WO2012014694A1 - Method for synthesis of feces-derived nucleic acid - Google Patents

Abstract

The purpose of the invention is to provide a method for synthesizing cDNA from RNA contained in feces by a reverse transcriptional reaction with high efficiency. The invention provides: a method for synthesizing a feces-derived nucleic acid, which comprises a washing step of washing RNA extracted from feces with a chaotropic salt and an alcohol and a reaction step of carrying out a reverse transcriptional reaction in a reaction solution containing a protein capable of binding to a single-stranded nucleic acid using the washed RNA as a template and using a reverse transcriptase having heat resistance and a low RNaseH activity; a method for synthesizing a feces-derived nucleic acid as mentioned above, wherein the reaction solution additionally contains an RNaseA inhibitor; and a feces-derived nucleic acid synthesis kit comprising a chaotropic salt, an alcohol, a reverse transcriptase having heat resistance and a low RNaseH activity and a a protein capable of binding to a single-stranded nucleic acid.

Description

Method for synthesizing fecal nucleic acid

The present invention relates to a method for efficiently synthesizing cDNA from RNA contained in feces by reverse transcription reaction.

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.

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.

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.

In the method described in Patent Document 2, it is important to cool the stool immediately after sampling in order to effectively prevent the stool sample from being altered. However, when stool collection is performed at home, such as in a medical examination, it is very difficult to cool a stool sample immediately after collection, which is not realistic. In addition, as performed in the method described in Patent Document 2 and the method described in Non-Patent Document 2, a method for removing impurities from stool, separating a cell having a target gene, and recovering a nucleic acid therefrom In addition to the problem that the process of separating the cells is complicated and leads to an increase in the cost of the test, the nucleic acid recovered after the process of separating the cells is low in yield and the loss of yield due to the process is large. There is also. For this reason, when recovering nucleic acid from stool, it is desirable to recover in a mixed state without distinguishing human-derived cells and bacteria-derived cells.

There is also a method of collecting nucleic acid directly 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 extracted directly from the prepared suspension, and the COX2 (cyclooxygenase-2) gene, which is an oncogene, is extracted. A method for detecting transcripts is disclosed.

JP 2005-514073 A Japanese National Patent Publication No. 11-511982 Special Table 2002-539765 Japanese Patent No. 4134047

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 (see Non-patent Document 3, for example). 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.
In addition, nucleic acid synthesis reaction inhibitors such as bile salts inhibit not only PCR but also reverse transcription reactions. Therefore, when nucleic acids are extracted from stool and used for amplification reactions such as reverse transcription and PCR, it is preferable to reduce the effects of these nucleic acid synthesis reaction inhibitors in order to improve reaction efficiency. .

However, when nucleic acids are directly collected from stool as in the methods described in Patent Documents 3 and 4, the collected nucleic acids are contaminated with stool compared to the method of collecting cells after separating them. There is a problem that a lot is brought in. For this reason, particularly when performing reverse transcription reaction, when a large amount of stool-derived RNA is brought into the reaction solution as a template, there is a problem that reaction inhibition occurs and a reaction product cannot be obtained.

An object of the present invention is to provide a method for efficiently synthesizing cDNA from RNA contained in feces by reverse transcription reaction.

As a result of earnest research to solve the above problems, the present inventor washed RNA extracted and collected from stool with a chaotropic salt and alcohol in advance before performing a reverse transcription reaction, and using the washed RNA as a template. The present inventors have found that the reaction efficiency can be improved by performing a reaction in the presence of a single-stranded nucleic acid binding protein using a reverse transcriptase having heat resistance and low RNase H activity, and the present invention has been completed.

That is, the present invention
(1) (a) a washing step of washing RNA extracted from stool with a chaotropic salt and alcohol; and (b) using a reverse transcriptase having heat resistance and low RNase H activity using the washed RNA as a template. A reaction step of performing a reverse transcription reaction in a reaction solution containing a single-stranded nucleic acid binding protein;
A method for synthesizing stool-derived nucleic acid,
(2) The method for synthesizing stool-derived nucleic acid according to (1), wherein the reaction solution further contains an RNase A inhibitor,
(3) The washing step (a) includes a step of first adding a chaotropic salt solution to RNA extracted from stool, then adding an alcohol solution, and then recovering RNA from the obtained RNA solution, (1) or the method for synthesizing a stool-derived nucleic acid according to (2),
(4) The washing step (a) includes a step of adding an alcohol solution containing a chaotropic salt to RNA extracted from stool and recovering RNA from the obtained RNA solution (1) or ( 2) The method for synthesizing the stool-derived nucleic acid described in
(5) The method for synthesizing a stool-derived nucleic acid according to any one of (1) to (4), wherein the RNA extracted from stool is RNA extracted from stool using a chaotropic salt,
(6) Before the washing step (a),
(A)
(I) adding an alcohol solution to feces to prepare a suspension;
(Ii) a step of recovering a solid component from the suspension; and (iii) a step of adding a chaotropic agent to the recovered solid component to prepare a suspension and extracting RNA from the solid component;
A method for synthesizing a stool-derived nucleic acid according to any one of (1) to (4),
(7) Before the washing step (a),
(A-1) adding a chaotropic salt solution to feces to prepare a suspension and extracting RNA; and (a-2) an alcohol solution or an alcohol solution further containing a chaotropic salt in the suspension. Recovering RNA from the suspension after adding
A method for synthesizing a stool-derived nucleic acid according to (1) or (2),
(8) The washing step (a)
(A-3) adding an alcohol solution to feces to prepare a suspension;
(A-4) recovering solid components from the suspension;
(A-5) adding a chaotropic agent to the recovered solid component to prepare a suspension, and extracting RNA from the suspension; and (a-6) further adding an alcohol solution to the suspension. Or a step of recovering RNA from the suspension after adding an alcohol solution containing a chaotropic salt;
The method for synthesizing stool-derived nucleic acid according to (1) or (2),
(9) A kit for synthesis of stool-derived nucleic acid having a chaotropic salt, alcohol, a reverse transcriptase having heat resistance and low RNase H activity, and a single-stranded nucleic acid binding protein,
Is to provide.

In the method for synthesizing stool-derived nucleic acid of the present invention, a larger amount of cDNA can be synthesized when cDNA is synthesized by reverse transcription reaction from RNA collected from stool containing a large amount of contaminants.

In Example 1, it is the figure which showed the expression level relative value of the COX2 gene in each sample at the time of using pseudo-feces origin RNA as a template. In Example 1, it is the figure which showed the relative expression level value of the COX2 gene in each sample at the time of using MKN45 cell sample origin RNA as a template. In Example 1, it is the figure which showed the expression level relative value of the COX2 gene in each sample at the time of using as a template RNA from E. coli containing MKN45 cell sample. In Example 1, it is the figure which showed the relative expression level value of the COX2 gene in each sample at the time of using bilirubin containing MKN45 cell sample origin RNA as a template. In Example 2, it is the figure which showed the expression level relative value of the COX2 gene at the time of using each reverse transcriptase. In Comparative example 1, it is the figure which showed the expression level relative value of the COX2 gene at the time of using each reverse transcriptase. In Example 3, it is the figure which showed the expression level relative value of the COX2 gene at the time of adding various SSB in a reaction liquid. In Example 4, it is the figure which showed the expression level relative value of the COX2 gene in each sample.

