WO2007116450A1

WO2007116450A1 - Nucleic acid extraction method and nucleic acid extraction kit

Info

Publication number: WO2007116450A1
Application number: PCT/JP2006/306699
Authority: WO
Inventors: Satoshi Ezaki; Yuki Shiraiwa; Ryuichiro Kurane
Original assignee: Biocosm Inc.
Priority date: 2006-03-30
Filing date: 2006-03-30
Publication date: 2007-10-18
Also published as: JPWO2007116450A1

Abstract

The object is to provide a nucleic acid extraction method which can treat any type of sample in a short time without using a reagent that needs to be handled carefully, can further reduce the influence on the subsequent analysis and is suitable for the detection of a trace amount of a sample. Particularly, it is intended to improve such a method for conveniently performing the procedures of a series of reaction system including the extraction of a nucleic acid, the amplification of the nucleic acid and the analysis of the nucleic acid in this order. The nucleic acid extraction method comprises the step of contacting a sample with deoxycholic acid, glycolic acid and a divalent cation chelator to release a nucleic acid.

Description

Specification

Nucleic acid extraction method and nucleic acid extraction kit

Technical field

[0001] The present invention relates to a nucleic acid extraction method.

Background art

[0002] In conventional nucleic acid extraction methods, many steps are required, such as destroying cell walls and cell membranes, removing them, denaturing proteins, and separating nucleic acids. The reagents used at this time included those requiring careful handling such as strongly alkaline sodium hydroxide solution and highly corrosive phenol solution.

[0003] Therefore, a method for purifying DNA, which is a mixture of microbial cells and phages that solves the problem, has been proposed (see, for example, Patent Document 1). Instead of the DNA concentration step using phenol and ethanol, a step of passing the sample through an ultrafiltration membrane is provided to remove impurities and concentrate the DNA.

[0004] According to the conventional nucleic acid extraction method described above, although the use of a phenol solution can be avoided, a strongly alkaline sodium hydroxide solution needs to be purified. As described above, there is a problem in that although it is possible to reduce the types of reagents that require careful handling, it is easy to handle all the reagents and is difficult to replace. Furthermore, microbial cells, specifically Escherichia coli, and a mixture of phages. Purifying DNA is relatively easy because it contains few contaminants and has few obstacles such as cell walls. In the case of application, it was difficult to provide a method for efficiently extracting nucleic acids regardless of the species of organisms, because the impurities contained in the structures and samples peculiar to the organisms became an obstacle. .

[0005] Furthermore, a reagent remaining in a nucleic acid sample extracted by using a conventional extraction method may interfere with subsequent analysis, requiring a further purification step and reducing analysis accuracy. There was a point. In particular, at present, in the field of biotechnology, it is recognized that a method for efficiently and intactly extracting genetic DNA from cells is essential for genetic testing of organisms. In addition, for rapid research, automation of gene extraction and other operations has been attempted. However, as the number of processes increases, the steps for purification such as centrifugation and suction filtration are complicated. Therefore, it was an obstacle to labor saving and downsizing of automatic genetic testing equipment. Furthermore, in the current technical situation, it is common to analyze a nucleic acid extract after amplifying the nucleic acid extract using a nucleic acid amplification technique, rather than subjecting the nucleic acid extracted from the test subject to direct analysis. ing . An analysis method using a nucleic acid amplification technique is also a general-purpose technique.

[0006] Therefore, in the polymerase chain reaction (hereinafter abbreviated as PCR) method, a method for efficiently amplifying nucleic acid in a sample such as stool and blood without passing through a nucleic acid extraction step has been proposed (for example, See Patent Documents 2, 3, 4, 5, 6, 7, and 8;). Specifically, by adding charged substances such as polyamines, polyions, surfactants, and DDT, the PCR reaction of dyes, proteins, sugars, or unknown contaminants contained in the sample is inhibited. It neutralizes positive and negative charged substances to suppress the effects of these PCR reaction inhibitors. However, these methods are not intended to rapidly destroy cells and extract nucleic acids.

