中で核酸合成反応を行う。核酸合成反応は、反応液に鉄キレート剤を添加する以外は、常法により行うことができる。本発明においては、核酸合成反応として、逆転写反応又はポリメラーゼを用いた塩基鎖伸長反応を行うことが好ましく、逆転写反応を行うことがより好ましい。糞便から回収されたＲＮＡを鋳型として逆転写反応を行った後、得られたｃＤＮＡを鋳型としてＰＣＲ等のポリメラーゼによる核酸伸長反応を行うことがより好ましい。 Using the collected nucleic acid as a template, a nucleic acid synthesis reaction is performed in a reaction solution containing an iron chelator. The nucleic acid synthesis reaction can be performed by a conventional method except that an iron chelator is added to the reaction solution. In the present invention, it is preferable to perform a reverse transcription reaction or a base chain extension reaction using a polymerase as the nucleic acid synthesis reaction, and more preferably a reverse transcription reaction. It is more preferable to perform a reverse transcription reaction using RNA collected from stool as a template, and then perform a nucleic acid extension reaction using a polymerase such as PCR using the obtained cDNA as a template.
　本発明における標的核酸とは、合成対象である核酸であり、ＰＣＲ等の通常核酸の解析に用いられる手法によって解析可能な程度に明らかになっている塩基配列を有する核酸であれば、特に限定されるものではない。本発明における標的核酸としては、哺乳細胞由来の核酸であることが好ましく、哺乳細胞由来のＲＮＡ（例えば、ｍＲＮＡ等）であることがより好ましい。 The target nucleic acid in the present invention is a nucleic acid to be synthesized, and is particularly limited as long as it has a nucleotide sequence that has been clarified to the extent that it can be analyzed by a method used for analysis of normal nucleic acids such as PCR. It is not something. The target nucleic acid in the present invention is preferably a nucleic acid derived from a mammalian cell, and more preferably an RNA derived from a mammalian cell (for example, mRNA or the like).
　例えば、適当な標的核酸を設定することにより、癌遺伝子等がコードされている塩基配列領域や、マイクロサテライトを含む塩基配列領域等の遺伝的変異の有無を検出することができ、これにより、癌の発症の有無を調べることができる。糞便試料から回収されたＤＮＡを用いた場合には、例えば、ＤＮＡ上のメチル化や、塩基の挿入、欠失、置換、重複、又は逆位等の変異を検出することができる。また、回収されたＲＮＡを用いた場合には、例えば、ＲＮＡ上の塩基の挿入、欠失、置換、重複、逆位、又はスプライシングバリアント（アイソフォーム）等の変異を検出することができる。また、ＲＮＡ発現量を検出することもできる。特に、ｍＲＮＡの発現解析、Ｋ－ｒａｓ遺伝子の変異解析、及びＤＮＡのメチル化の解析等を行うことが好ましい。なお、これらの解析は、当該分野において公知の方法により行うことができる。また、Ｋ－ｒａｓ遺伝子変異解析キット、メチル化検出キット等の市販の解析キットを用いてもよい。 For example, by setting an appropriate target nucleic acid, it is possible to detect the presence or absence of a genetic variation such as a base sequence region encoding an oncogene or a base sequence region containing a microsatellite. The presence or absence of onset can be examined. When DNA recovered from a stool sample is used, for example, methylation on the DNA, base insertion, deletion, substitution, duplication, or inversion mutation can be detected. Further, when the recovered RNA is used, for example, a base insertion, deletion, substitution, duplication, inversion, or splicing variant (isoform) mutation on the RNA can be detected. Moreover, the RNA expression level can also be detected. In particular, it is preferable to perform mRNA expression analysis, K-ras gene mutation analysis, DNA methylation analysis, and the like. These analyzes can be performed by methods known in the art. A commercially available analysis kit such as a K-ras gene mutation analysis kit or a methylation detection kit may also be used.
　本発明においては、糞便から回収される核酸であることから、大腸、小腸、胃等の消化管細胞由来の核酸を標的核酸とすることが好ましく、大腸剥離細胞由来の核酸を標的核酸とすることがより好ましい。 In the present invention, since it is a nucleic acid recovered from feces, it is preferable to use a nucleic acid derived from gastrointestinal cells such as the large intestine, small intestine, stomach, etc. as a target nucleic acid, and a nucleic acid derived from a large intestine exfoliated cell as a target nucleic acid. Is more preferable.
