WO2007052765A1 - Rnaの抽出方法及びrnaの検出方法 - Google Patents
Rnaの抽出方法及びrnaの検出方法 Download PDFInfo
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- WO2007052765A1 WO2007052765A1 PCT/JP2006/322010 JP2006322010W WO2007052765A1 WO 2007052765 A1 WO2007052765 A1 WO 2007052765A1 JP 2006322010 W JP2006322010 W JP 2006322010W WO 2007052765 A1 WO2007052765 A1 WO 2007052765A1
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- Prior art keywords
- rna
- sample
- solution
- treatment
- rnase
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Classifications
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- 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
-
- 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
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- 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
- C12Q2523/00—Reactions characterised by treatment of reaction samples
- C12Q2523/10—Characterised by chemical treatment
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- 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
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/101—Temperature
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- 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
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/119—Reactions demanding special reaction conditions pH
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- 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
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/125—Specific component of sample, medium or buffer
Definitions
- the present invention relates to a method for inactivating an RNase present in a sample or the like, from an RNA inclusion (cell, fungus, bacterium, virus, etc.) present in the sample, or from the sample.
- the present invention relates to a method for easily and stably extracting RNA from separated RNA inclusions, a method for detecting the RNA, and a reagent used in those methods.
- the present invention relates to an RNA amplification method, and more particularly to an RNA amplification method by a reverse transcription-polymerase chain reaction (hereinafter abbreviated as RT-PCR) method.
- RT-PCR reverse transcription-polymerase chain reaction
- RNA inclusions cells, fungi, bacteria, viruses, etc.
- RNA inclusions cells, fungi, bacteria, viruses, etc.
- RNA inclusions are separated and collected from the test substance, and then RNA is extracted from the RNA inclusions and the extracted RNA is purified.
- a process is required.
- RNases are ubiquitous and are extremely difficult to inactivate. For this reason, when purifying RNA from RNA inclusion bodies in biological samples, RNase control (inhibition of activity) and RNase removal must be performed in the RNA extraction process from the inside of RNA inclusions. A very rigorous and cumbersome method was needed.
- RNA is extracted and extracted with phenol or phenol / chloroform. Purification methods are used. Recently, a method in which an ion exchange resin, glass filter, glass beads, magnetic beads, or a reagent having a protein agglutinating action is used in the process of RNA extraction and purification has been reported. Extraction and purification of RNA is, Chomczynski & Sacchi (1987) Analytical Biochemistry, 162: 156- 159.
- the RT-PCR method is a method of amplifying cDNA by PCR after converting RNA into complementary DNA (cDNA) using Reverse Transcriptase.
- the RT-PCR method is used as one of the most sensitive and highly quantitative analysis methods today because it can quantitatively analyze even small amounts of RNA. For example, detection of viruses carrying RNA as a gene, quantitative detection of mRNA, analysis of expression genes by sequencing of mRNA, and analysis and production of expression products by cloning of cDNA are essential. Sena, become technology!
- the PCR method followed by the RT reaction can amplify the target DNA fragment hundreds of thousands of times by repeating the DNA synthesis reaction between primers sandwiching a specific region in the DNA strand. Is the method.
- the PCR method is disclosed in Japanese Patent Laid-Open No. 61-274697, which is an invention of Maris et al.
- RNA is easily degraded by RNase that is universally present in all biological samples.
- RNA inclusions cells, fungi, bacteria, viruses and the like (hereinafter referred to as RNA inclusions) are separated from the test substance, and then RNA is removed from the RNA inclusions.
- a process of extraction and purification is required.
- US Pat. No. 6,825,340 and US Pat. No. 6,777,210 include a heat treatment in the presence of a reducing agent to inactivate RNase as well as from cultured cells after washing with PBS. RNA extraction and RT-PCR are disclosed.
- Japanese Laid-Open Patent Publication No. 2001-29078 discloses a direct RT-PCR from a sample containing an RNA inclusion.
- JP-A-2004-301684 discloses a dilution for norovirus specimen using an alkaline buffer and an antigen-antibody reaction using the dilution. Norovirus detection is disclosed.
- Non-Patent Document 1 Chomczynski and Sacchi, “Analytical Bar Analytical Biochemistry, 1987, 162, p. 156-159
- Non-patent document 2 Joseph. Sambrook and David W. Russell, “Molecules” Cloning: Laboratory Manual Third Edition (2001)
- Patent Document 1 Japanese Patent Laid-Open No. 61-274697
- Patent Document 2 U.S. Patent No. 6825340
- Patent Document 3 US Patent No. 6777210
- Patent Document 4 Japanese Patent Laid-Open No. 2001-29078
- Patent Document 5 Japanese Unexamined Patent Application Publication No. 2004-301684
- RNA is always exposed to the risk of degradation by RNases that are ubiquitously present in any environment in which the organism exists, as well as in vivo. Therefore, when RNA is extracted from within the RNA inclusions, it must be treated with a rapid RNase inactive enzyme, as well as in a rigorous manner that does not contaminate RNase during or after the purification process. Operation and management are required.
- RNA in a sample is purified using conventional methods, it is often difficult to remove contaminants or the amount of RNA recovered in the sample is not constant. In particular, when the target RNA content in the sample is low, subsequent RNA analysis may be difficult. Also, these purification methods are complicated and time consuming, and there is a high chance of contamination during operation. For these reasons, conventional purification methods require skill. Therefore, in order to solve these problems, a simpler and more effective sample pretreatment method has been desired.
- the purpose of the present invention is a sample such as a biological sample, excrement sample, and environmental sample, or a biological sample, excrement-derived sample, environment-derived sample obtained by separating RNA inclusions from the sample, etc. It is intended to provide a method for inactivating RNases that are universally present in samples.
- the purpose of the present invention is a sample such as a biological sample, excrement sample, environmental sample, or a sample such as a biological sample obtained by separating RNA inclusions from the sample, a sample derived from excrement, an environment-derived sample, Provide a method for efficiently extracting RNA from RNA inclusions in food There is.
- An object of the present invention is to easily extract the sample force RNA, further suppress the action of an inhibitory substance on the nucleic acid synthesis reaction, and efficiently amplify the RNA in the sample. It is intended to provide a method for detecting RNA present in a sample quickly and stably.
- An object of the present invention is to provide a treatment reagent that can be used in these methods.
- the present inventors have performed the above-mentioned book by inactivating RNase in a biological sample and extracting RNA from the RNA inclusion body in one step, and subsequently performing RNA amplification.
- the inventors have found that the object of the invention can be achieved and have completed the present invention.
- the following relates to a method for inactivating RNase.
- the following RNase deactivation method comprises inactivating a RNase under heating conditions using an alkaline treatment reagent containing at least a reducing agent for a sample containing RNase. This is an enzyme deactivation method.
- a method of inactivating the RNase by maintaining the mixture under the heating condition to inactivate the RNase.
- the alkalinity of the treatment reagent is such that the pH of the mixture becomes 8.1 or more (at 25 ° C) when mixed with the sample to form a mixture. Under heating conditions of 30 ° C or higher
- V A method for inactivating the RNase using the treatment reagent.
- the treatment reagent includes a Tris buffer solution, a Good buffer solution, a borate buffer solution, and an alkaline buffer that also has a group strength that is a carbonate buffer solution.
- RNA-degrading enzyme which is contained in the treatment reagent as sodium hydroxide and / or potassium hydroxide ImM ⁇ : LOO mM as the alkaline substance.
- the thiol type reducing agent is a general term for reducing agents having a thiol group.