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. Specific examples include bile acids, bile salts, bilirubin, mucopolysaccharides and the like.

The method for synthesizing stool-derived nucleic acid of the present invention (hereinafter sometimes referred to as the synthesis method of the present invention) comprises a washing step of washing RNA extracted from stool with a chaotropic salt and alcohol, and a RNA after washing. And a reaction step of performing a reverse transcription reaction in a reaction solution containing a single-stranded nucleic acid binding protein using a reverse transcriptase having heat resistance and low RNase H activity as a template.

That is, in the synthesis method of the present invention, RNA extracted from stool is washed with a chaotropic salt and alcohol in advance, and then the RNA after washing is used as a template, using a reverse transcriptase having specific properties, and then single-stranded. By performing the reverse transcription reaction in the presence of the nucleic acid binding protein, the reaction efficiency of the reverse transcription reaction can be dramatically improved. The reason why such an effect is obtained is not clear, but it is presumed that the influence of the stool-derived inhibitory substance can be significantly reduced as described below.

In the RNA extracted from stool, inhibitors contained in stool are brought in. By washing the extracted RNA with a solution containing a chaotropic salt or an alcohol solution, the introduced inhibitory substance can be efficiently washed away.

When collecting RNA directly from stool without separating cells from stool, it is difficult to completely remove the inhibitor even if it is washed with chaotropic salt or alcohol. For this reason, some inhibitory substances remain in the RNA recovered after washing.

As countermeasures against such harmful effects of inhibitors, reverse transcriptase with higher reverse transcription efficiency can be used, but even if a highly active enzyme is selected, a reaction product may be obtained depending on the amount of inhibitor brought in. It may not be possible. In addition, proteins such as BSA (bovine serum albumin) are generally used as mitigating agents for PCR reaction inhibitors, but when stool-derived RNA is used, conventionally used mitigating agents are used. In many cases, the reverse transcription reaction efficiency is not improved only by adding an agent to the reaction solution.

In the present invention, the effect of an inhibitory substance in stool is conventionally achieved by performing a reverse transcription reaction in a reaction solution containing a single-stranded nucleic acid binding protein using a reverse transcriptase having heat resistance and low RNase H activity. It can be significantly reduced. Thus, the combined use of a heat-resistant and low RNase H reverse transcriptase and a single-stranded nucleic acid binding protein is particularly effective in reducing the influence of stool-derived inhibitors and improving the reaction efficiency. There is a finding for the first time by the present inventors.

Hereinafter, it demonstrates for every process.
First, as a washing step, RNA extracted from stool is washed with a chaotropic salt and alcohol. Specifically, an RNA solution is prepared by adding chaotropic salt and alcohol to RNA extracted from stool, and then RNA is recovered from the RNA solution to obtain RNA washed with chaotropic salt and alcohol. be able to.

In the washing step, chaotropic salt and alcohol may be added to RNA, respectively, or an alcohol solution containing chaotropic salt may be prepared in advance and added thereto. In addition, when the chaotropic salt and the alcohol are added separately, which one is added first can be appropriately determined in consideration of the RNA extraction method from feces. In the present invention, it is preferable to add an alcohol after adding a chaotropic salt first, or add an alcohol solution containing a chaotropic salt.

The chaotropic salt added to RNA extracted from feces in the washing step is not particularly limited, and any chaotropic salt used in the technical field may be used. As such chaotropic salts, for example, guanidine salts such as guanidine hydrochloride, guanidine thiocyanate, and guanidine isothiocyanate are preferable. One type of chaotropic salt may be used, or two or more types of chaotropic salts may be used in combination.

The chaotropic salt is preferably added as a chaotropic salt solution dissolved in an appropriate solvent. As the solvent used in this case, for example, water, citrate buffer, phosphate buffer, Tris buffer, or the like can be used. In particular, since RNA is a substance that is very easily degraded, it is preferable to use a buffer containing an RNase inhibitor such as guanidine thiocyanate or guanidine hydrochloride. In addition, a chaotropic salt-containing alcohol solution (alcohol solution containing a chaotropic salt) can be prepared by using alcohol as the solvent.

The concentration of the chaotropic salt solution added to the RNA extracted from stool is not particularly limited as long as it is a concentration that can exert the inhibitor cleaning effect, and the type of chaotropic salt and the RNA solution obtained after the addition The concentration can be determined as appropriate in consideration of the concentration of the chaotropic salt, alcohol, the type of alcohol, and the like.

As the alcohol added to RNA extracted from stool in the washing step, alcohol having a linear structure and being liquid at room temperature, for example, 15 to 40 ° C. is used. In the present invention, an alcohol having a solubility in water of 12% by weight or more is preferable, an alcohol having a solubility in water of 20% by weight or more is more preferable, and an alcohol having a solubility in water of 90% by weight or more. More preferred is an alcohol that can be mixed with water in any proportion. Examples of alcohols that can be mixed with water at an arbitrary ratio include methanol, ethanol, n-propanol, and 2-propanol.

Specifically, there are methanol, ethanol, propanol, butanol, mercaptoethanol and the like which are water-soluble alcohols. The propanol may be n-propanol or 2-propanol. The butanol may be 1-butanol (water solubility 20% by weight) or 2-butanol (water solubility 12.5% by weight). The alcohol used in the present invention is more preferably ethanol, propanol, or methanol from the viewpoints of availability, handleability, safety, and the like. In particular, ethanol is particularly useful in screening tests such as periodic medical checkups because it has the highest safety and can be easily handled at home. In addition, as alcohol added to RNA extracted from feces, only 1 type may be sufficient and you may add in combination of 2 or more types.

The RNA extracted from stool may be added directly as alcohol, or may be added as an alcohol solution diluted with an appropriate solvent. The amount of alcohol to be added and the concentration and amount of the alcohol solution are particularly limited as long as they are added so as to have a concentration capable of exhibiting an inhibitor cleaning effect in the RNA solution obtained after the addition. Instead, it can be appropriately determined in consideration of the type of alcohol, the amount of RNA solution obtained after the addition, the type of chaotropic salt added, and the like.

For example, when a chaotropic salt-containing alcohol solution is added to RNA extracted from feces, the alcohol concentration in the chaotropic salt-containing alcohol solution is preferably 25% or more, and more preferably 35% or more. In the present invention and the present specification, “%” means “volume%” unless otherwise specified.

The RNA used for the washing step may be RNA extracted from the collected cells after separating and collecting desired cells from the stool, but may be directly extracted from the stool without performing the cell separation operation. RNA is preferred. This is because when RNA is extracted directly from stool, the amount of the stool-derived inhibitor is increased compared to when extracted from separated and collected cells, and thus the effects of the present invention are more remarkably exhibited. .