　特に、新生物性転化（癌を含む）のマーカー遺伝子や炎症性消化器疾患のマーカー遺伝子由来の核酸を標的核酸とすることが好ましく、大腸癌のマーカー遺伝子由来の核酸を標的核酸とすることがより好ましい。なお、「遺伝子由来の核酸」とは、当該遺伝子のゲノムＤＮＡや、ｍＲＮＡ等の発現産物を意味する。該新生物性転化を示すマーカーとして、例えば、Ｃｏｘ－２遺伝子、癌胎児性抗原（ＣＥＡ）、シアリルＴｎ抗原（ＳＴＮ）等の公知の癌マーカーや、ＡＰＣ遺伝子、ｐ５３遺伝子、Ｋ－ｒａｓ遺伝子等の変異の有無等がある。また、ｐ１６、ｈＭＬＨＩ、ＭＧＭＴ、ｐ１４、ＡＰＣ、Ｅ－ｃａｄｈｅｒｉｎ、ＥＳＲ１、ＳＦＲＰ２等の遺伝子のメチル化の検出も、大腸疾患の診断マーカーとして有用である（例えば、Lind et al.、「A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines」、Molecular Cancer、２００４年、第３巻第２８章参照。）。一方、炎症性消化器疾患を示すマーカーとして、例えば、Ｃｏｘ－２遺伝子由来核酸等がある。なお、Ｃｏｘ－２遺伝子由来核酸は、新生物性転化を示すマーカーとしても用いられる。 In particular, a nucleic acid derived from a marker gene for neoplastic transformation (including cancer) or a marker gene for inflammatory gastrointestinal diseases is preferably used as a target nucleic acid, and a nucleic acid derived from a marker gene for colon cancer is preferably used as a target nucleic acid. More preferred. The “gene-derived nucleic acid” means an expression product such as genomic DNA or mRNA of the gene. Examples of the marker showing neoplastic conversion include known cancer markers such as Cox-2 gene, carcinoembryonic antigen (CEA), sialyl Tn antigen (STN), APC gene, p53 gene, K-ras gene, etc. The presence or absence of mutations. Furthermore, detection of methylation of genes such as p16, hMLHI, MGMT, p14, APC, E-cadherin, ESR1, and SFRP2 is also useful as a diagnostic marker for colorectal diseases (for example, LindLet al., “A CpG island hypermethylation, profile, of primary, colorectal, carcinomas, and colon, cancer, cell lines, Molecular Cancer, 2004, Vol. 3, Chapter 28). On the other hand, examples of markers that indicate inflammatory digestive tract diseases include Cox-2 gene-derived nucleic acids. The Cox-2 gene-derived nucleic acid is also used as a marker indicating neoplastic transformation.
　本発明の合成方法においては、鉄キレート剤の存在下で核酸合成反応を行うことにより、反応効率を高めることができ、結果として安定した反応を行うことを可能とする。このため、本発明の合成方法により、夾雑物の多い糞便から回収された核酸中の標的核酸について、高精度であり、かつ信頼性も高い結果を得ることができる。例えば、本発明の合成方法を用いることにより、信頼性の高い糞便由来ＲＮＡの遺伝子発現解析の結果を得ることができる。こうして得られた結果は、臨床検査のために好適に用いられる。 In the synthesis method of the present invention, by performing a nucleic acid synthesis reaction in the presence of an iron chelator, the reaction efficiency can be increased, and as a result, a stable reaction can be performed. For this reason, with the synthesis method of the present invention, it is possible to obtain a highly accurate and highly reliable result for the target nucleic acid in the nucleic acid recovered from stool containing a large amount of contaminants. For example, by using the synthesis method of the present invention, highly reliable results of gene expression analysis of stool-derived RNA can be obtained. The result thus obtained is preferably used for clinical examination.
　例えば、本発明の合成方法を用いることにより、糞便中のＣｏｘ－２遺伝子等の大腸癌のマーカー遺伝子由来の核酸を精度よく合成することができる。こうして得られた合成産物は、大腸癌検出に利用することができる。すなわち、本発明の合成方法を用いることにより、臨床検査のための情報を提供することができる。 For example, by using the synthesis method of the present invention, a nucleic acid derived from a marker gene for colorectal cancer such as Cox-2 gene in feces can be synthesized with high accuracy. The synthetic product thus obtained can be used for colorectal cancer detection. That is, information for clinical examination can be provided by using the synthesis method of the present invention.