- the thiol-type reducing agent power dithiothreitol and mercaptoethanol power are selected from the group consisting of the above-mentioned RNase deactivation methods.
- the method for deactivating RNA-degrading enzyme wherein the reducing agent is contained in the treatment reagent at a concentration of 0. ImM to a saturated concentration.
- the RNase deactivation method wherein the sample is selected from the group consisting of a biological sample, a biological sample, an environmental sample, and an environmental sample.
- the R is selected from the group consisting of a waste sample and a waste-derived sample.
- RNA inclusion body is selected from the group power of cells, fungi, bacteria, and RNA viruses.
- RNA virus is selected from the group consisting of retrovirus, norovirus (SRSV), rotavirus, and hepatitis C virus (HCV). Law.
- SRSV norovirus
- HCV hepatitis C virus
- the retrovirus is an AIDS virus.
- the pH of the mixture containing the RNase in the solution containing at least the reducing agent and the mixture of the sample and the reducing agent is 8.1 or more at 25 ° C.
- RNA-degrading enzyme comprising subjecting said RNA-degrading enzyme to deactivation by subjecting said pH-adjusted mixed solution to heating conditions.
- the inactivation of RNase in the above method is performed by subjecting the sample to an alkaline environment having a reducing agent in the pH of 8.1 or higher.
- the following (1) to (11) relate to RNA extraction methods.
- the extraction method of the present invention uses an alkaline treatment reagent containing at least a reducing agent for a sample containing RNA inclusion bodies and RNase, and inactivates the RNase under heating conditions. And RNA extraction from the RNA inclusion body.
- RNA extraction from the inside of an RNA inclusion means that RNA contained in the membrane structure is extracted by destroying the membrane structure of the RNA inclusion and exposed to the environment outside the membrane. Defined as In addition, performing any treatment on the exposed RNA or the external environment exposed to the exposed RNA is not included in the definition of extraction in the present invention.
- a method of extracting RNA comprising the step of deactivating the RNase and extracting RNA from RNA inclusions by maintaining the mixture under the heating condition.
- the alkalinity of the treatment reagent is such that the pH of the mixture becomes 8.1 or more (at 25 ° C) when mixed with the sample to form a mixture.
- RNA extraction according to (1) wherein the treatment reagent includes a Tris buffer solution, a Good buffer solution, a borate buffer solution, and an alkaline buffer that also has a group strength that can be a carbonate buffer.
- RNA extraction method according to (1) or (2), wherein the treatment reagent contains an alkaline substance selected from the group power of hydroxide, ammonia, and amine.
- RNA of the RNA wherein the hydroxide is sodium hydroxide and / or potassium hydroxide Extraction method.
- the thiol-type reducing agent is a general term for reducing agents having a thiol group.
- RNA extraction method according to any one of the above, wherein dithiothreitol is included in the treatment reagent as ImM ⁇ : LOOmM as the reducing agent.
- RNA The method for extracting RNA according to any one of (1) to (4), wherein the sample is selected from the group consisting of a biological sample, a biological sample, an environmental sample, and an environmental sample. .
- the sample is selected from a group of excrement samples and excrement-derived samples.
- RNA extraction method according to any one of to (5).
- RNA extraction method according to any one of (1) to (6), wherein the RNA inclusion body is selected from a group power consisting of cells, fungi, bacteria, and RNA viruses.
- the RNA virus is a retrovirus, norovirus (SRSV), rotavirus, and
- RNA according to (7) wherein hepatitis C virus (HCV) force is selected.
- HCV hepatitis C virus
- RNA virus is a retrovirus
- the retrovirus is AIDS virus (HIV).
- An RNA extraction method comprising the steps of deactivating the RNA degrading enzyme and extracting RNA from the RNA inclusion body strength by subjecting the pH-adjusted mixed solution to heating conditions. That is, inactivation of RNAse and RNA extraction in the above (1) to (10) are performed by subjecting the sample to an alkaline environment of pH 8.1 or higher in the presence of a reducing agent.
- the following (12) to (22) relate to an RNA detection method.
- the RNA detection method of the present invention comprises the steps of inactivating the RNase under heating conditions using an alkaline treatment reagent containing at least a reducing agent for a sample containing an RNA inclusion body and an RNase.
- RNA extracted from the RNA inclusion body internal force is extracted, a sample treatment solution is obtained, and the sample treatment solution and the amplification reaction solution are mixed to perform an RNA amplification reaction.
- RNA extraction from the inside of an RNA inclusion is defined as the removal of RNA contained in the membrane structure by breaking the membrane structure of the RNA inclusion and exposing it to the environment outside the membrane. To do. Further, any treatment for the exposed RNA or the external environment to which the exposed RNA is exposed is not included in the definition of extraction in the present invention.
- RNA detection method in which the sample treatment solution and the amplification reaction solution are mixed to perform an RNA amplification reaction
- the degree of alkalinity of the treatment reagent is such that the pH of the mixture becomes 8.1 or more (at 25 ° C) when mixed with the sample to form a mixture.
- the treatment reagent includes a Tris buffer solution, a Good buffer solution, a borate buffer solution, and an alkaline buffer that also has a carbonate buffer solution strength.
- RNA detection method according to (12) or (13), wherein the treatment reagent contains an alkaline substance selected from the group power of hydroxide, ammonia, and amine.
- RNA detection method described above, wherein the hydroxide is sodium hydroxide and / or potassium hydroxide.
- RNA detection method wherein the alkaline substance is contained in the treatment reagent at a concentration of 0. ImM to a saturated concentration.
- the thiol-type reducing agent is a general term for reducing agents having a thiol group.
- RNA detection method as described above, wherein the thiol-type reducing agent power dithiothreitol and mercaptoethanol power are selected.
- RNA detection method wherein the reducing agent is contained in the treatment reagent at a concentration of 0. ImM to a saturated concentration.
- RNA detection method as described above, wherein dithiothreitol is contained in the treatment reagent as ImM ⁇ : LOOmM as the reducing agent.
- RNA detection method according to any one of (12) to (15), wherein the sample is selected from the group consisting of a biological sample, a biological sample, an environmental sample, and an environmental sample.
- the sample is selected from a group of excrement samples and excrement-derived samples.
- RNA detection method according to any one of (12) to (17), wherein the RNA inclusion body is selected from the group power of cells, fungi, bacteria, and RNA viruses.
- RNA virus is a retrovirus, norovirus (SRSV), rotavirus, and
- RNA detection method according to (19), wherein when the RNA virus is a retrovirus, the retrovirus is AIDS virus (HIV).
- RNA detection method according to any one of (12) to (20), wherein the RNA is mRNA.
- the mixed solution of the sample processing solution and the amplification reaction solution further includes an additive that selects a sulfated polysaccharide, a polyamine, albumin, and a group force that also has a nonionic surfactant power.
- RNA detection method RNA detection method.
- RNA detection method described above wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene sorbitan monolaurate and polyoxyethylene octylphenol ether.
- RNA detection method described above, wherein the treatment reagent further contains a sulfated polysaccharide.
- RNA is extracted from the RNA inclusion body, and a sample processing solution containing the extracted RNA is prepared.
- An RNA detection method comprising a step of mixing the sample treatment solution and an amplification reaction solution to perform an RNA amplification reaction.
- the deactivation of RNAse and RNA extraction in the above methods (12) to (21) are performed by subjecting the sample to an alkaline environment of pH 8.1 or higher in which a reducing agent is present.
- the following relates to processing reagents for samples containing RNases.
- a reagent for treating a sample containing an RNA-degrading enzyme comprising at least an alkaline substance and / or an alkaline buffer and a reducing agent.