In addition, when RNA is directly recovered from stool without performing cell separation operation, nucleic acids of all species contained in the stool, mainly nucleic acids derived from animals excreting the stool, and intestinal resident Nucleic acids derived from bacteria such as fungi are simultaneously extracted and recovered from feces. Thereby, it is possible to efficiently extract and collect RNA derived from mammalian cells that are contained only in a relatively small amount in feces. 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.

The RNA extracted from the stool subjected to the washing step may be RNA extracted from solid components such as cells in the stool. For example, it may be a suspension in which RNA is extracted into a liquid phase from cells or the like contained in stool by adding and mixing a nucleic acid extraction solution to stool, and a solid component from the suspension. It may be a relatively clear extract from which RNA has been removed, or an RNA solution recovered and purified from these suspensions or crude extracts.

As a nucleic acid extraction solution added to feces to prepare a suspension in which RNA is extracted in the liquid phase, proteins in the solid component are denatured, and mammalian cells and intestinal resident bacteria in the solid component The solution is not particularly limited as long as the nucleic acid can be eluted from a cell such as a nucleic acid extraction solution, and any solution used in the technical field may be used. For example, a solution obtained by adding a compound usually used as a protein denaturant such as a chaotropic salt, an organic solvent, or a surfactant as an active ingredient to an appropriate solvent can be used as a solution for nucleic acid extraction. These active ingredients may be a combination of two or more.

Examples of the chaotropic salt that can be an active ingredient of the nucleic acid extraction solution include guanidine hydrochloride, guanidine thiocyanate, and guanidine isothiocyanate.

As an organic solvent that can be an active ingredient of a solution for nucleic acid extraction, phenol is preferable. Phenol may be neutral, but is preferably acidic. When acidic phenol is used, RNA can be selectively extracted into the aqueous layer rather than DNA.

As a solvent for preparing a nucleic acid extraction solution by adding these active ingredients, for example, water, citrate buffer, phosphate buffer, Tris buffer, or the like can be used.

It is also preferable to treat stool with alcohol before extracting RNA from the solid component. When stool is suspended in alcohol, the inhibitory substance contained in the solid component of stool is eluted in alcohol. At this time, since nucleic acids such as RNA remain in the solid component, the liquid phase of the suspension is removed, and RNA is extracted and collected from the remaining solid component. More reduced RNA can be recovered.

In particular, alcohol has not only an effect of removing and eluting inhibitory substances, but also an effect of stably storing nucleic acids in stool. This is because, due to the dehydration action of alcohol, the cellular activities of mammalian cells, microorganisms, etc. contained in the stool are remarkably reduced, and due to the protein denaturation action of alcohol, the protease, DNase, RNase in stool It is presumed that it is obtained because the activity of various degrading enzymes such as That is, by suspending stool with alcohol, the inhibitory substance can be eluted and removed while suppressing degradation of nucleic acid in the stool.

As the alcohol added to the stool before RNA extraction from the solid component, the same alcohols listed as those that can be added in the washing step can be used. Further, it may be added directly to feces as alcohol, or may be added as an alcohol solution diluted with an appropriate solvent. If the amount of alcohol added to the stool, or the concentration or amount of the alcohol solution is such that the suspension can be added at a concentration that can achieve the nucleic acid stabilization effect and the inhibitory substance elution removal effect, It is not particularly limited, and can be determined appropriately in consideration of the type of alcohol, the amount of stool (solid component amount) and the mixing ratio of the added chaotropic salt solution or liquid component such as alcohol. In addition, when the alcohol concentration in the suspension is sufficiently high, the alcohol component quickly penetrates into the whole stool, so that the inhibitory substance elution removal effect and the nucleic acid stabilization effect can be quickly achieved.

A lot of inhibitory substances are eluted in the liquid phase of the suspension obtained by mixing the stool with alcohol or an alcohol solution. For this reason, it is preferable to extract RNA by removing the liquid phase from the suspension and adding a nucleic acid extract to the collected solid components. The method for recovering the solid component can be appropriately selected from separation methods usually used when separating the liquid component and the solid component. For example, the suspension may be centrifuged, and then the supernatant may be removed to remove the solid component derived from stool, which is a precipitate. The suspension may be filtered and filtered on the filter surface. You may carry out by the filtration method which collect | recovers the remaining stool origin solid components.

After removing the alcohol component and recovering the solid component, the recovered solid component may be washed with an appropriate buffer or the like before adding the nucleic acid extract. Examples of the buffer include the aforementioned chaotropic salt-containing alcohol solution and a buffer solution whose pH is maintained within the range of 2 to 7.5.

Moreover, it is preferable that the suspension obtained by mixing the stool with alcohol or an alcohol solution is stored for a predetermined time before collecting the solid components. By storing the suspension, it is possible to increase the amount of the inhibitory substance that is eluted and removed from the stool-derived solid component. The time for storing the suspension can be appropriately determined in consideration of the type and concentration of the alcohol, the ratio of the stool-derived component in the suspension, the storage temperature, and the like. In the synthesis method of the present invention, storage for 1 hour or longer is preferable, storage for 12 hours or longer is more preferable, storage for 24 hours or longer is further preferable, and storage for 72 hours or longer is particularly preferable. Moreover, you may preserve | save for 168 hours or more.

The inhibitory substance elution removal effect by alcohol is higher when the temperature is higher than when the temperature at which the suspension is stored is low. Specifically, the storage temperature of the suspension obtained by mixing stool with alcohol or an alcohol solution is preferably 4 ° C. or higher, and more preferably 20 ° C. or higher. However, the storage temperature is preferably 50 ° C. or lower. This is because if the alcohol is stored for a long time under a high temperature condition of 50 ° C. or more, the concentration of the alcohol in the suspension may be lower than a concentration sufficient for the effect due to volatilization or the like.

When RNA in the liquid phase of a suspension containing a solid component derived from stool is subjected to a washing step as RNA extracted from stool, specifically, a chaotropic salt or alcohol was added to the suspension. Thereafter, the RNA that has been washed can be recovered by recovering the RNA present in the liquid phase of the obtained RNA solution in a suspended state.

Before recovering the RNA in the liquid phase, the denatured protein may be removed from the suspension.
The quality of the recovered RNA can be improved by removing the previously denatured protein before recovering the RNA. The protein can be removed from the suspension 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.

The method for recovering RNA in the liquid phase can be performed by a known method such as ethanol precipitation or cesium chloride ultracentrifugation. Moreover, after adsorbing RNA in a liquid phase on an inorganic support, the nucleic acid can be recovered by eluting the adsorbed nucleic acid from the inorganic support using a certain volume of solvent. In addition, RNA can be recovered from the liquid phase of the suspension after the addition of chaotropic salt or alcohol using a commercially available kit such as a nucleic acid extraction kit.

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.