[0007] In addition, a technique for extracting nucleic acid from a biological sample by using a surfactant and a chelating agent that avoids the use of a reagent that requires careful handling is known. Specifically, in a method of extracting nucleic acid from a biological sample by capturing it on a nonwoven fabric, or a method of extracting nucleic acid from a biological sample by capturing it on a polysaccharide carrier such as dextran sulfate, pretreatment of these nucleic acid extractions It is reported that cells, bacteria, etc. are destroyed by surfactants, chelating agents, reducing agents, proteolytic enzymes, etc., and the use of cholic acid as a surfactant is exemplified (for example, Patent Document 9, 10 reference.) while ₀ tooth force, these methods, which require special equipment such as a nonwoven fabric or column, surfactants and chelate agent is utilized in the pretreatment stage of the cell disruption Furthermore, it is assumed that a process such as cleaning of the carrier is necessary after capture. In addition, a method for extracting nucleic acids from biological samples derived from hard tissues by reacting proteinase K in the presence of potassium chloride, anionic surfactant, and thiol compound was reported. The use of cholic acid as an agent is exemplified (for example, see Patent Document 11).

Here, it is known that the above-mentioned surfactants such as cholic acid and chelating agents are used for nucleic acid extraction, but on the other hand, they may also inhibit various enzyme reactions such as nucleic acid amplification reactions. It was known. For this reason, when nucleic acids are extracted using these substances, a series of reactions following nucleic acid amplification and subsequent analysis are performed without steps such as purification and dilution of nucleic acid samples. There is a problem that the nucleic acid after extraction cannot be used in the reaction system. However, none of the above-mentioned methods gives any knowledge to the solution of the problematic problems.

[0009] In view of the above problems, the present inventors have developed a nucleic acid extraction method using cholic acids having a specific reagent configuration (see Non-Patent Document 1). Specifically, a reaction solution that has doxycholic acid and glycocholic acid power is added to the test sample so as to have lmM and 10 mM, respectively, followed by reaction that also has proteinase 1: and EDTA power. After adding the solution, the nucleic acid is extracted from the test sample by heating at 70 ° C for 10 minutes and then at 94 ° C for 10 minutes.

Patent Document 1: Japanese Patent Application Laid-Open No. 5-236963

Patent Document 2: Japanese Patent Laid-Open No. 6-270661

Patent Document 3: JP-A-8-173198

Patent Document 4: JP-A-9 9967

Patent Document 5: Japanese Patent Laid-Open No. 9-238687

Patent Document 6: Japanese Patent Laid-Open No. 2000-93175

Patent Document 7: Japanese Patent Laid-Open No. 2001-258556

Patent Document 8: Japanese Patent Laid-Open No. 2001-258562

Patent Document 9: Japanese Patent Laid-Open No. 2006-55079

Patent Document 10: Japanese Patent Laid-Open No. 2003-235555

Patent Document 11: Japanese Unexamined Patent Application Publication No. 2004-201558

Non-Patent Document 1: Yuki Oizumi et al., Detection and Species Identification of Cryptosporidium by DNA Microarray, Water and Wastewater, August 1, 2004, No. 46, No. 8, pp. 685-692

Disclosure of the invention

Problems to be solved by the invention

[0010] The above-described conventional method of the present inventors is a useful method capable of performing processing in a short time without using a reagent having careful handling regardless of the type of the test sample. It has been confirmed. In addition, the above-described conventional method of the present inventors should examine the above-mentioned recent technical situation and suppress the influence on the reaction system such as analysis after nucleic acid extraction including nucleic acid amplification reaction. However, depending on the reaction system, it was found that the nucleic acid amplification reaction may not proceed effectively. Therefore, the nucleic acid extract may need to be diluted when amplifying the nucleic acid after nucleic acid extraction, and the analysis accuracy of a very small amount of sample may not be sufficient. Furthermore, it was also found that heat treatment at a high temperature of 94 ° C may cause degradation of long-chain DNA and reduce the analysis accuracy. In addition, there was a demand for a place where it was easy to incorporate a nucleic acid extract into the reaction system without changing the PCR conditions optimized for each analysis target!