　また、鉄キレート剤を、核酸合成反応に用いられる試薬とキット化することにより、本発明の合成方法をより簡便に行うことができる。このような核酸合成用キットとしては、鉄キレート剤としてＨＩＤＳ、又はＨＩＤＳとＥＤＴＡとを備えることが好ましい。核酸合成（核酸増幅）反応に用いられる試薬としては、例えば、酵素（核酸合成酵素）、バッファー、プライマー等が挙げられる。例えば、市販の逆転写反応用キットやＰＣＲ用キット等に、鉄キレート剤を付加させたものを、本発明の核酸合成用キットとすることができる。 Moreover, the synthesis method of the present invention can be carried out more easily by making an iron chelating agent into a kit with a reagent used in a nucleic acid synthesis reaction. Such a nucleic acid synthesis kit preferably includes HIDS or HIDS and EDTA as an iron chelator. Examples of the reagent used for the nucleic acid synthesis (nucleic acid amplification) reaction include an enzyme (nucleic acid synthase), a buffer, a primer, and the like. For example, a product obtained by adding an iron chelator to a commercially available kit for reverse transcription reaction or PCR can be used as the nucleic acid synthesis kit of the present invention.
　また、本発明の核酸合成用キットは、糞便から核酸を回収するために用いられる試薬類を備えていてもよい。このような試薬類としては、例えば、糞便に添加する保存用溶液、核酸の抽出用溶液、無機支持体、無機支持体からの溶出用溶媒等が挙げられる。また、市販の核酸抽出用キットに、鉄キレート剤を付加させたものを、本発明の核酸合成用キットとすることもできる。なお、本発明においては、混合の手間を省くことができるため、無機支持体からの溶出用溶媒に予め鉄キレート剤を溶解させておくことが好ましい。 Moreover, the kit for nucleic acid synthesis of the present invention may be provided with reagents used for recovering nucleic acid from stool. Examples of such reagents include a storage solution added to feces, a nucleic acid extraction solution, an inorganic support, a solvent for elution from the inorganic support, and the like. Moreover, what added the iron chelating agent to the commercially available nucleic acid extraction kit can also be used as the nucleic acid synthesis kit of the present invention. In the present invention, it is preferable to dissolve the iron chelating agent in advance in the solvent for elution from the inorganic support, because the labor of mixing can be saved.
　次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。なお、ＭＫＮ４５細胞は、常法により培養したものを用いた。 Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples. MKN45 cells used were cultured by a conventional method.
［実施例１］擬似大腸癌患者由来糞便から抽出したＲＮＡ中のＣｏｘ－２遺伝子由来核酸の合成
　本発明の合成方法により、擬似大腸癌患者由来糞便試料から抽出したＲＮＡ中のＣｏｘ－２遺伝子由来核酸を合成し検出した。
　擬似大腸癌患者由来糞便試料（大腸癌患者から採取された糞便の擬似試料）は、健常人の糞便に、大腸癌細胞と同様にＣｏｘ－２遺伝子を高発現する培養細胞株であるＭＫＮ４５細胞を混合したものを用いた。具体的には、健常人の糞便５ｇに生理食塩水を等量添加し、よく混ぜて均一化した後、糞便試料１ｇ中にＭＫＮ４５細胞が５×１０５個含むように調整した細胞ペレットを、当該糞便試料に混合した。 [Example 1] Synthesis of nucleic acid derived from Cox-2 gene in RNA extracted from stool derived from pseudo-colon cancer patient According to the synthesis method of the present invention, derived from Cox-2 gene in RNA extracted from stool sample derived from pseudo-colorectal cancer patient Nucleic acids were synthesized and detected.
A stool sample derived from a pseudo-colorectal cancer patient (a stool pseudo-sample collected from a colorectal cancer patient) contains MKN45 cells, a cultured cell line that highly expresses the Cox-2 gene, in the same manner as colorectal cancer cells. A mixture was used. Specifically, after adding an equal amount of physiological saline to 5 g of normal human feces, mixing well and homogenizing, a cell pellet adjusted so that 5 × 10 5 MKN45 cells are contained in 1 g of the fecal sample, The stool sample was mixed.