In addition, when recovering RNA from a liquid phase, it is also preferable to selectively recover RNA over DNA. For example, by eluting RNA into the aqueous layer preferentially over DNA by the phenol / chloroform method using acidic phenol, the amount of DNA contamination can be reduced by performing a precipitation method using ethanol precipitation or an inorganic support. Reduced RNA can be recovered.
In addition, RNA can be selectively recovered by cesium chloride ultracentrifugation. In addition, for example, in US Pat. No. 5,155,018, Gillespie et al. Isolated and biologically active RNA from biological sources including RNA, DNA and other cellular contents. A method of purification is disclosed. In the method, the raw material containing RNA is brought into contact with particles made of silica gel containing a material such as finely crushed glass. The binding buffer from which RNA is adsorbed to the particles is an acidified solution containing a chaotropic salt. Under such conditions, RNA binds to the silica material, but not DNA, so that RNA can be selectively recovered. Japanese Patent No. 04036625 describes that RNA can be selectively adsorbed and recovered by changing the concentration of guanidine salt and ethanol. In these methods, in an arbitrary step after RNA is extracted from a solid component derived from stool, a chaotropic salt solution, an alcohol solution, or a chaotropic salt-containing alcohol solution is added, and then RNA is recovered to obtain DNA. Washed RNA with a reduced amount of contamination can be recovered.

Moreover, the solid extract was removed from the suspension obtained by adding the nucleic acid extraction solution to the stool by centrifugation or the like, and the crude extract containing only the liquid phase containing RNA was used as RNA extracted from the stool. It can also be subjected to a washing process. Specifically, the washed RNA can be recovered by adding chaotropic salt or alcohol to the crude extract and then recovering the RNA in the liquid phase. Similarly, washed RNA can be recovered by adding a chaotropic salt solution, alcohol solution, or chaotropic salt-containing alcohol solution to RNA purified by a conventional method, and then recovering RNA in the liquid phase. In addition, the recovery method of RNA in a liquid phase can use the method similar to the method quoted when a chaotropic salt or alcohol is added to the suspension.

In the step of extracting RNA from stool, when chaotropic salt is used or alcohol is used, the RNA extracted from stool may already contain chaotropic salt or alcohol. For example, when a chaotropic salt solution is used as a nucleic acid extraction solution, or when feces are stored in an alcohol solution before extracting RNA from solid components, chaotropic salt or alcohol remains in the extracted RNA. May have. Also in this case, the inhibitor can be washed and removed more effectively by washing with a chaotropic salt or alcohol.

Further, the washing treatment in the washing step is a treatment of recovering RNA from a liquid phase containing chaotropic salt or alcohol after directly contacting chaotropic salt or alcohol with RNA extracted from the solid component of stool. For this reason, in the process of extracting RNA from stool, when chaotropic salt or alcohol is used, and when RNA extracted from a solid component is subjected to a treatment that contacts chaotropic salt or alcohol, the treatment is performed. Can be the cleaning treatment in the cleaning step of the present invention.

For example, when RNA is extracted from a solid component using a chaotropic salt solution as a nucleic acid extraction solution, the extracted RNA directly contacts the chaotropic salt in the liquid phase, and thus RNA is recovered from the liquid phase. As a result, the RNA washed with the chaotropic salt can be recovered without adding a new chaotropic salt.

Specifically, a chaotropic salt solution is added to feces to prepare a suspension, RNA is extracted, and then an alcohol solution or an alcohol solution containing a chaotropic salt is added to the suspension containing the chaotropic salt. After that, RNA is recovered from the suspension. Thereby, RNA washed with chaotropic salt and alcohol can be recovered.

Also, after adding an alcohol solution to feces and preparing a suspension, a solid component is recovered from the suspension, and a chaotropic salt solution is added to the solid component separated from the alcohol component, and then suspended again. Prepare a suspension. After RNA is extracted from the solid component by the action of the chaotropic salt, an alcohol solution or an alcohol solution containing a chaotropic salt is further added to the suspension, and then the RNA is recovered from the suspension to thereby collect the chaotropic salt. And RNA washed with alcohol can be recovered.

The stool from which RNA to be subjected to the synthesis method of the present invention is extracted is not particularly limited as long as it is an animal, but is preferably derived from a mammal, preferably from a human. More preferably. For example, human feces collected for periodic medical examinations and diagnosis are preferable, but feces such as livestock and wild animals may be used. Moreover, although it may be preserved for a certain period after collection, it is preferably immediately after collection. Further, the collected feces are preferably those immediately after excretion, but may be those that have passed time after excretion.

In the washing step, the RNA washed with the chaotropic salt and alcohol is preferably RNA extracted from 10 mg to 1 g of stool as a weight, for example. If the amount of stool becomes too large, the handleability and the like may be reduced. On the other hand, when the amount of stool is too small, the number of mammalian cells such as large intestine exfoliated cells contained in the stool becomes too small, and thus the necessary RNA amount may not be recovered. Moreover, since feces are heterogeneous, it is preferable to collect from a wide range of feces when collecting feces in order to avoid the influence of the localization of mammalian cells.

After the washing step, as a reaction step, in a reaction solution containing a single-stranded nucleic acid binding protein using a reverse transcriptase having heat-resistant and low RNase H activity using the washed RNA collected in the washing step as a template Perform reverse transcription reaction with. By combining a reverse transcriptase with specific properties and a single-stranded nucleic acid-binding protein, both the template RNA and reverse transcriptase are sufficiently stabilized, so the reduction in reaction efficiency due to inhibitors can be suppressed. it is conceivable that. The reverse transcription reaction can be performed by a conventional method except that a reverse transcriptase having heat resistance and low RNase H activity is used and a single-stranded nucleic acid binding protein is added to the reaction solution.

The reverse transcriptase used in the present invention is an enzyme having heat resistance. In the present invention, “heat-resistant reverse transcriptase” means a reverse transcriptase having resistance to heat inactivation. Specifically, it is an enzyme that retains at least 50% or more of its enzyme activity even when heated at 90 ° C. for 30 seconds.

The reverse transcriptase used in the present invention is an enzyme having a low RNase H activity. In the present invention, “reverse transcriptase having low RNase H activity” means that the RNase H activity of the enzyme is wild type or wild type Moloney murine leukemia virus (M-MLV), avian myeloblastosis virus (AMV) or It refers to a reverse transcriptase that is less than about 20% of the RNase H activity of an RNase H + enzyme such as Rous sarcoma virus (RSV) reverse transcriptase.

Specific examples of the reverse transcriptase having heat resistance and low RNase H activity include M-MLV H-reverse transcriptase and the like. In particular, SuperScript (registered trademark) III manufactured by Invitrogen is most preferable. The enzyme is a single-base mutant of RNase H- of M-MLV reverse transcriptase.

In the present invention, the “single-stranded nucleic acid binding protein” is a protein that mainly binds to single-stranded DNA rather than double-stranded DNA regardless of the base sequence. Specific examples of the single-stranded nucleic acid binding protein that can be used in the present invention include, for example, T4 gene 32.
protein (manufactured by Promega), E.I. Examples include SSB derived from E. coli, Thermos aquaticus SSB exhibiting heat resistance, methane bacterium (Methanococcus jannaschii), SSB derived from Sulfolobus sulfatalicus, and the like. These single-stranded nucleic acid binding proteins may be used alone or in combination.