[0011] Therefore, the object of the present invention is to allow processing to be performed in a short time without using a carefully handled reagent regardless of the type of test sample, and to affect the subsequent analysis. Further, the present invention is to provide a nucleic acid extraction method which is further reduced and suitable for detection of a small amount of sample. In particular, the inventor's conventional nucleic acid extraction method was improved in order to facilitate the nucleic acid amplification following the nucleic acid extraction and the sequence of reaction systems following the subsequent analysis.

Means for solving the problem

[0012] In order to achieve this object, the characteristic means of the nucleic acid extraction method of the present invention comprises the step of bringing a test sample into contact with a chelating agent of deoxycholic acid, glycolic acid, and divalent cation to liberate nucleic acid. It is in having.

[0013] In the above characteristic means, the concentration force of the deoxycholic acid is preferably 0.01 to 10 mM, the concentration force of the daricholic acid is preferably 0.1 to 100 mM, or a proteolytic enzyme is used. It is preferable to have a step of releasing nucleic acid by contacting with the test sample, or it is preferable to have a step of performing heat treatment at 25 to 95 ° C for 5 to 120 minutes, or It is preferable to have a step of adding a magnesium salt and a buffer solution following the nucleic acid releasing step.

And it is preferable that the said test sample is a sample containing microorganisms.

[0014] To achieve this object, the nucleic acid extraction kit of the present invention is characterized in that it contains deoxycholate, glycolic acid, and a divalent cation chelating agent.

[0015] In the above feature configuration, it is preferable to include deoxycholic acid so that the final concentration is 0.01 to 10 mM when used, and glycolic acid is included so that the final concentration is 0.1 to 100 mM when used. Or contain proteolytic enzymes Preferably, it preferably contains a magnesium salt and a buffer.

[0016] The nucleic acid extraction method of the present invention focuses on bile acid, which is one of digestive fluids possessed by mammals. The main components of bile acids are cholic acids such as taurocholic acid, deoxycholic acid and glycocholic acid. Cholic acids also have properties as surfactants and are highly useful substances that are also used as cell membrane disrupting reagents for the extraction of membrane proteins. However, since cholic acids inhibit the enzyme reaction, when they are used in the enzyme reaction, they must be removed after cell destruction or diluted. The inventors, on the other hand, used a cholic acid, deoxycholic acid, in combination with glycolic acid, so that one kind of cholic acid did not give any strength to the subsequent enzymatic reaction and maintained stability. It was found that nucleic acids can be extracted efficiently in the state. Furthermore, it has also been found that the nucleic acid released from the cell can be protected by the nucleolytic enzyme force by the coexistence of the chelating agent in the nucleic acid extraction reaction, thereby achieving efficient nucleic acid extraction. The nucleic acid extraction method of the present invention is constructed by optimizing the nucleic acid extraction reagents and the application concentration, thereby achieving efficient nucleic acid extraction.

[0017] The molecular chemical mechanism of cell destruction of deoxycholic acid and dalicholic acid is presumed as follows. Cell membranes are also composed of phospholipids and protein power. Dexiolic acid and glycolic acid can penetrate into the lipid bilayer of phospholipids of this cell membrane, making the membranes fragile and phospholipids and protein components by surface activity. Dissolve. This mechanism may be common to all living organisms, whether animals, plants or microorganisms. Therefore, this method is considered to be effective for a wide range of biological specimens.

[0018] Here, deoxycholic acid and glycolic acid are easier to handle than phenolic solutions and strong alkaline solutions. At the same time, the sample used in the subsequent reaction system such as nucleic acid amplification reaction is purified because it is configured not to interfere with the enzyme reaction after nucleic acid extraction by limiting cholic acid to one kind of doxycholic acid in combination with glycolic acid. No process is required.