　得られた擬似大腸癌患者由来糞便試料を１００μＬずつ３本の１．５ｍＬチューブに分注した。これらの３本の１．５ｍＬチューブに、グアニジン塩を含むＲＬＴバッファーを１ｍＬずつ添加し、Ｖｏｒｔｅｘで攪拌した。攪拌後に遠心分離処理を行い、上清３００μＬを分取し、さらに等量の７０％エタノールを加えて混合した。得られた混合液を、ＲＮｅａｓｙ　ｍｉｄｉ　ｋｉｔ（Ｑｉａｇｅｎ社製）のＲＮＡ回収用カラムに通し、添付のプロトコールに従って該ＲＮＡ回収用カラムの洗浄操作及びＲＮＡ溶出操作を行うことにより、ＲＮＡを５０μＬのＴｏｔａｌ　ＲＮＡ溶液として回収した。３本のチューブに回収されたＴｏｔａｌ　ＲＮＡ溶液を混合して一のＴｏｔａｌ　ＲＮＡ溶液とし、当該溶液のＲＮＡ濃度を測定し、回収された全ＲＮＡ量を定量した。 The obtained stool samples derived from pseudo colorectal cancer patients were dispensed into three 1.5 mL tubes of 100 μL each. To these three 1.5 mL tubes, 1 mL each of RLT buffer containing guanidine salt was added and stirred with Vortex. Centrifugation was performed after stirring, 300 μL of the supernatant was collected, and an equal amount of 70% ethanol was added and mixed. The obtained mixed solution is passed through an RNA recovery column of RNeasy midi kit (manufactured by Qiagen), and the RNA recovery column is subjected to washing operation and RNA elution operation according to the attached protocol, whereby 50 μL of total RNA is obtained. It was collected as a solution. The total RNA solution collected in three tubes was mixed to form one total RNA solution, the RNA concentration of the solution was measured, and the total RNA amount collected was quantified.
　回収されたＴｏｔａｌ　ＲＮＡを鋳型として、ＨＩＤＳ、ＥＤＴＡ、又はＢＳＡの存在下で、逆転写反応を行い、ｃＤＮＡを合成した。コントロールとして、ＨＩＤＳ、ＥＤＴＡ、及びＢＳＡのいずれも添加しない条件下で逆転写反応を行った。各サンプルの反応液中のＨＩＤＳ、ＥＤＴＡ、及びＢＳＡ濃度を、表１に示す。また、ＲＮＡ濃度の測定結果に基づき、各反応液中に１μｇのＲＮＡが添加されるように、逆転写反応の反応液を調製した。逆転写酵素として、Ｍ－ＭＬＶ（ＴａＫａＲａ社製）を用いた。 Using the recovered total RNA as a template, a reverse transcription reaction was performed in the presence of HIDS, EDTA, or BSA to synthesize cDNA. As a control, a reverse transcription reaction was performed under the condition where none of HIDS, EDTA, and BSA was added. Table 1 shows the concentrations of HIDS, EDTA, and BSA in the reaction solution of each sample. Further, based on the measurement result of the RNA concentration, a reaction solution for the reverse transcription reaction was prepared so that 1 μg of RNA was added to each reaction solution. M-MLV (TaKaRa) was used as a reverse transcriptase.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　得られたｃＤＮＡをテンプレートとしてリアルタイムＰＣＲを行い、Ｃｏｘ－２遺伝子の発現産物（ｍＲＮＡ）の検出を行った。リアルタイムＰＣＲのプライマーは、アプライドバイオシステム社製のＣｏｘ－２プライマープローブＭＩＸ（カタログＮｏ：Ｈｓ００１５３１３３＿ｍ１）を用いた。具体的には、０．２ｍＬの９６ウェルＰＣＲプレートに、各ｃＤＮＡを１μＬずつ分取した。その後、各ウェルに８μＬの超純水と１０μＬの核酸増幅試薬「ＴａｑＭａｎ　ＧｅｎｅＥｘｐｒｅｓｓｉｏｎ　Ｍａｓｔｅｒ　Ｍｉｘ」（アプライドバイオシステム社製）を添加し、さらに、１μＬのＣｏｘ－２プライマープローブＭＩＸ（アプライドバイオシステム社製）をそれぞれ添加して混合し、ＰＣＲ反応液を調製した。該ＰＣＲプレートを、ＡＢＩリアルタイムＰＣＲ装置に設置し、９５℃で１０分間処理した後、９５℃で１分間、５６．５℃で１分間、７２℃で１分間の熱サイクルを４０サイクル行った後、さらに７２℃で７分間処理することにより、経時的に蛍光強度を計測しながらＰＣＲを行った。 Real-time PCR was performed using the obtained cDNA as a template, and the expression product (mRNA) of the Cox-2 gene was detected. As a primer for real-time PCR, Cox-2 primer probe MIX (Catalog No: Hs00153133_m1) manufactured by Applied Biosystems was used. Specifically, 1 μL of each cDNA was dispensed into a 0.2 mL 96-well PCR plate. Thereafter, 8 μL of ultrapure water and 10 μL of nucleic acid amplification reagent “TaqMan GeneExpression Master Mix” (Applied Biosystems) were added to each well, and 1 μL of Cox-2 primer probe MIX (Applied Biosystems) was further added. Were added and mixed to prepare a PCR reaction solution. The PCR plate was placed in an ABI real-time PCR apparatus, treated at 95 ° C. for 10 minutes, and then subjected to 40 cycles of thermal cycling at 95 ° C. for 1 minute, 56.5 ° C. for 1 minute, and 72 ° C. for 1 minute. Further, PCR was performed while measuring the fluorescence intensity over time by treating at 72 ° C. for 7 minutes.