The single-stranded nucleic acid binding protein used in the present invention in the reverse transcription reaction may be a non-heat-resistant single-stranded nucleic acid binding protein, but is preferably a heat-resistant single-stranded nucleic acid binding protein. The term “heat-resistant single-stranded nucleic acid binding protein” means a protein whose binding activity to single-stranded DNA has resistance to inactivation against heat. A single-stranded nucleic acid binding protein that retains at least 50% or more of its binding activity even when heated for seconds.

In the present invention, it is preferable to further add an RNase A inhibitor to the reaction solution for the reverse transcription reaction. By performing a reverse transcription reaction in the presence of an RNase A inhibitor, the influence of a fecal-derived inhibitor obtained by using a single-stranded nucleic acid-binding protein and a reverse transcriptase having heat resistance and low RNase H activity is reduced. The effect can be further enhanced. Examples of the RNase A inhibitor include Ribonclease Inhibitor, Cloned (manufactured by Invitrogen), Ribonclease Inhibitor (manufactured by TaKaRa), and the like.

The cDNA synthesized by the synthesis method of the present invention can be subjected to various analyzes in the same manner as DNA obtained by other methods. The presence or absence of a gene mutation can detect, for example, a mutation such as insertion, deletion, substitution, duplication, inversion, or splicing variant (isoform) of a base on RNA. In addition, RNA expression level can be detected (mRNA expression analysis). 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 may also be used.

Since the cDNA synthesized by the synthesis method of the present invention is derived from stool, it is preferably used for analyzing RNA derived from gastrointestinal cells such as the large intestine, small intestine, stomach, etc. It is more preferable to use it.

In particular, it is preferably used for analyzing RNA derived from a marker gene for neoplastic transformation (including cancer) or a marker gene for inflammatory digestive organ disease, and used for analyzing RNA derived from a marker gene for colorectal cancer. It is more preferable. The “gene-derived RNA” means an expression product such as mRNA of the gene. Examples of the marker showing neoplastic conversion include known cancer markers such as COX2 (cyclooxygenase-2) gene, carcinoembryonic antigen (CEA), sialyl Tn antigen (STN), APC gene, p53 gene, K- The presence or absence of mutations such as ras gene. 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, as a marker indicating inflammatory digestive organ disease, for example, there is COX2 gene-derived RNA. Cox-2 gene-derived RNA is also used as a marker indicating neoplastic conversion.

The synthesis method of the present invention can significantly reduce the influence of the stool-derived inhibitory substance and increase the reaction efficiency of the reverse transcription reaction, and as a result, a stable reaction can be performed. For this reason, a highly reliable result can be obtained by using the cDNA obtained by the synthesis method of the present invention for nucleic acid analysis such as gene expression analysis of stool-derived RNA. The detection result thus obtained is suitably used for clinical examination.

Moreover, the synthesis method of the present invention can be carried out more easily by making a kit of reagents used in the synthesis method of the present invention. Such a stool-derived nucleic acid synthesis kit includes, for example, a kit comprising a chaotropic salt, alcohol, a heat-resistant and low RNase H activity reverse transcriptase, and a single-stranded nucleic acid binding protein. The kit may further include a reagent used for RNA extraction from stool or reverse transcription reaction. Examples of such a reagent include a nucleic acid extraction solution, an inorganic support, a solvent for elution from the inorganic support, a reverse transcription reaction buffer, a primer, and dNTP.

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.

<Preparation of pseudofecal RNA>
By using SDS / phenol extraction method, guanidine / ethanol extraction method, and guanidine / ethanol / silica extraction method, RNA is extracted from a stool sample derived from a pseudo-colorectal cancer patient (a stool pseudo-sample collected from a colorectal cancer patient). Extraction and collection were performed to prepare pseudofecal RNA.

A stool sample derived from a pseudo-colon cancer patient was prepared as follows. First, an equal amount of physiological saline was added to 5 g of normal human stool, mixed well, and homogenized. Then, 200 × g centrifugation treatment was performed to precipitate impurities, and the supernatant was collected. This stool supernatant was mixed with a cell pellet adjusted so as to contain 1 × 10 ⁵ MKN45 cells, and this was used as a stool sample derived from a simulated colorectal cancer patient. MKN45 cells are derived from gastric cancer, but are cultured cell lines that highly express the COX2 gene in the same manner as colon cancer cells.

The prepared stool sample derived from a pseudo colorectal cancer patient is divided into three 1.5 mL tubes of 300 μL each, and RNA is extracted by SDS / phenol extraction method, guanidine / ethanol extraction method, or guanidine / ethanol / silica extraction method, respectively. Recovered.

In the SDS / phenol extraction method, first, 300 μL of AE buffer (50 mM CH ₃ COONa, 10 mM EDTA, pH 5.3) is added and suspended in a tube containing 300 μL of a stool sample derived from a pseudo colorectal cancer patient. Then, 30 μL of 10% SDS solution was added to perform vortex. The tube was further vortexed with an equal amount of phenol, and then heated at 65 ° C. for 4 minutes. Thereafter, the tube was immediately cooled using liquid nitrogen. The frozen contents of the tube were thawed at room temperature and then centrifuged at 14000 rpm for 2 minutes, and the upper layer was collected in another 1.5 mL tube. Chloroform was added to the 1.5 mL tube and mixed (phenol / chloroform treatment), and then centrifuged again at 14000 rpm for 2 minutes, and the upper layer was collected in another 1.5 mL tube. To the obtained upper layer, 30 μL of 3M CH ₃ COONa and an equal amount of isopropanol were added and mixed, followed by centrifugation at 4 ° C., 14000 rpm for 10 minutes to obtain a pellet (ethanol precipitation). The pellet was rinsed with a 75% ethanol solution, dried, and then dissolved in 50 μL of distilled water as phenol / chloroform-extracted RNA.

In the guanidine / ethanol extraction method, first, 300 μL of guanidine thiocyanate buffer (RLT buffer, manufactured by QIAGEN) was added to a tube containing 300 μL of a stool sample derived from a pseudo colorectal cancer patient, and after performing Vortex, etc. A volume (about 600 μL) of 70% ethanol was added and vortexed to obtain a suspension. Thereafter, 300 μL each of the suspension was dispensed into four 1.5 mL tubes, and 2.5 times the volume of 99.5% ethanol solution and 60 μL of 3M CH ₃ COONa were added and mixed. Centrifugation was performed at 4 ° C., 14000 rpm for 10 minutes to obtain a pellet (ethanol precipitation). The obtained pellets in the four tubes were combined into one, rinsed with a 75% ethanol solution, dried, and then dissolved in 50 μL of distilled water as guanidine / ethanol-extracted RNA.

In the guanidine / ethanol / silica extraction method, RNeasy mini kit (manufactured by QIAGEN) was used. First, 300 μL of an RTL buffer (a buffer containing a guanidine salt, manufactured by Qiagen) was added to a tube containing 300 μL of a stool sample derived from a pseudo colorectal cancer patient, and stirred with Vortex. After stirring, an equal amount of 70% ethanol solution added and mixed is passed through the RNA recovery column of RNeasy midi kit (manufactured by Quiagen), and the RNA recovery column is washed and eluted according to the attached protocol. The RNA was recovered as a 50 μL RNA solution. The RNA was guanidine / ethanol / silica extracted RNA.