- the inactivation method of the RNA degrading enzyme, the RNA extraction method according to any one of (1) to (11), or the RNA detection method according to any one of (12) to (22) Contains at least an RNase containing alkaline substances and / or alkaline buffers and reducing agents for use in Sample processing reagent.
- an RNase that is universally present in a sample such as a biological sample or an environmental sample, or a sample such as a biological sample obtained by separating RNA inclusions from the biological sample or the like.
- a method of deactivation can be provided.
- RNA is efficiently extracted from RNA inclusions present in samples such as biological samples and environmental samples, or samples such as biological samples obtained by separating RNA inclusions therefrom.
- a method of extraction can be provided.
- RNase present in a sample can be easily and stably obtained by performing inactivation of RNase in the sample and RNA extraction from the RNA inclusion body in one step. Can be amplified. Further, by suppressing the action of an inhibitor on nucleic acid synthesis, it becomes possible to amplify RNA present in the sample more simply, stably, efficiently and rapidly. This can provide a simple, “stable”, efficient and rapid method for detecting RNA in a sample.
- a treatment reagent that can be used in these methods can be provided.
- FIG. 1 shows the treatment of a sample obtained by adding RNA inclusions to human serum using distilled water or three kinds of treatment reagents having different compositions in Example 1.
- FIG. 4 is an electrophoretogram showing the results of RNA detection by subsequent RNA amplification.
- FIG. 2 shows the results of Example 2 in which the sample treated with distilled water or three kinds of processing reagents having different compositions in Example 1 was stored in a refrigerator for 1 day.
- FIG. 4 is an electrophoretogram showing the results of RNA detection by subsequent RNA amplification.
- FIG. 3 is a graph showing the relationship between the temperature and time of heat treatment and the amount of RNA detected obtained in Example 3.
- FIG. 4 is a graph showing the relationship between the heat treatment time at 85 ° C. and the amount of RNA detected obtained in Example 4.
- FIG. 5 shows the relationship between the temperature and time of the heat treatment and the RNA detection amount obtained in Example 5. It is the graph which showed the relationship.
- FIG. 6 shows the results of treatment of a sample obtained by adding RNA inclusion bodies to human serum with 15 treatment reagents having different compositions in Example 8, followed by RNA amplification.
- FIG. 6 is an electrophoretogram showing the results of detecting RNA.
- FIG. 7 shows that in Example 9, a sample obtained by adding RNA inclusion bodies to human serum was treated with one of the treatment reagents in Example 8 under various heating conditions, and thereafter FIG. 6 is an electrophoretogram showing the results of detecting RNA by performing RNA amplification.
- FIG. 8 shows a treatment sample obtained by adding RNA inclusion bodies to human serum in Example 9, using a treatment reagent containing one of the treatment reagents in Example 8 and further containing EGTA.
- FIG. 3 is an electrophoretogram showing the results of detecting RNA by performing RNA amplification under various heating conditions and then performing RNA amplification.
- FIG. 9 shows a case where eight treatment reagents having different compositions of NaOH concentration were used in the stool sample solution mixed with the pseudo-norovirus positive stool sample in Example 10.
- FIG. 3 is an electrophoretogram showing the results of RNA detection after treatment and subsequent RNA amplification.
- FIG. 10 shows a treatment using seven treatment reagents each having a different DTT concentration in the stool sample solution mixed with the pseudo-norovirus positive stool sample in Example 11.
- FIG. 2 is an electrophoretogram showing the results of detecting RNA by performing RNA amplification after that.
- Fig. 11 is an electrophoretogram showing the results of RNA detection performed in Example 12 without RNA purification on infected stool samples having different virus concentrations.
- FIG. 12 is an electrophoretogram showing the results of RNA detection performed by purifying RNA in Norovirus-infected stool samples with different virus concentrations in Example 12.
- FIG. 13 is an electrophoretogram showing the results of RNA detection using Norovirus-infected stool samples derived from 18 different specimens infected with Norovirus in Example 13.
- FIG. 13 is an electrophoretogram showing the results of RNA detection using Norovirus-infected stool samples derived from 18 different specimens infected with Norovirus in Example 13.
- FIG. 14 shows the results of RNA detection using norovirus-uninfected stool samples derived from 10 different specimens not infected with Norovirus in Example 13, respectively. It is an electropherogram.
- FIG. 15 shows a stool sample liquid mixed with pseudo-norovirus-positive stool sample in Example 14, which is treated with a treatment reagent under various heating conditions, and then RNA amplification is performed. It is an electrophoretic diagram which shows the result of having detected RNA by this.
- FIG. 16 is a graph showing the results of quantification of amplified RNA by real-time PCR in Example 14.
- FIG. 17 is a photograph showing a state after the sample specimen was mixed with 15 types of processing reagents and heat-treated in Example 8.
- the upper row shows the results of using DTT OmM treatment reagent. From left, [1], [2], [3], [4], [5], [6], [7] It is the result using.
- the bottom row shows the results using DTT 20 mM treatment reagent from the left [8], [9], [10], [11], [12], [13], [14], [15] It is a result using the processing reagent.
- the RNase deactivation method and RNA extraction method of the present invention are realized in an alkaline environment and in the presence of a reducing agent.
- the RNA detection method of the present invention includes a step of inactivating RNase in a sample and RNA extraction from the RNA inclusion body, and a step of performing an RNA amplification reaction.
- RNA can be amplified directly from a sample without special purification of RNA.
- the present invention can be applied to any sample to be processed as long as it can contain RNase.
- samples include biological samples, biological samples, environmental samples, environmental samples, excrement samples, excrement samples, and the like.
- an RNA inclusion is a structure surrounded by a membrane structure and having RNA therein. Specifically, it refers to cells, fungi, bacteria, viruses and the like.
- the cells include leukocytes derived from blood, cerebrospinal fluid and the like, oral mucosal cells and the like.
- the cells include food-derived cells, exfoliated cells from the body, and the like.
- RNA such as mRNA
- viruses include RNA viruses.
- RNA viruses include retroviruses (AIDS virus (HIV), etc.), norovirus (SRSV), rotavirus, hepatitis C virus (HCV), etc.
- Examples of biological samples include animal and plant tissues and body fluids.
- Body fluids include blood samples, cerebrospinal fluid, saliva, milk, and the like.
- the blood sample includes whole blood, plasma, serum and the like.
- the biological sample includes a sample obtained by subjecting the biological sample to some treatment.
- Examples of environmental samples include any sample containing air, soil, water, etc., as long as they contain RNA inclusions.
- the environment-derived sample includes a sample obtained by treating the environmental sample.
- the excreta includes urine, feces, vomit, and the like.
- the excrement sample includes the excretion itself excreted from the living body, or the excrement itself suspended in water, physiological saline, pH buffer solution or the like.
- Examples of the living body include humans, livestock, insects, and all other animals.
- the excrement-derived sample includes a sample obtained by subjecting the excrement sample to some treatment.
- An example of a treatment that may be performed on the sample includes a recovery treatment of RNA inclusion bodies.
- a method for recovering RNA inclusions any method can be used as long as it can separate RNA inclusions from the sample. For example, centrifugation / ultracentrifugation operation, filtration / ultrafiltration operation; a method using a coprecipitation agent such as polyethylene glycol, an adsorbent bile body such as an antibody, etc. in combination with the operation; A separation method using a membrane or the like is used. RNase can remain with either method The present invention is effective in the case of recovery treatment of a characteristic RNA inclusion.
- the sample in the present invention is allowed to contain a substance that inhibits the RNA amplification reaction.