That is, the nucleic acid extraction method comprising the step of bringing a test sample into contact with a chelating agent of deoxycholic acid, glycolic acid, and a divalent cation according to the nucleic acid extraction method of the present invention is careful. It is possible to perform the processing in a short time without using a reagent having a proper handling. In addition, the nucleic acid extract can be easily incorporated into a subsequent reaction system such as a nucleic acid amplification reaction. Therefore, the nucleic acid extraction method of the present invention is considered to be greatly useful for automation and labor saving of a series of reaction systems including a nucleic acid extraction reaction and subsequent analysis. In turn, labor saving and downsizing of automated genetic testing equipment can be realized.

[0020] Further, when the concentration of the deoxycholic acid in contact with the test sample is 0.01 to 10 mM, or when the concentration of the daricholic acid is 0.1 to 100 mM, an enzyme reaction after nucleic acid extraction is included. The nucleic acid can be reliably extracted without hindering the reaction system, which is the most efficient.

[0021] In addition, when the step of bringing a proteolytic enzyme into contact with the test sample is used, the protein mixed in the sample is degraded, so that the molecular structure of the membrane is completely destroyed, and the cells can be more reliably destroyed. .

[0022] If there is a step of adding a magnesium salt and a buffer solution after releasing the nucleic acid, it can be more easily incorporated into a subsequent reaction system such as a nucleic acid amplification reaction.

[0023] Further, when nucleic acid is released from the cell, it is often heated.

When the temperature is 30 to 70 ° C, the destruction of the cell membrane is promoted by the thermal fluctuation at the same time as the decomposition of the protein without decomposing the released nucleic acid.

[0024] Samples containing microbial cells may be difficult to extract quickly and efficiently due to obstacles such as a robust structure peculiar to the organism or a multi-layered membrane structure.

Even in the case of these microorganisms, the present method can be applied. Therefore, by adopting this method, it is possible to achieve high speed and high accuracy in the medical field such as diagnosis of diseases and in the inspection of environmental pollution.

[0025] In carrying out the above-described nucleic acid extraction method, using a nucleic acid extraction kit containing a chelating agent of deoxycholic acid, glycolic acid and divalent cation capable of carrying out this method, a reagent having careful handling is not used. Processing can be performed in a short time. In addition, the nucleic acid extract can be easily incorporated into a subsequent reaction system such as a nucleic acid amplification reaction. Therefore, it is considered to be very useful for automation and labor saving including the nucleic acid extraction reaction and the subsequent analysis, and greatly contributing to labor saving by preparing necessary reagents as a kit. Can do.

[0026] Furthermore, the nucleic acid extraction kit contains deoxycholic acid so that the final concentration is 0.01 to 10 mM at the time of use, and glycolic acid is included so that the final concentration is 0.1 to 100 mM at the time of use. Thus, the nucleic acid can be extracted reliably without inhibiting the reaction system including the enzyme reaction after the nucleic acid extraction.

[0027] Furthermore, when the nucleic acid extraction kit contains a proteolytic enzyme, the protein mixed in the sample is degraded, so that the cells can be more reliably destroyed.

Furthermore, when the nucleic acid extraction kit contains a magnesium salt and a buffer, it can be easily incorporated into a subsequent reaction system such as a nucleic acid amplification reaction.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] Hereinafter, embodiments of the present invention will be described. First, the nucleic acid extraction method of the present invention is based on the above-described conventional nucleic acid extraction method of the present inventors (“Water and wastewater”, No. 46, No. 8, pages 685 to 692).

).

In the present application, “nucleic acid” is used as a concept including DNA, RNA, and derivatives thereof.