<Preparation of RNA used for comparison>
A cell sample (MKN45 cell sample) adjusted with PBS so that 1 × 10 ⁵ MKN45 cells are contained, and added to the cell sample so as to contain 1 × 10 ⁸ E. coli cells cultured in a liquid medium. Three types, E. coli-containing MKN45 cell sample, and those obtained by adding bilirubin to the cell sample to a final concentration of 1 mg / μL (bilirubin-containing MKN45 cell sample) were used as comparison targets.
From these cell samples, RNA was extracted and collected by the SDS / phenol extraction method or the guanidine / ethanol extraction method in the same manner as the stool samples derived from pseudo colorectal cancer patients.

<Reverse transcription reaction>
A reverse transcription reaction was performed using each of the obtained RNAs as a template. Each RNA was quantified, and 1 μg of RNA was added to each reaction solution based on the measurement result of the RNA concentration.
Specifically, as shown in Table 1, phenol / chloroform as a template for each of pseudo-feces-derived RNA, MKN45 cell sample-derived RNA, E. coli-containing MKN45 cell sample-derived RNA, and bilirubin-containing MKN45 cell sample-derived RNA M-MLV (manufactured by TakaRa), which is non-thermostable and has high RNaseH activity, or SuperScript (registered trademark) III (manufactured by Invitrogen) having low thermostability and low RNaseH activity as reverse transcriptase using extracted RNA or guanidine / ethanol-extracted RNA 16 kinds of reaction solutions (samples) were prepared by changing the presence or absence of addition of 40 ng / μL T4 gene 32 protein (Promega) and the presence or absence of addition of RNase A inhibitor (TaKaRa). Perform a reverse transcription reaction It was synthesized cDNA. T4 gene 32 protein is a single-stranded nucleic acid binding protein (hereinafter referred to as SSB). In Table 1, in the column of “RNA extraction method”, “+” indicates that RNA extracted by the extraction method is added as a template, and “−” indicates that RNA extracted by the extraction method is used as a template. It means that there was no. In the column of “reverse transcription reaction”, “+” means that the substance was added to the reaction solution for reverse transcription reaction, and “−” means that it was not added.

<Measurement of expression level of COX2 gene>
Real-time PCR was performed using the obtained cDNA as a template, and the expression product (mRNA) of the COX2 gene was detected. As a primer for real-time PCR, COX2 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 COX2 primer probe MIX (Applied Biosystems) was added. The mixture was 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.

Fluorescence intensity measurement results were analyzed, and the amount of cDNA synthesized from the COX2 gene mRNA in each sample (COX2 gene expression level) was examined. Of the 16 types of samples shown in Table 1, guanidine / ethanol extracted RNA was used as a template, M-MLV reverse transcriptase was used, and the expression level of sample 12 to which neither SSB nor RNase A inhibitor was added was defined as 1. The relative value of the expression level of other samples was calculated. The calculation results are shown in FIGS. FIG. 1 shows the results when pseudo stool-derived RNA was used as a template, FIG. 2 shows the results when MKN45 cell sample-derived RNA was used as a template, and FIG. 3 shows the case where E. coli-containing MKN45 cell sample-derived RNA was used as a template. FIG. 4 shows the results when RNA derived from a bilirubin-containing MKN45 cell sample was used as a template.

As a result, in all of FIGS. 1 to 4, when other conditions were the same, SuperScript (registered trademark) III was used rather than using M-MLV (samples 1 to 4 and 9 to 12). In the cases (Samples 5-8, 13-16), the expression level of the COX2 gene was higher.

On the other hand, when MKN45 cell sample-derived RNA was used as a template (FIG. 2) and when E. coli-containing MKN45 cell sample-derived RNA was used as a template (FIG. 3), when other conditions were the same, phenol / chloroform There is almost no difference between using extracted RNA (samples 1 to 8) and guanidine / ethanol extracted RNA (samples 9 to 16), and differences in RNA extraction methods have little effect on reverse transcription. It turned out not to give. Moreover, neither the presence or absence of the addition of SSB nor the presence or absence of the addition of the RNase A inhibitor was observed to affect the expression level.

On the other hand, when pseudo-fecal RNA was used as a template (FIG. 1), guanidine · RNA was extracted more than when phenol / chloroform-extracted RNA was used (samples 1 to 8) when the other conditions were the same. The expression level of the COX2 gene was higher when ethanol-extracted RNA was used (samples 9 to 16). Even when bilirubin-containing MKN45 cell sample-derived RNA was used as a template (FIG. 4), the same tendency as when pseudo-feces-derived RNA was used as a template was observed.

From these results, when performing a reverse transcription reaction using RNA containing fecal-derived inhibitory substances such as bilirubin as a template, the RNA is preliminarily obtained by using a chaotropic salt and alcohol when the RNA is recovered. It is clear that the treatment with the tropic salt and alcohol can reduce the influence of the inhibitor introduced into the reaction solution of the reverse transcription reaction.

Furthermore, when pseudofecal RNA was used as a template (FIG. 1), SSB was added only when guanidine / ethanol extracted RNA was used as a template and SuperScript (registered trademark) III was used (samples 13 to 16) ( The expression level of the COX2 gene was clearly higher by the addition of sample 14), the addition of RNase A inhibitor (sample 15), and the combined use of both (sample 13) than when none was added (sample 16). Among them, compared to the sample 16 to which none of them was added, the expression level of the COX2 gene of the sample 14 to which SSB was added was about 5 times, and the sample 13 to which both were used was about 6 times as much.

On the other hand, when bilirubin-containing MKN45 cell sample-derived RNA is used as a template (FIG. 4), guanidine / ethanol extracted RNA is used as a template, SuperScript (registered trademark) III is used, and SSB is added (samples 13 and 14). ), The COX2 gene expression level was higher than when SSB was not added (samples 15 and 16). However, as can be seen from the comparison of Samples 13 and 14, or 15 and 16, unlike the case of using pseudofeces-derived RNA as a template, the effect of the presence or absence of the addition of an RNase A inhibitor was hardly observed.

That is, the effect of adding the RNase A inhibitor is not observed when the bilirubin-containing MKN45 cell sample-derived RNA is used as a template, and the effect of adding SSB is more than that when the bilirubin-containing MKN45 cell sample-derived RNA is used as a template. It was significantly larger when pseudo-fecal RNA was used as a template. The difference is that when bilirubin-containing MKN45 cell sample-derived RNA was used as a template, only one kind of inhibitor was introduced into the reaction solution, whereas pseudofecal-derived RNA was used as a template. In some cases, it is presumed that various inhibitors derived from stool were brought into the reaction solution.