- a substance that inhibits the RNA amplification reaction are usually contained in samples such as biological samples, biological samples, environmental samples, environmental samples, excrement samples, excrement samples, and the like.
- Substances that inhibit the RNA amplification reaction include substances that exist inside or outside the cell, such as pigments, proteins, sugars, and unknown contaminants present in biological samples.
- the sample may be subjected to an alkaline environment containing at least a reducing agent.
- an alkaline mixed solution in which at least the reducing agent and the sample are finally mixed is prepared, there is no limitation on the river page to be mixed.
- the mixed solution since the mixed solution is to be subjected to heating conditions (item 3 described later), the preparation operation of the mixed solution and the heating operation do not matter.
- the mixed solution should be in a state where the sample is mixed in an alkaline solution containing at least a reducing agent when subjected to heating conditions.
- one or both of the sample and the processing reagent can be heated, and then both can be mixed. That is, the mixed solution may be subjected to heating conditions at the same time as being prepared.
- a mixed solution of a sample and a processing reagent may be prepared at room temperature, and the obtained mixed solution may be subjected to heating conditions.
- the treatment reagent is usually used as an aqueous solution.
- mixed at room temperature with a solution and a sample containing at least a reducing agent, resulting reducing agent - the P H of the sample mixture was adjusted (described later), pH adjusted reductant - sample mixture heated condition You may use below.
- the treatment reagent itself having the following composition is not used, but the pH-adjusted reducing agent-sample mixture corresponds to the above-mentioned sample-treatment reagent mixture.
- this pH-adjusted reducing agent sample mixture shall also be included.
- the pH of the mixture of the treatment reagent and the sample (treatment reagent-sample mixture) can be pH 8.1 or higher, for example, pH 8.1 to 11.1 at 25 ° C. Depending on the sample, a pH of 9.0 to 11.1 may be preferred.
- Such a case includes a case where a sample of excrement (especially a stool sample) or a sample derived therefrom is used as a sample.
- an alkaline buffer and / or an alkaline substance may be included in the processing reagent.
- the alkaline buffer is not particularly limited as an alkaline buffer.
- Examples of the alkaline buffer include Tris buffer solution, Good buffer solution, borate buffer solution, and carbonate buffer solution.
- the buffer that constitutes the Good buffer is not particularly limited, and examples include Tricine, MOPS, HEPES, and CHES.
- hydroxide As an alkaline substance contained in the processing reagent, hydroxide, ammonia, and amine power may be selected.
- hydroxide examples include sodium hydroxide and potassium hydroxide.
- amines examples include trishydroxymethylaminomethane. These can be used alone or in combination of two or more.
- the concentration of the alkaline substance in the treatment reagent is different depending on the type of alkaline substance, the type and concentration of the sample, the mixing ratio with the sample, etc. 0. ImM to saturated concentration (saturated concentration at room temperature), preferably ImM to saturation concentration (saturation concentration at room temperature).
- a thiol-type reducing agent As a reducing agent contained in the processing reagent, a thiol-type reducing agent may be used.
- the thiol type reducing agent is a general term for reducing agents having a thiol group.
- thiol type reducing agent examples include dithiothreitol (DTT) and mercaptoethanol.
- DTT dithiothreitol
- mercaptoethanol is usually 2-mercaptoethanol.
- the concentration of the reducing agent in the treatment reagent varies depending on the type of reducing agent, the type and concentration of the sample, the mixing ratio with the sample, and the like. Preferably, it can be set to ImM-saturation concentration (saturation concentration at room temperature).
- the concentration of the reducing agent in the mixture of the treatment reagent and the sample can be, for example, 0. ImM to LM, preferably ImM to LOOmM. 0 if the sample is a blood sample. In some cases, it is more preferable that the concentration be from 05 mM to 20 mM. In addition, when the sample is an excrement sample or an excrement-derived sample, it may be more preferable to use 2.5 mM to 25 mM depending on the type of reducing agent, the type of the sample, and the like!
- the processing reagent may further include a chelating agent.
- RNA hydrolysis is known to be promoted by divalent metal ions. Therefore, it is effective to add chelating agents (EGTA, EDTA, etc.) that chelate divalent metal ions to the treatment reagent.
- chelating agents EGTA, EDTA, etc.
- the treatment reagent may further contain a sulfated polysaccharide.
- the difference in the amount of RNase in the sample, the difference in the fragility of the membrane structure due to the difference in the type of RNA inclusions, and the pH due to the difference in the amount of alkaline substances used It is not particularly limited because it differs depending on the difference.
- the treatment time can be, for example, about 60 minutes for 1 second, preferably 30 seconds to 30 minutes, and more preferably about 30 seconds to 15 minutes.
- the treatment temperature is preferably 30 ° C or higher.
- the processing temperature is, for example, from 30
- the processing temperature is 60 ° C or higher, for example, 60 ° C or higher and 100 ° C or lower, preferably 70 ° C to 90 ° C, more preferably 80 ° C force is also about 85 ° C. It can be.
- the processing time can be 30 seconds to 5 minutes.
- both the inactivation of RNase contained in the sample and the extraction of RNA from the RNA inclusion body are realized.
- RNA extraction occurs simultaneously with RNase inactivation or subsequent to RNase inactivation.
- both of the above treatments can be performed by a simple operation of only the heat treatment using the above treatment reagent, so that RNA can be rapidly and stably extracted.
- RNase is denatured so that the enzyme active site does not function. Even under heating conditions, RNases are usually stable to heat and are not easily inactivated. However, such a RNase can be inactivated by a heating operation using the treatment reagent of the present invention.
- RNA from inside the RNA inclusion body means that the membrane structure of the RNA inclusion body is destroyed, and the RNA contained in the membrane structure is exposed to the environment outside the membrane. RNases that existed in the environment outside the RNA inclusion membrane are inactivated by the same treatment reagent. For this reason, exposed RNA has a very high risk of degradation by nature! Despite exposure to the RNA envelope outer membrane environment, the risk of degradation is extremely low. For this reason, in the present invention, in order to achieve RNA extraction from the inside of the RNA inclusion body, it is not necessary to immediately purify the exposed RNA as long as the RNA is exposed to the environment outside the RNA inclusion body. A can exist stably.
- a sample treatment solution in which RNase is inactivated and RNA is exposed is obtained.
- the sample treatment solution can be used in various processes. For example, it can be used for steps such as RNA amplification and hybridization performed for RNA analysis.
- RNase is inactivated. For this reason, since RNA is stably contained, it can use for said process without performing any treatment.
- Even if the sample processing solution obtained by the method of the present invention is obtained by subjecting it to some further processing, it can be used for the above-mentioned steps. For example, treatment for adjusting pH such as neutralization, and RNA purification treatment such as centrifugation and RNA isolation can be mentioned.
- RNase is inactivated, and a sample treatment solution with exposed RNA is obtained.
- the obtained sample treatment solution can be used for the preparation of an amplification reaction solution.
- the sample treatment solution used for the amplification reaction solution may be obtained without any treatment after the heat treatment, or the supernatant obtained by performing a centrifugation after the heat treatment. Or as a filtrate obtained by performing filtration. May be.
- the sample treatment solution is mixed with the RNA amplification reaction solution to be the final reaction solution.
- the pH of the mixture of the sample treatment solution and the amplification reaction solution is If it deviates from the reaction conditions, it is necessary to adjust the pH of the mixture to be within the optimum conditions at an appropriate stage between the heat treatment and the start of the amplification reaction.
- a person skilled in the art can appropriately determine the optimum pH and the pH adjustment method.