[0031] The nucleic acid extraction method according to the present invention includes a step of contacting a test sample with a chelating agent of deoxycholic acid, glycolic acid and divalent cation. The divalent cation chelating agent is not limited as long as it can form a stable complex salt by coordinating with metal ions in the sample, particularly magnesium ions, but ethylenediamine tetraacetic acid (hereinafter referred to as `` EDTA ''). (Omitted) is a good example. Preferably, the deoxycholate is added to the test sample in a state where the deoxycholate concentration is 0.01 to 10 mM, the glycomonophosphate is 100 to 100 mM, the EDTA is 0.01 to 200 mM, more preferably 10 to 100 mM. Contact glycolic acid and EDTA

[0032] Thereby, the nucleic acid can be efficiently extracted and the extracted nucleic acid can be protected. Cell membranes and the like are dissolved by utilizing the surface-active action of deoxycholic acid and dalicholic acid, in particular, deoxycholic acid, whereby nucleic acids inside the cells are released into the solution. At the same time, the released nucleic acid is protected from nucleolytic enzymes such as DNA degrading enzymes by the action of glycolic acid and EDTA. Specifically, glycolic acid is hydroxyacetic acid. It is also called a low molecular weight compound of an electrolyte that protects nucleic acids from DNA-degrading enzymes around the nucleic acids. Then, the magnesium-requiring DNA-degrading enzyme is inactivated by chelating magnesium ions in the solution with the chelating agent. This process is continued until the cell membrane of the test sample is sufficiently destroyed. For preparing a small sample such as a PCR sample, a contact time of about 5 to 60 minutes is sufficient.

[0033] In the conventional method proposed by the present inventors described above, two types of cholic acids, deoxycholic acid and dalcocholic acid, were used. However, according to the nucleic acid extraction method of the present invention, deoxycholic acid, It is limited to only one type, and it is configured to coexist with glycolic acid and divalent cation chelating agent. In other words, the method of the present invention is configured by restricting the use of cholic acids, which are inhibitors of enzyme reactions such as DNA polymerase, and it is possible to minimize the influence on subsequent analysis. . Therefore, the nucleic acid extract obtained by the nucleic acid extraction method of the present invention can be directly applied to subsequent analysis such as nucleic acid amplification reaction without passing through means for removing enzyme reaction inhibitor such as dilution. It can be suitably used even when the extraction target is a very small amount.

[0034] In order to ensure the destruction of the cell membrane of the test sample, a step of bringing the proteolytic enzyme into contact with the test sample can be further provided. As a result, protein components such as cell membranes of cells in the test sample are decomposed, and the release of nucleic acids inside the cells into the solution is further promoted. This step can be performed simultaneously with the step of bringing the above-mentioned deoxycholic acid, glycolic acid and divalent cation chelating agent into contact with the test sample. For example, proteinase K is suitable as the proteolytic enzyme, and for example, 0.01 to 10 mAU is added thereto.

[0035] After the above step, heat treatment is performed to decompose the protein component. The heat treatment is performed at 25 to 95 ° C for 5 to 120 minutes. As a result, the protein component in the sample is decomposed. In the conventional method proposed by the present inventors described above, the heat treatment is performed at 70 ° C. for 10 minutes and then at 94 ° C. for 10 minutes. In the method of the present invention, the heating temperature is lowered. In addition, the heating time is shortened. This makes it possible to suppress the degradation of long-chain DNA by heating under milder conditions than the conventional method, and to further improve the nucleic acid extraction efficiency and the detection sensitivity of subsequent analysis. [0036] In order to facilitate direct application to a subsequent analysis such as a nucleic acid amplification reaction, a step of bringing a magnesium salt and a pH buffer solution into contact with a test sample can be further provided. The magnesium salt is not limited as long as it does not interfere with the stability of nucleic acid and the subsequent analysis, but is preferably magnesium salt. The magnesium salt is preferably added in an amount corresponding to the binding amount of the chelating agent added previously. The buffer solution is not limited as long as it does not inhibit the stability of nucleic acid and the subsequent analysis, but preferably has a buffer capacity in the range of pH 6 to 10, particularly preferably a TAPS buffer solution. Preferably, magnesium chloride is adjusted to 0.1 to 50 mM, TAPS buffer is adjusted to 0.01 to 200 mM, pH 7 to 9, and added. Here, the magnesium salt and the buffer solution may be added alone, or may be added in a form in which the magnesium salt is dissolved in the buffer solution.