　蛍光強度の計測結果を分析して、各サンプル中のＣｏｘ－２遺伝子の発現量を調べた。
ＨＩＤＳ等が無添加のコントロール（表１中、サンプル１７）の発現量を１として、他のサンプルの発現量の相対値を算出した。算出結果を図１のグラフに示した。
　この結果、１０ｍＭ～１Ｍの範囲でＨＩＤＳを添加したサンプルでは、他の条件と比べてＣｏｘ－２遺伝子の発現量が４倍以上であった。一方で、ＥＤＴＡにも３価の鉄イオン（Ｆｅ３＋）をキレートする効果がある。また、ＨＩＤＳを１Ｍと高濃度に添加した場合でも、特に反応が阻害されている様子は観察されなかったことから、ＨＩＤＳの特徴として効果を示す濃度範囲が広いことが明らかとなった。但し、ＥＤＴＡは０．１Ｍを添加した場合には検出される発現量を増大させる効果が見られたが、１～１０ｍＭの範囲ではその効果はほとんど観察されず、また、１Ｍでは逆に反応が阻害されてしまった。また、ＢＳＡを添加した場合には、ほとんど効果が観察されなかった。
　ＨＩＤＳとＥＤＴＡを併用した場合には、各５ｍＭを添加した場合には、ＨＩＤＳ単独で添加した場合と同様に効果が観察されたが、各０．５ｍＭを添加した場合には効果が観察されず、各５０ｍＭ以上を添加した場合には、ＥＤＴＡ単独で添加した場合と同様に反応阻害が観察された。 The measurement result of the fluorescence intensity was analyzed, and the expression level of the Cox-2 gene in each sample was examined.
The relative value of the expression level of other samples was calculated with the expression level of the control without addition of HIDS and the like (sample 17 in Table 1) being 1. The calculation results are shown in the graph of FIG.
As a result, in the sample to which HIDS was added in the range of 10 mM to 1 M, the expression level of the Cox-2 gene was 4 times or more compared to other conditions. On the other hand, EDTA also has an effect of chelating trivalent iron ions (Fe 3+ ). In addition, even when HIDS was added at a high concentration of 1M, it was revealed that the reaction was not particularly inhibited, so that the concentration range showing the effect as a feature of HIDS was wide. However, EDTA had an effect of increasing the detected expression level when 0.1M was added, but the effect was hardly observed in the range of 1 to 10 mM, and the reaction was reversed at 1M. I was blocked. When BSA was added, almost no effect was observed.
When HIDS and EDTA were used in combination, when 5 mM was added, the same effect was observed as when HIDS was added alone, but when 0.5 mM was added, no effect was observed. When 50 mM or more of each was added, reaction inhibition was observed as in the case of adding EDTA alone.