- the optimum pH condition as described later, when a substance that inhibits the RNA amplification reaction is present in the reaction system, the pH may be adjusted to an alkaline range in order to suppress the action of the inhibitor. It is valid.
- the optimum pH refer to Japanese Patent No. 3494509 and Japanese Patent No. 3452717.
- RNA amplification reaction method any method can be used as long as it is a method for performing RNA amplification, including RT-PCR method.
- the composition of the amplification reaction solution is not particularly limited and can be appropriately determined by those skilled in the art.
- RT-PCR When RT-PCR is performed as an RNA amplification reaction, the form is such that a mixture of a sample treatment solution and an RT reaction solution is prepared in a tube, the RT reaction is performed in the tube, and the RT reaction is performed.
- a reaction form (Two tube-Two step) that is performed by adding a part of the product to the PCR reaction solution prepared in another tube and carrying out the PCR reaction; the sample treatment solution and the RT reaction solution in the tube Prepare a mixture, perform an RT reaction in the tube, add a PCR reaction solution to the RT reaction product in the tube, and perform a PCR reaction (One tube-Two step); Also, prepare both RT reaction solution and PCR reaction solution in the tube, mix with sample treatment solution, and perform the RT reaction and PCR reaction in a continuous manner (One tube-One step).
- the RNA amplification reaction solution to be mixed with the sample treatment solution may be an RT reaction solution or a mixture of an RT reaction solution and a PCR reaction solution, depending on the above execution form. May be a liquid.
- Any known RT reaction solution can be used without limitation. Usually, pH buffer, salts, primers, deoxyribonucleotides, and reverse transcriptase are included.
- the above-mentioned salts may be changed to other salts as appropriate, using the power of MgCl, KC1, etc.
- a primer refers to an oligonucleotide that serves as a starting point for synthesis during cDNA synthesis.
- the reverse transcriptase used in the RT reaction means an enzyme that can reverse transcribe RNA into cDNA.
- Reverse transcriptases include reverse transcriptases derived from avian retroviruses such as Rous associated virus (RAV) and Avian myeloblastosis virus (AMV); reverse transcriptases derived from mouse retroviruses such as Moloney murine leukemia virus (MMLV); And forces including Tth DNA polymerase derived from Thermus thermophilus are not limited to these.
- Any known PCR reaction solution can be used without limitation. Usually, pH buffer solution, salts, primers, deoxyribonucleotides, and thermostable DNA polymerase are included. The above-mentioned salts can be used with MgCl, KC1, etc.
- a primer is an oligonucleotide that serves as a starting point for synthesis during nucleic acid amplification.
- the thermostable DNA polymerase used in PCR means a polymerase with excellent thermostability that synthesizes DNA based on a primer.
- Suitable thermostable DNA polymerases include Taq DNA polymerase from Thermus aquaticus; Tth DNA polymerase from Thermus thermophilus; KOD DNA polymerase, Pfo DNA polymerase, Pwo DNA polymerase from Pyrococcus; and these thermostable DNA polymerases The power to raise the mixture etc. It is not limited only to these.
- Tth DNA polymerase has both RT and PCR activity! /, When RT-PCR is performed in One tube-One step, it can be covered with one kind of enzyme. It has features that can be done.
- the substance that inhibits the RNA amplification reaction includes, for example, a dye in a biological sample.
- a dye in a biological sample examples include substances that exist inside and outside the cell, such as seed proteins and sugars.
- an additive capable of selecting a sulfated polysaccharide and a polyamine power can be used in the present invention.
- sulfate-polysaccharides examples include heparin, dextran sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, funolan, sulfate diagarose, carrageenan, borhuilan, fucoidan, sulfated curdlan, and their Salt power can also be selected and used. Among these, heparin and its salt, dextran sulfate and its salt are preferable.
- the sulfated polysaccharides can be used alone or in combination of several kinds.
- the sulfate polysaccharide may be contained in the reaction system during the RNA amplification reaction. Therefore, the sulfated polysaccharide is, for example, any one of the processing reagent used for the heat treatment, the sample treatment liquid after the heat treatment, the amplification reaction liquid, and the mixture of the sample treatment liquid and the amplification reaction liquid. Can be added.
- the effective concentration range varies depending on the molecular weight of the sulfated polysaccharide, the presence of the amplification reaction inhibitor, and the like.
- heparin an example of a sulfated polysaccharide
- a sulfated polysaccharide such as heparin
- the amount used thereof is the above-mentioned amount except for the amount of sulfated polysaccharide itself that inhibits RT reaction and PCR reaction.
- the amount that suppresses the action of an inhibitor of the RT reaction and PCR reaction is not particularly limited and is acceptable.
- specific amounts relating to sulfated polysaccharides the above-mentioned matters relating to sulfated polysaccharides are described in JP-A-2000-93176.
- the amount of heparin used is, for example, 0.1 g / ml or more, preferably 0.3 g / mL to 50 in the final reaction solution in which the sample treatment solution and the RNA amplification reaction solution are mixed. It is better to add g / mL.
- Polyamine is a general term for hydrocarbons having two or more primary or secondary amino groups. Certain types of polyamines are present in the living body and are abundant in tissues where protein and nucleic acid synthesis is active, and have various physiological effects. In the present invention, The polyamine is not particularly limited as long as it is a hydrocarbon having two or more primary or secondary amino groups in one molecule, which does not necessarily require such action. Specific examples of polyamines include ethylene diamine, trimethylene diamine, spermine, penremidine, diethylene triamine, triethylene tetramine, tetraethylene pentamine and pentaethylene hexamine.
- the polyamine may be contained in the reaction system during the RNA amplification reaction. Therefore, the polyamine can be stored in, for example, any of the sample processing solution after the heat treatment, the amplification reaction solution, and the mixture of the sample processing solution and the amplification reaction solution.
- the above-mentioned matters concerning polyamines are described in detail in JP-A-6-2777061, and the amount of polyamine used can also be referred to.
- an additive selected from albumin (Bovine Serum Albumin; BSA) and a nonionic surfactant is added to the final mixture of the sample treatment solution and the RNA amplification reaction solution. It can be further contained in the reaction solution. These additives may be used together with the above polyamine.
- Albumin is a general term for a group of soluble proteins contained in animal cells, plant cells, and body fluids. Representative examples include ovalbumin, lactalbumin in milk, serum albumin, wheat and wheat leucosin, and ricin in castor bean (castor) seeds. Of these, serum albumin is particularly preferred, and ushi serum albumin is particularly preferred. However, it is not limited to these albumins. Albumin may be contained in the reaction system during the RNA amplification reaction. Therefore, albumin can be used in any of the power of the sample treatment solution, amplification reaction solution after heat treatment and pH adjustment, and the mixture of the sample treatment solution and amplification reaction solution after heat treatment and pH adjustment. be able to.
- albumin is uniformly contained in the final reaction solution, and the state (for example, when albumin is added to the sample treatment solution after heat treatment and pH adjustment and the RNA amplification reaction solution is mixed without stirring).
- the state for example, when albumin is added to the sample treatment solution after heat treatment and pH adjustment and the RNA amplification reaction solution is mixed without stirring.
- albumin is uniformly contained in the final reaction solution, and the state (for example, when albumin is added to the sample treatment solution after heat treatment and pH adjustment and the RNA amplification reaction solution is mixed without stirring).
- the above-mentioned matters concerning albumin are described in detail in JP-A-2001-8685, and the amount of albumin used can also be referred to.
- nonionic surfactants include polyoxyethylene sorbitan monolaurate. And polyoxyethylene octylphenol ether.