From these results, the effect of improving the reaction efficiency obtained by adding an SSB or RNase A inhibitor to the reaction solution of the reverse transcription reaction is remarkably exhibited when a fecal-derived inhibitor is brought into the reaction solution. I found out. That is, reverse transcription using reverse transcriptase with heat resistance and low RNase H activity using RNA with reduced amount of stool-derived inhibitor as a template by using chaotropic salt and alcohol when recovering from stool It is clear that cDNA can be efficiently synthesized from stool-derived RNA by adding SSB to the reaction solution. Moreover, it can be said that by adding an RNase A inhibitor in addition to SSB, it is possible to more effectively avoid the influence of an inhibitor specific to feces having various characteristics other than bilirubin.

The influence of the RNase H activity of the reverse transcriptase used in the reaction when cDNA was synthesized using RNA in feces from healthy individuals as a template was examined.

<Extraction of fecal RNA>
First, RNA was extracted from feces derived from healthy individuals by the guanidine / ethanol extraction method in the same manner as in Example 1. Specifically, an equal amount of guanidine thiocyanate buffer (RLT buffer, manufactured by QIAGEN) was added to a stool sample collected from a healthy person, and after vortexing, an equal amount of 70% ethanol was added and vortex was added. To obtain a suspension. Thereafter, 300 μL each of the suspension was dispensed into four 1.5 mL tubes, and 2.5 times the volume of 99.5% ethanol solution and 60 μL of 3M CH ₃ COONa were added and mixed. Centrifugation was performed at 4 ° C., 14000 rpm for 10 minutes to obtain a pellet (ethanol precipitation). After the pellets in the four tubes obtained were combined into one, rinsed with a 75% ethanol solution, dried, and dissolved in 50 μL of distilled water, an RNA sample extracted from feces and did. The RNA sample was quantified and the RNA concentration was measured.

<Reverse transcription reaction and measurement of COX2 gene expression level>
Using the obtained RNA sample as a template, AMV with high RNase H activity (Avian Myeloblastosis Virus) Reverse Transcriptase (manufactured by Promega) (hereinafter referred to as AMV), RiverScript IV with low RNase H activity (manufactured by WakoNa H), A reverse transcription reaction was performed using each of three thermostable reverse transcriptases of SuperScript III (derived from M-MLV, manufactured by Invitrogen). In each reaction solution, an RNA sample is added so that 1 μg of RNA is added, and Taq SSB (manufactured by Bio Academia), which is a heat-resistant SSB, is added to 500 ng / μL, and an RNase A inhibitor ( The product was prepared according to the instruction manual attached to each reverse transcriptase, except that Taunit (manufactured by TaKaRa) was added to 0.5 units / μL. Each prepared reaction solution was reacted as described in the instruction manual for each reverse transcriptase to synthesize cDNA.
Thereafter, in the same manner as in Example 1, real-time PCR was performed using the obtained cDNA as a template, and the expression product (mRNA) of the COX2 gene was detected.

Also, using the obtained RNA sample as a template, reverse transcription reaction was carried out in the absence of RNase A inhibitor using AMV, ReverseScript IV, or SuperScript III, respectively. Specifically, each reaction solution was prepared by adding an RNA sample so that 1 μg of RNA was added, and further adding Taq SSB (manufactured by Bio Academia) to 500 ng / μL. It was prepared according to the instruction manual attached to the reverse transcriptase. Each prepared reaction solution is reacted as described in the instruction manual for each reverse transcriptase, and in the same manner as in Example 1, real-time PCR is performed using the obtained cDNA as a template to express the COX2 gene. Product (mRNA) was detected.

The measurement result of fluorescence intensity was analyzed, and the expression level of the COX2 gene in each reaction solution was examined. As a result, regardless of whether or not RNase A inhibitor was added to the reaction solution, the expression level was the lowest when AMV with high RNase H activity was used, and the expression level was highest when SuperScript III was used. With the expression level when AMV was used in the presence of RNase A inhibitor as 1, the relative value of the expression level when other reverse transcriptase was used was calculated. The calculation results are shown in FIG. In spite of using the same RNA sample as a template, when RNase A inhibitor is added to the reaction solution, it is approximately 4.8 times when SuperScript III is used compared to when AMV is used. In that case, the expression level was about 3.5 times higher.

In addition, when any reverse transcriptase was used, the amount of COX2 gene expressed was greater when RNase A inhibitor was added to the reaction solution than when it was not added. In the case of using AMV, assuming that the expression level when RNaseA inhibitor was added to the reaction solution was 1, it was about 0.7 when RNaseA inhibitor was not added. Similarly, when SuperScript III or ReverseScript IV was used, the expression level when RNase A inhibitor was not added was about 95% of the expression level when added.

In addition, RNA was extracted from feces derived from healthy individuals by the guanidine / ethanol / silica extraction method in the same manner as in Example 1. Using the obtained RNA samples as templates, the above three types of reverse transcriptases were used, respectively. In the same manner as described above, reverse transcription reaction was performed in a reaction solution to which RNase A inhibitor was added, real-time PCR was performed using the obtained cDNA as a template, and the expression product (mRNA) of the COX2 gene was detected. As a result, similarly to the case of using RNA extracted by the guanidine / ethanol extraction method, the expression level was the lowest when AMV was used, and the expression level was highest when SuperScript III was used. Assuming that the expression level when using AMV was 1, the relative value of the expression level when using other reverse transcriptases was calculated. The expression level when using ReverseScript IV was 4.1, and SuperScript III The expression level when using was 4.6.

From these results, when RNA extracted from feces is detected by reverse transcription reaction and subsequent nucleic acid amplification reaction such as PCR, such as SuperScript (registered trademark) III and RiverScript (registered trademark) IV, It is clear that it can be detected with high sensitivity by using a reverse transcriptase that exhibits thermostability and low RNase H activity, and that detection sensitivity can be further enhanced by carrying out the reverse transcription reaction in the presence of an RNase A inhibitor. It is.

[Comparative Example 1]
Using RNA extracted from cultured cells considered to have few inhibitors as a template, and measuring the expression levels of cDNA synthesis and COX2 gene expression products using three types of reverse transcriptase in the same manner as in Example 2, The influence of RNase H activity of the reverse transcriptase used in the reaction was examined.
Specifically, RNA was extracted from the cell pellet adjusted to contain 1 × 10 ⁵ MKN45 cells by the guanidine / ethanol extraction method in the same manner as in Example 1, and this was used as an RNA sample. Using the obtained RNA sample as a template, in the same manner as in Example 2, a reverse transcription reaction was performed using AMV, ReverseScript IV, or SuperScript III, and real-time PCR was performed using the obtained cDNA as a template to express the COX2 gene. Product (mRNA) was detected.

The measurement result of the fluorescence intensity was analyzed, and the expression level of the COX2 gene in each reaction solution was examined. As a result, as in Example 2, the expression level was the lowest when AMV was used, and the expression level was highest when SuperScript III was used, but there was not much difference in the expression level.
Assuming that the expression level when using AMV is 1, and the relative value of the expression level when using other reverse transcriptases is calculated, the expression level when using SuperScript III or RiverScript IV is about 1.6 to It was only 1.7. The calculation results are shown in FIG. From these results, when RNA extracted from cultured cells with a small amount of inhibitor is used as a template, even when reverse transcriptase with low RNase H activity is used, reverse transcriptase with high RNase H activity is used. It is clear that the reaction efficiency is not improved much compared to.