　これらの結果から、ＨＩＤＳ等の鉄キレート剤の存在下で逆転写反応等の核酸合成反応を行うことにより、糞便から抽出された核酸を鋳型とした場合の反応効率を飛躍的に改善し得ることが明らかである。また、添加剤の種類と濃度によって効果に差が見られ、特に、ＨＩＤＳ等の鉄イオンへの特異性の高いキレート剤に、ＥＤＴＡのような比較的多くのイオン種をキレートし得るキレート剤を適当量併用することによっても、高い反応効率改善効果が得られることも分かった。 From these results, by performing nucleic acid synthesis reaction such as reverse transcription reaction in the presence of iron chelating agents such as HIDS, the reaction efficiency when nucleic acid extracted from feces is used as a template can be dramatically improved. Is clear. In addition, there is a difference in effect depending on the type and concentration of the additive, and in particular, a chelating agent that can chelate a relatively large number of ionic species such as EDTA to a chelating agent having high specificity to iron ions such as HIDS. It was also found that a high reaction efficiency improvement effect can be obtained by using an appropriate amount together.
　本発明の合成方法を用いることにより、核酸合成反応を利用して、糞便中の標的核酸を高精度に合成し、検出することができるため、特に臨床検査等の分野において利用が可能である。 By using the synthesis method of the present invention, a target nucleic acid in stool can be synthesized and detected with high accuracy using a nucleic acid synthesis reaction, and thus can be used particularly in the field of clinical examinations.

Claims (10)

  1. 　糞便から回収された核酸を鋳型とする核酸合成反応を、鉄キレート剤を含有する反応液中で行う、糞便中の標的核酸の合成方法。 A method for synthesizing a target nucleic acid in stool, in which a nucleic acid synthesis reaction using a nucleic acid collected from stool as a template is carried out in a reaction solution containing an iron chelator.
  2. 　前記鉄キレート剤が、３－ヒドロキシ－２，２’－イミノジコハク酸４ナトリウムである、請求項１に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to claim 1, wherein the iron chelating agent is tetrasodium 3-hydroxy-2,2'-iminodisuccinate.
  3. 　前記反応液が、さらにＥＤＴＡを含む、請求項１又は２に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to claim 1 or 2, wherein the reaction solution further contains EDTA.
  4. 　前記核酸合成反応が、逆転写反応、又はポリメラーゼを用いた核酸増幅反応である請求項１～３のいずれか一項に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to any one of claims 1 to 3, wherein the nucleic acid synthesis reaction is a reverse transcription reaction or a nucleic acid amplification reaction using a polymerase.
  5. 　前記糞便から回収された核酸が、動物より採取された糞便又はその固形成分に、核酸抽出用溶液を添加して混合した後、当該抽出用溶液から回収された核酸である、請求項１～４のいずれか一項に記載の糞便中の標的核酸の合成方法。 The nucleic acid recovered from the stool is a nucleic acid recovered from the extraction solution after the nucleic acid extraction solution is added to and mixed with the stool collected from the animal or a solid component thereof. The method for synthesizing a target nucleic acid in stool according to any one of the above.
  6. 　前記標的核酸が、哺乳細胞由来の核酸である、請求項１～５のいずれか一項に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to any one of claims 1 to 5, wherein the target nucleic acid is a nucleic acid derived from a mammalian cell.
  7. 　前記標的核酸が、新生物性転化を示すマーカー又は消化器系疾患を示すマーカーである、請求項６に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to claim 6, wherein the target nucleic acid is a marker indicating neoplastic transformation or a marker indicating digestive system disease.
  8. 　前記標的核酸が、Ｃｏｘ－２遺伝子由来核酸である、請求項７に記載の糞便中の標的核酸の合成方法。 The method for synthesizing a target nucleic acid in stool according to claim 7, wherein the target nucleic acid is a Cox-2 gene-derived nucleic acid.
  9. 　前記標的核酸を大腸癌のマーカー遺伝子由来の核酸として請求項１～８のいずれか一項に記載の糞便中の標的核酸の合成方法行い、
    　当該合成方法の産物を検出する
    ことを含む、大腸癌検査のための情報提供方法。     The method for synthesizing a target nucleic acid in stool according to any one of claims 1 to 8, wherein the target nucleic acid is a nucleic acid derived from a colon cancer marker gene,
    A method for providing information for colorectal cancer testing, comprising detecting a product of the synthesis method.
  10. 　糞便からの核酸抽出用溶液；
    　　核酸合成酵素及びバッファーを含む、核酸増幅試薬；及び
    　　鉄キレート剤を有する、核酸合成用キット。     Solution for nucleic acid extraction from feces;
    A nucleic acid synthesis kit comprising a nucleic acid amplification reagent comprising a nucleic acid synthase and a buffer; and an iron chelator.
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