- Polyoxyethylene sorbitan monolaurate includes polyoxyethylene sorbitan (20) monolaurate (Tween 20).
- polyoxyethylene octyl ether include polyoxyethylene (9) octyl ether (nodette P-4O (NP40)) and polyoxyethylene (10) octyl ether (Triton X 100).
- RNA amplification method As a procedure of the RNA amplification method, a sample is heat-treated using the above-described treatment reagent, the obtained sample treatment solution and the reaction solution are mixed, and the pH is adjusted appropriately. An amplification reaction can be performed based on the method.
- the reaction is performed for about 30 minutes to 1 hour at a reaction temperature suitable for the selected primer and reverse transcriptase.
- PCR DNA is converted into single-stranded DNA by heat denaturation; an annealing step that amplifies the region-neutralizing primer; and DNA in the presence of deoxyribonucleotides.
- the region sandwiched between the primers is amplified by repeating the three steps of the polymerization step, which is performed by applying a polymerase to the primer extension reaction.
- the RNase inactivation method and RNA extraction method of the present invention RNase inactivation in a biological sample and RNA extraction from the RNA inclusion body can be performed, so that the RNA inclusion in the biological sample can be purified.
- a simple and stable RNA sample treatment solution can be obtained without performing the step.
- the influence of adsorption and embedding by extracted contaminants such as proteins contained in biological samples can be suppressed for the extracted RNA. Therefore, the RNA extraction method of the present invention is effective for the subsequent detection and analysis of RNA.
- the sample treatment solution can be used for subsequent processes such as RNA detection and analysis without any treatment or by performing minimal treatment such as dilution, pH adjustment, and addition of additives. be able to.
- the present invention can be used as a pre-step for RNA purification.
- RNA detection method of the present invention RNase inactivation in a biological sample and extraction of RNA contained in the RNA inclusion body are performed in a sample in a simple 'stable' and efficient manner. It becomes possible to amplify RNA. Even when nucleic acid synthesis inhibitors are included in the sample treatment solution, the effects of inhibitors on nucleic acid synthesis can be reduced or suppressed by dilution, pH adjustment, and addition of appropriate additives to the amplification reaction solution. Simple and stable 'Efficiently and efficiently amplifies RNA present in the sample.
- RNA viruses such as retrovirus (AIDS virus (HIV)), norovirus (SRSV), rotawinoles, type C, which are hidden in a biological sample
- HCV Hepatitis virus
- fungi, bacteria, etc. fungi, bacteria, etc.
- mutant cells eg, cancer cells, etc.
- the analysis of expressed genes by detecting mRNA transcribed in cells and sequencing, and the analysis and production of expression products by cloning of cDNA can be performed easily and rapidly. Can be done.
- the present invention is applied to environmental samples such as air, soil, and water, it is considered possible to develop the method for testing microorganisms in environmental samples.
- RNA extracted by the treatment reagent of the present invention can be stored in the present treatment reagent, stored after neutralization, or the like.
- a model sample prepared by adding RNA inclusions to human serum (containing RNase) is used as a sample, and distilled water (for comparison), NaOH aqueous solution (for comparison), and DTT aqueous solution are used.
- a solution (for comparison) or NaOH-DTT aqueous solution as a treatment reagent of the present invention was added to the sample and heat-treated, and then RNA extraction was confirmed.
- Ambion Armored RNA Hepatitis C Virus (Genotype 2b) Catalog #: 42011 was used as the RNA inclusion.
- Human serum and Armored RNA Hepatitis C Virus solution An equal volume (v / v) mixed model specimen was prepared as a sample. Prepare four 4 ml samples in a 0.5 ml tube. (1) Distilled water (for comparison), (2) 10 mM NaOH aqueous solution (for comparison), (3) 10 mM DTT aqueous solution (For comparison) or (4) 16 ⁇ l of an aqueous solution containing 10 mM NaOH and 10 mM DTT as a treatment reagent of the present invention was collected and heated at 85 ° C. for 1 minute.
- RT-PCR was performed using primers specific to HCV RNA, using each sample treatment solution after heat treatment as a saddle.
- RT-PCR was performed with 1 ⁇ l of the sample treatment solution per 50 1 reaction solution.
- the primer for the RT reaction uses an oligonucleotide with a base sequence complementary to HCV RNA.
- an oligonucleotide with a base sequence complementary to the cDNA synthesized in the RT reaction is added. I went.
- the RNA-derived product in the RT-PCR of this experiment is 244 bp.
- the primer sequences used are as follows. (5, primer) 5, -CTTCACGCAGAAAGCGTCTAGCCATGGCGT-3, (SEQ ID NO: 1)
- the RT reaction solution contained 10 mM Tris-HC1, 35 mM KC1, 1.5 mM MgCl, 200 ⁇ M each of dATP.
- the RT reaction was performed at 55 ° C for 30 minutes. After the reaction, it was treated at 95 ° C for 5 minutes to inactivate reverse transcriptase.
- PCR was performed by adding 20 pmol of 5 'primer and 1.25 units of Taq DNA polymerase (Platinum Taq: Invitrogen, CA, USA) to the RT reaction solution.
- Taq DNA polymerase Platinum Taq: Invitrogen, CA, USA
- PCR was performed at 94 ° C for 2 minutes, followed by polymerization at 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 60 seconds, and finally at 72 ° C for 7 minutes. .
- FIG. 1 shows the electropherogram of the amplified product.
- M is a size marker (250 ng of ⁇ X174-RF DNA cut with Hindi)
- 1, 2, 3 and 4 are distilled water (for comparison), 10 mM NaOH aqueous solution (for comparison), 10 mM, respectively. It is the result of using DTT aqueous solution (for comparison) and lOmM NaOH-lOmM DTT aqueous solution (treatment reagent of this invention).
- Example 2 shows the electropherogram of the amplified product.
- M is a size marker (250 ng ⁇ X174-RF DNA cut with Hindi)
- 1, 2, 3 and 4 are distilled water (for comparison), lOmM NaOH aqueous solution (for comparison), lOmM DTT, respectively. It is the result of using an aqueous solution (for comparison) and lOmM NaOH-lOmM DTT aqueous solution (treatment reagent of the present invention).
- RNA after extraction with the treatment reagent of the present invention is stably present.
- RNA of serum can be analyzed by the treatment reagent of the present invention. Therefore, it was confirmed that by using the treatment reagent of the present invention, RNA can be extracted by a simple operation such as the strength of RNA inclusion contained in a biological sample or the like.
- HCV positive about lOOIU / ml
- PEG aqueous solution (Roche Diagnostics Inc. “Amplicon (R) HBV monitor for sample processing” HBV SOL A included in “Reagent for use”, and the same as in Examples 4, 5, 6, and 7).
- FIG. 3 is a graph showing the average value at each temperature of Data 1, with the vertical axis representing absorbance and the horizontal axis representing heating time.
- RNA can be detected by heating for about 15 seconds, and the heating time can be appropriately selected according to the heating temperature.
- total OD was measured from the same sample as in Example 4 above by the procedure of the quantitative method shown in the package insert of the Amplcore (R) HCV v2.0 kit. . In the comparative example, this operation was further repeated 5 times, and a total of 6 measurements were performed. Each measurement result is 0.75, 0.76, 1.14, 0.77, 1.30, and 1.06 in terms of total OD (absorbance), which is an HCV signal, and the average of these six measurements is 0.96.
- the method of the Amplicon (R) HCV v2.0 kit package insert performed in Comparative Example 1 obtains 1,000 ⁇ L of RNA extract from 100 ⁇ L of plasma.