[0037] Thus, the nucleic acid extract obtained by the nucleic acid extraction method of the present invention can be directly applied to subsequent analysis such as nucleic acid amplification reaction, and optimized nucleic acid amplification reaction conditions according to individual detection targets. It can be easily incorporated into the reaction system without changing. In other words, Taq DNA polymerase and other DNA polymerases used in nucleic acid amplification reactions are magnesium-requiring enzymes, which can eliminate the effects of previously added chelating agents, and can be used in later reactions such as PCR. When used in the system, the nucleic acid extract can be applied as it is without changing the reaction conditions optimized for each reaction system, particularly the magnesium ion concentration. As a result, a reaction environment suitable for the expression of the activity of an enzyme such as DNA polymerase can be formed, and the analysis accuracy is improved. Here, the use of a magnesium salt is preferred, but a metal salt that is suitable for forming an enzyme reaction environment, such as iron or manganese salt, that is suitable for the formation of the reaction environment of the enzyme, or a magnesium salt that does not affect the magnesium salt. It can be configured to be added in place of the salt or in combination. By adding a pH buffer solution, pH adjustment in subsequent analysis is facilitated. Particularly in PCR, it is easy to adjust the DNA polymerase to be used to the optimum pH, and after the previously added reagent group. The influence on the reaction system can be eliminated. Therefore, the nucleic acid extract can be easily incorporated into the reaction system without changing the reaction conditions optimized for each detection target, particularly the magnesium ion concentration in PCR, the type of DNA polymerase, and the like.

[0038] By configuring as described above, the nucleic acid extract obtained by the nucleic acid extraction method of the present invention Can be directly incorporated into the subsequent reaction system. That is, in the nucleic acid extract obtained by the nucleic acid extraction method of the present invention, contamination of reaction inhibitory substances that inhibit subsequent analysis is minimized. Therefore, it is not necessary to provide a process for removing the reaction inhibitory substance including dilution of the nucleic acid extract required in the conventional method. As a result, it is possible to save labor and to minimize the loss of the nucleic acid sample, and to reduce the decrease in detection sensitivity due to an increase in the total liquid volume. Therefore, it is possible to meet market demands for reliably detecting a small amount of various detection objects without taking time and effort. The analytical reaction system suitable for incorporating the nucleic acid extract obtained by the nucleic acid extraction method of the present invention is not particularly limited, but preferred examples include amplification and detection of nucleic acid by a nucleic acid amplification method. Examples of nucleic acid amplification methods include PCR, LCR (ligase chain reaction) and the like, which can be particularly preferably used for PCR with Taq DNA polymerase, which is a general-purpose technology, and its application range is wide.

[0039] This method can be applied to a sample containing all living organisms regardless of whether they are microorganisms, animals, or plants. Therefore, the test sample is a concept that includes any organism, that is, an environment estimated to contain nucleic acid, a biological sample, or the like.

[0040] Specifically, examples of the test sample containing microorganisms include samples containing all bacteria, yeasts, protozoa, protozoa, etc. regardless of whether they are gram-positive or gram-negative. Microbial samples may be difficult to extract rapidly and efficiently due to the obstruction of a robust structure peculiar to the organism and the layered membrane structure, etc. Even in the case of such microorganisms, it is applicable. Therefore, by adopting the nucleic acid extraction method of the present invention, rapid and high accuracy can be achieved even in the medical field such as disease diagnosis and in the examination of environmental pollution. In particular, test samples containing microorganisms can be used for examining environmental pollution by microorganisms, or in the field of bioremediation, pollutants in the environment such as soil and water to be purified. It can be used to confirm the existence of assimilating bacteria.