The influence of the heat resistance of SSB added to the reaction solution when synthesizing cDNA using stool RNA derived from healthy persons as a template was examined.
Using the RNA sample extracted from stool in Example 2 as a template and the reverse transcriptase SuperScript III used in Example 2 in the same manner, reaction solutions were prepared so that each of the following four types of SSBs had respective concentrations.
・ T4 gene 32 protein (non-heat resistant, manufactured by Bio Academia), 500 ng / μL
E. E. coli SSB (non-heat resistant, manufactured by Bio Academia), 500 ng / μL
-ET SSB (heat resistance, New England Bio Lab), 4 ng / μL
-Taq SSB (heat resistance, manufactured by Bio Academia), 500 ng / μL

Specifically, each reaction solution was added to the SuperScript III, except that the RNA sample was added so that 1 μg of RNA was added, and various SSBs were added to each concentration. Prepared according to the instructions. Each prepared reaction solution was reacted as described in the instruction manual for SuperScript III, and in the same manner as in Example 1, real-time PCR was performed using the obtained cDNA as a template to obtain an expression product of COX2 gene ( mRNA) was detected.

The measurement result of fluorescence intensity was analyzed, and the expression level of the COX2 gene in each reaction solution was examined. The expression level when T4 gene 32 protein was used was 1, and the relative value of the expression level when other SSB was used was calculated. The calculation results are shown in FIG. As a result, when ET SSB and Taq SSB were used even though the same RNA sample was used as a template, T4 gene 32 protein and E.I. The expression level of the COX2 gene was higher than when E. coli SSB was used. From these results, when reverse transcription reaction was performed using RNA extracted from feces as a template, the reaction efficiency was higher by adding heat-resistant SSB to the reaction solution than when non-heat-resistant SSB was added. It is clear that can be improved.

When cDNA was synthesized using RNA in stool from a healthy person as a template, the reaction efficiency when BSA was further added to the reaction solution was examined.
Using the RNA sample extracted from stool in Example 2 as a template, and using the reverse transcriptase SuperScript III used in Example 2 as well, BSA (manufactured by Promega) was added to a concentration of 400 ng / μL, and 40 ng / μL. 4 reaction solutions (samples 17 to 20) were prepared by changing the conditions of whether or not T4 gene 32 protein (Promega) was added and whether or not RNase A inhibitor (TaKaRa) was added. To synthesize cDNA. Table 2 shows the presence or absence of addition of T4 gene 32 protein (“SSB” in the table), BSA, and RNase A inhibitor in each sample. In Table 2, “+” means that the substance was added to the reaction solution for the reverse transcription reaction, and “−” means that it was not added. Samples 17 to 20 correspond to the conditions in which BSA was added to Samples 13 to 16 of Example 1, respectively, in the RNA extraction conditions and the composition of the reaction solution for the reverse transcription reaction.

Reverse transcription reaction was performed in the four types of reaction solutions of Samples 17 to 20 in the same manner as in Example 1. Real-time PCR was performed using the obtained cDNA as a template to detect the expression product (mRNA) of the COX2 gene. It was. The measurement result of the fluorescence intensity was analyzed, and the expression level of the COX2 gene in each reaction solution was examined. As a result, similar to Samples 13 to 16 of Example 1, when both SSB and RNase A inhibitor were added to the reaction solution (Sample 17), the expression level was the highest, and both SSB and RNase A inhibitor were added. When there was no sample (sample 20), the expression level was the smallest. Moreover, among SSB and RNase A inhibitors, the amount of expression was higher when only SSB was added to the reaction solution (sample 18) than when only RNase A inhibitor was added (sample 19).

When the pseudofecal RNA derived from Example 1 was used as a template (FIG. 1), the expression level of sample 12 was 1, and the relative value of the expression levels of samples 17 to 20 was calculated. The calculation results are shown in FIG. As reference values, the relative expression levels of samples 13 to 16 in FIG. 1 are also shown in the same graph. As a result, in the samples ( samples 13, 14, 17, and 18) in which SSB was added to the reaction solution, a tendency that the expression level of the COX2 gene slightly decreased was observed by further adding BSA. On the contrary, in the samples to which SSB was not added to the reaction solution ( samples 15, 16, 19, and 20), the expression level hardly changed depending on whether or not BSA was added.

By using the synthesis method of the present invention, cDNA can be efficiently synthesized from RNA contained in stool, so that it can be used not only in academic research but also in fields such as clinical tests using stool as a specimen. is there.

Claims (9)

1. (A) a washing step of washing RNA extracted from stool with a chaotropic salt and alcohol; and (b) one using a reverse transcriptase having heat resistance and low RNase H activity using the washed RNA as a template. A reaction step of performing a reverse transcription reaction in a reaction solution containing a strand nucleic acid binding protein;
   A method for synthesizing stool-derived nucleic acid.
2. The stool-derived nucleic acid synthesis method according to claim 1, wherein the reaction solution further contains an RNase A inhibitor.
3. The washing step (a) comprises a step of first adding a chaotropic salt solution to RNA extracted from stool, then adding an alcohol solution, and then recovering RNA from the obtained RNA solution. 2. A method for synthesizing a stool-derived nucleic acid according to 2.
4. The stool-derived origin according to claim 1 or 2, wherein the washing step (a) includes a step of adding an alcohol solution containing a chaotropic salt to RNA extracted from stool and recovering RNA from the obtained RNA solution. Nucleic acid synthesis method.
5. The method for synthesizing a stool-derived nucleic acid according to any one of claims 1 to 4, wherein the RNA extracted from the stool is an RNA extracted from the stool using a chaotropic salt.
6. Before the washing step (a),
   (A)
   (I) adding an alcohol solution to feces to prepare a suspension;
   (Ii) a step of recovering a solid component from the suspension; and (iii) a step of adding a chaotropic agent to the recovered solid component to prepare a suspension and extracting RNA from the solid component;
   The method for synthesizing a stool-derived nucleic acid according to any one of claims 1 to 4, further comprising an RNA extraction step having:
7. Before the washing step (a),
   (A-1) adding a chaotropic salt solution to feces to prepare a suspension and extracting RNA; and (a-2) an alcohol solution or an alcohol solution further containing a chaotropic salt in the suspension. Recovering RNA from the suspension after adding
   The stool-derived nucleic acid synthesis method according to claim 1 or 2, wherein
8. The washing step (a)
   (A-3) adding an alcohol solution to feces to prepare a suspension;
   (A-4) recovering solid components from the suspension;
   (A-5) adding a chaotropic agent to the recovered solid component to prepare a suspension, and extracting RNA from the suspension; and (a-6) further adding an alcohol solution to the suspension. Or a step of recovering RNA from the suspension after adding an alcohol solution containing a chaotropic salt;
   The stool-derived nucleic acid synthesis method according to claim 1 or 2, wherein
9. A stool-derived nucleic acid synthesis kit comprising chaotropic salt, alcohol, heat-resistant and low RNase H activity reverse transcriptase, and single-stranded nucleic acid binding protein.

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