- 100 L of RNA extract was obtained from 100 ⁇ L of plasma (that is, the RNA extract obtained in Example 4 was 10 times more concentrated). Therefore, since the total OD in Comparative Example 1 is 0.96, if the total OD in Example 4 is 9.6, it can be said that the sensitivity equivalent to that of the conventional method was obtained.
- Example 4 the total OD in the range of 9 to 10 was obtained by the heat treatment time of 80 to 160 seconds. From this, the method of the present invention represented by Example 4 is comparative example 1 It is thought that the sensitivity not inferior to the conventional method illustrated in (1) was obtained.
- HCV RNA can be detected by heating for a long time, for example, 5 minutes or more, even at a temperature of 60 ° C or lower.
- HCV RNA can be detected by heating for a short time, for example, 30 seconds to 3 minutes, even at a heating temperature higher than 85 ° C.
- the heating time depends A stable signal with low sensitivity was obtained, and it was shown to be highly sensitive. Therefore, it can be said that 80 ° C. to 85 ° C. is a particularly preferable temperature condition under the conditions shown in Examples 3 to 5.
- the heating time can be 30 seconds to 10 minutes, more preferably 30 seconds to 5 minutes, and still more preferably 80 seconds to 160 seconds.
- Example 6 PEG aqueous solution was added to a total of 4 plasma samples, 3 types of plasma samples known to be HCV positive (approximately 100, 500, 5000 IU / ml) and a sample of plasma samples known to be HCV negative, and centrifuged. After that, the obtained precipitate was used as a sample.
- the PEG aqueous solution precipitate from plasma, not only viruses but also many plasma components are precipitated, and RNase is also present in them. For each sample, RNase was inactivated and RNA was extracted from the RNA inclusion body according to the method of the present invention, and RT-PCR was performed using primers specific for HCV RNA.
- HCV concentration (IU / ml) 0 100 500 5000
- HCV TOD value 0.06 0.58 4.01 51.63 [0143] As shown in Data 4 above, a signal was obtained in the positive specimen, and it was possible to detect HCV RNA. This result indicates that RNase was inactivated by the method of the present invention, and HCV RNA was extracted from the inside of the HCV virus, resulting in an RT-PCR type.
- RNA samples known to be HIV positive approximately 700 copies / ml
- plasma samples known to be HIV negative were used as a sample.
- Each specimen was centrifuged with an aqueous PEG solution, and the resulting precipitate was used as a sample.
- RNase was inactivated and RNA was extracted from the RNA inclusion body according to the method of the present invention, and RT-PCR was performed using primers specific for HIV RNA.
- sample treatment solution 50 1 in the tube is mixed with 50 ⁇ L of the master mix from the Amplicore® HIV Monitor vl.5 kit (Roche Diagnostats ) prepared in a separate tube, and GeneAmp9600 (Applied Systems)
- RT-PCR was carried out in accordance with the attached document of Amplicor (R) HIV monitor vl.5 kit. Even after RT-PCR, HIV-1 signal was quantified according to the prescribed procedure according to the package insert. The TOD value for HIV copy / ml is shown in Data 5 below.
- HIV TOD value 0.05 1.10 As shown in Data 5 above, a signal was obtained in the positive sample, and it was possible to detect HIV RNA.
- RNA inclusion body-1 TSM III noffer solution was prepared by mixing the above RNA inclusion body-1 TSM III noffer solution and human serum in a volume ratio of 1: 1.
- concentration of the above RNA inclusion-TSM III noffer solution is defined as “73,000 IU / mL when 5 (v / v)% of the plasma is added to the plasma” and mixed with serum at a 1: 1 (volume ratio). 730IU / ⁇ L.
- RNA detection gene amplification was first performed using Amplicor HCV v2.0 amplification reagent set (Roche Diagnostics). In the RT-PCR temperature program, the number of cycles of PCR reaction according to the manufacturer's recommended method was 38.
- RNA in a sample could be extracted with a processing reagent containing an alkaline substance and a reducing agent.
- a 200 L plastic tube was mixed with 2 L of a model sample and 8 ⁇ L of each processing reagent (15 types) shown in Table 6 and heat-treated at 85 ° C for 3 minutes. Add 90 ⁇ L of TE Buffer (pH 8.0) to this, and add 5 ⁇ L of this to AMPVRYMIX (Roche Diagnostics: HCV Master Mix v2 included in the AMPLICORE HCV v2.0 amplification reagent set) .0 and HCV manganese test solution mixed at a ratio of 7: 1) were mixed with 5 L and RT-PCR was performed. Table 6 also shows the pH of each processing reagent (before adding the model sample) and the pHO deviation of the model sample-processing reagent mixture (after adding the model sample) at 25 ° C).
- FIG. 6 shows an agarose electrophoresis photograph.
- Fig. 17 shows photographs showing the condition after heat treatment under each condition.
- the top row in Fig. 17 shows the results using the DTT OmM treatment reagent. From left, [1], [2], [3], [4], [5], [6], [7] It is the result using a processing reagent.
- the bottom row shows the results of using DTT 20 mM treatment reagent from the left [8], [9], [10], [11], [12], [13], [14], [15] It is a result using the processing reagent.
- RNA hydrolysis may have occurred because the extracted RNA force was exposed to both high heating temperature (85 ° C) and pH (10.1) under high conditions.
- the following conditions should be adjusted. That is, the pH is lowered by adjusting the NaOH concentration and the type and concentration of the buffer agent (preferably the conditions [10] to [14] described above); the temperature is lowered (for example, by not heating the RNA It has been confirmed by the present inventors that detection is possible); or a chelating agent that chelates divalent ions such as EGTA is added without changing the temperature and NaOH concentration (Example 9 below). ).
- RNA could not be detected in the neutral range may be due to the adsorbed and embedded RNA in the contaminated component such as denatured protein.
- the reason why RNA could not be detected under the condition [15] is presumed to be due to RNA hydrolysis not due to the influence of denatured protein, as described above.
- This example is an example showing that EGTA reduces the hydrolysis of RNA under hot alkaline conditions. It is known that divalent metal ions promote RNA hydrolysis. Depending on the sample targeted by the present invention, since it contains divalent metal ions, it is effective to add chelating agents (EGTA, etc.) that chelate them to the processing reagent.
- chelating agents EGTA, etc.
- the temperature in the heat treatment is 65 ° C to 100 ° C, more preferably 70 ° C to 100 ° C. It has been found that it can be set at ° C, more preferably between 70 ° C and 95 ° C.
- RT-PCR RT reactions Ampdirect (R) Plus (P / N: 241-08800-98: Shimadzu Corporation), 0.4 ⁇ pseudo-norovirus RNA reverse primer (5,- ACTGACAATTTCATCAT CACC-3 ': SEQ ID NO: 3) and 3.75U
- AMV reverse transcriptase mixed RT-PCR reaction solution 25 ⁇ L is mixed with the above sample treatment solution 20 L, under conditions of 42 ° C for 1 hour. Reaction was performed.
- RNA forward primer 5, -TGGAATTCCATCGCCCACTGG-3, SEQ ID NO: 4
- Nova Taq TM Hot Start DNA Polymerase EMD Biosciences
- Example 10 Eight types of processing reagents ⁇ -1 to ⁇ -8 having the compositions described in Table 7 below were prepared as processing reagents, and the above operation was performed for each of the eight types. .
- A-2 to A-8 are processing reagents in the present invention
- A-1 is a processing reagent prepared for comparison.
- Lane M is molecular weight (hindi digest of ⁇ X174 RF DNA)
- Lane 1 is the result of using treatment reagent A-1
- Lane 2 is the result of using treatment reagent A-2
- Lane 3 is the result.
- Lane 4 is the result using treatment reagent A-4
- Lane 5 is the result using treatment reagent A-5
- Lane 6 is the result using treatment reagent A-6
- Lane 7 shows the results using treatment reagent A-7
- lane 8 shows the results using treatment reagent A-8.
- Example 10 The same operations as in Example 10 were performed except that seven treatment reagents B-1 to B-7 having the following compositions were prepared and used as treatment reagents, respectively.
- B-2 to B-7 are processing reagents in the present invention
- B-1 is a processing reagent prepared for comparison.
- Fig. 10 shows the electrophoretogram obtained in Example 11.
- lane M is a molecular weight marker (hindi digest of ⁇ X174 RF DNA)
- lane 1 is the result using treatment reagent B-1
- lane 2 is the result using treatment reagent B-2
- lane 3 is the treatment
- Reagent B-3 Lane 4 is the result of using Reagent B-4
- Lane 5 is the result of using Reagent B-5
- Lane 6 is the result of using Reagent B-6
- Lane 7 shows the results using treatment reagent B-7.
- NaOH in the treatment reagent is 20 mM to 60 mM
- DTT is
- the stool of a person infected with Norovirus was suspended in physiological saline at a concentration of 20% (w / v) and centrifuged for 5 minutes in a microcentrifuge to obtain a supernatant.
- the stool of healthy norovirus-negative individuals was suspended in physiological saline at a concentration of 20% (w / v), and the suspension was centrifuged for 5 minutes using a microcentrifuge to obtain a supernatant. .
- the supernatant derived from the stool of the infected person was diluted 10-fold with the supernatant derived from the stool of the healthy person to prepare six types of stool sample solutions D-1 to D-6. Specifically, the noble Shakuritsu 1 ⁇ D-1, D-2 dilution 10 times, D-3 of the dilution ratio 10 twice, 10 3 times dilution of D-4, D- The dilution ratio of 5 is 10 4 times, and the dilution ratio of D-6 is 10 5 times.
- a treatment reagent having a composition of 30 mM NaOH, 20 mM DTT, and lOmM EGTA was used.
- RNA forward primer 5, -TGGAATTCCATCGCCCACTGG-3, SEQ ID NO: 4
- Nova Taq TM Hot Start DNA Polymerase EMD Biosciences
- Lane 1 is the result of using fecal sample solution D-1
- Lane 2 is the result of using fecal sample solution D-2
- Lane 3 is the result of using fecal sample solution D-3
- Lane 4 is the result of using fecal sample solution D-3.
- sample solution D-4 shows the result of using fecal sample solution D-5
- lane 6 shows the result of using fecal sample solution D-6.
- Lane 7 shows the result of the same operation except that negative control (Negative Control), that is, stool of non-norovirus-infected healthy subjects was used instead of stool of norovirus-infected subjects.
- Lane M is a molecular weight marker (Hindi digest of ⁇ X174 RF DNA).
- E-1 to E-6 were used as controls for solutions D-1 to D-6.
- E-1 is a purified RNA solution corresponding to D-1 (1x)
- E-2 is a purified RNA solution corresponding to D-2 (10x)
- E-3 is compatible with D-3
- E-4 is a purified RNA solution corresponding to D-4 (10 3 times)
- E-5 is a purified RNA solution corresponding to D-5 (10 4 times)
- E- 6 is a purification RN a solution corresponding to D-6 (10 5 times).
- Fig. 12 shows the electropherogram obtained by ⁇ 2>.
- Lane 1 is the result using purified RNA solution E-1
- Lane 2 is the result using purified RNA solution E-2
- Lane 3 is the result using purified RNA solution E-3
- Lane 4 is the purified RNA solution.
- lane 5 shows the result using purified RNA solution E-5
- lane 6 shows the result using purified RNA solution E-6
- Lane 7 shows the result of the same operation except that negative control (Negative Control), that is, fecal stool that is not infected with norovirus, was used instead of fecal stool that was not infected with norovirus.
- Lane ⁇ is a molecular weight marker (Hindi digest of ⁇ X174 RF DNA).
- Example 12 1> For 18 kinds of norovirus positive stool derived from 18 different specimens (Specimen Nos. 1 to 18) infected with Norovirus, the same procedure as in Example 12 1> was performed. The obtained electropherogram is shown in FIG. In Fig. 13, the numbers in the lanes correspond to the sample numbers. Lane M is a molecular weight marker (Hindi digest of ⁇ X174 RF DNA).
- treatment reagents with the composition of 30 mM NaOH, 20 mM DTT, and lOmM EGTA as treatment reagents, heat treatment at various temperatures from 20 ° C to 100 ° C and for various times from 1 minute to 60 minutes The same operation as in Example 10 was performed except that the above was performed.
- Example 14 An electropherogram obtained by Example 14 is shown in FIG. In FIG. 15, five lanes correspond to the case where the heat treatment time is 1 min (min), 5 min, 15 min, 30 min, and 60 min, and the heat treatment temperature for each lane is 25 ° C (for comparison), 35 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85 ° C, 90 ° The results for C, 95 ° C, and 100 ° C are shown.
- RNA was quantified by real-time PCR. Specifically, lO X SYBR (TM) Green I (Molecular Probes) was added to the obtained RT-PC R reaction solution, followed by preheating at 95 ° C for 5 minutes as a temperature program. Then, a cycle of 92 ° C., 30 seconds, 58 ° C., 30 seconds, and 72 ° C., 1 minute was performed for 30 cycles, and then a polymerization of 72 ° C., 7 minutes was performed.
- the fluorescence intensity at the 30th cycle is shown in FIG. In FIG. 16, the horizontal axis represents the heat treatment temperature (° C.), and the vertical axis represents the fluorescence intensity (relative fluorescence intensity: RFU).
- SEQ ID Nos: 1 to 5 are synthetic primers.
- an RNase that is universally present in a sample such as a biological sample or an environmental sample, or a sample such as a biological sample obtained by separating RNA inclusions therefrom, or the like. Deactivation Can be provided.
- RNA is efficiently extracted from RNA inclusions present in samples such as biological samples and environmental samples, or samples such as biological samples obtained by separating RNA inclusions therefrom.
- a method of extraction can be provided.
- RNase present in a sample can be easily and stably obtained by performing inactivation of RNase in the sample and RNA extraction from the RNA inclusion body in one step. Can be amplified. Further, by suppressing the action of an inhibitor on nucleic acid synthesis, it becomes possible to amplify RNA present in the sample more simply, stably, efficiently and rapidly. This can provide a simple, “stable”, efficient and rapid method for detecting RNA in a sample.
- a treatment reagent that can be used in these methods can be provided.
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US12/092,067 US20090269745A1 (en) | 2005-11-02 | 2006-11-02 | Rna extraction method and rna detection method |
JP2007542815A JP4735645B2 (ja) | 2005-11-02 | 2006-11-02 | Rnaの検出法 |
EP06822928A EP1944364B1 (en) | 2005-11-02 | 2006-11-02 | Rna extraction method and rna detection method |
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JP2005-319333 | 2005-11-02 | ||
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EP1944364A1 (en) | 2008-07-16 |
JPWO2007052765A1 (ja) | 2009-04-30 |
KR20080066727A (ko) | 2008-07-16 |
EP1944364A4 (en) | 2009-06-24 |
US20090269745A1 (en) | 2009-10-29 |
JP4735645B2 (ja) | 2011-07-27 |
EP1944364B1 (en) | 2012-09-12 |
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