[0041] Further, if the microorganism contained in the test sample is cryptosporidium, it can be used for the safety test of tap water and the like, so it is very useful in epidemiological viewpoint. The Cryptosporidium is a human belonging to the order of the spore genus Coccidium. It is a parasitic protozoan that parasitizes the digestive tract of domestic animals such as pigs and mammals such as pigs and mice, and when humans are infected with this protozoa, watery or mucous feces with abdominal pain are the main features. Symptoms that persist for about 3 days to a week, sometimes with vomiting and fever. Patients infected with this protozoa, the power of infected animals The enormous number of oocysts excreted together with feces is a source of infection to new individuals, so the transmission path of cryptosporidium is diverse. Cryptosporidium oocysts present in water are resistant to chlorine and are not effective in removing water from normal water treatment because chlorine disinfection is ineffective in water treatment. Large-scale outbreaks can occur through

[0042] Various methods have been proposed for determining the type and amount of cryptosporidium, and there has also been a method of extracting nucleic acids from oocysts and identifying and quantifying them by PCR. In this method, after freezing and thawing, sonication, etc., the sporozoites are removed from the oocysts, and then the sporozoites are dissolved with a protease, or known nucleic acid purification such as phenol extraction / ethanol precipitation. It was common to do so by processing. For this reason, each type of treatment is complicated and takes time, and in the process of nucleic acid purification, there is a problem in that the loss of nucleic acid is inevitably caused, resulting in a decrease in detection sensitivity.

[0043] However, when the decapsulation process by this method was observed with a microscope, it was confirmed that not only the oocysts were destroyed, but also the sporozoites coming out of them were destroyed. Therefore, in the present method, when the test sample is cryptosporidium, the operation in the test is simplified and rapid analysis is possible, and the analysis accuracy is improved. Is.

[0044] In addition, examples of animal-derived samples include hair roots, blood containing blood cells, feces, etc. If animal-derived test sample nucleic acids such as biopsy samples containing some animal cells are extracted, For example, it can be used for examinations in the medical field such as diagnosing diseases, and a rapid and highly accurate diagnosis method can be provided.

[0045] Examples of plant-derived samples include seeds, fruits, seed coats, stems, leaves, roots and the like. In conventional methods, robust structures such as cell walls may be an obstacle, and it may be difficult to extract nucleic acids quickly and efficiently from plant cells. Even in such cases, this method can be applied. It can be used to identify brands. In addition, these uses are examples and the above-mentioned thing It is not limited to.

[0046] Here, in the case of extracting nucleic acid from an organism having a cell wall such as a plant cell or yeast mold, Gram-positive bacteria, a test sample and a chelating agent of deoxycholic acid, glycolic acid, and divalent cation Before the contact, it is preferable to provide a pretreatment step for decomposing the cell wall. Since the composition of the cell wall differs depending on the species, cell wall degrading enzymes (for example, cellulase, zymolyase, lysozyme, etc.) corresponding to each composition are brought into contact with the test sample to lyse the cell wall. However, this method can be applied without causing cell wall degrading enzymes to act on these organisms.

[0047] In carrying out the nucleic acid extraction method of the present invention, each step (including the subsequent analysis step) can be performed by a mechanical device and automated. In such a case, or when a person performs an operation, for the convenience of the person, a reagent containing deoxycholic acid, glycolic acid, and a divalent cation chelating agent should be supplied as a nucleic acid extraction kit. Can do. In this kit, mix deoxycholic acid to a final concentration of 0.01 to 10 mM at the time of use, glycolic acid to 0.1 to 100 mM, and a divalent cation chelating agent to 0.01 to 200 mM. Is preferred. In particular, it is preferable to mix deoxycholic acid so that it may become 0.01-0.5 mM, and glycolic acid so that it may become 0.1-10 mM. Furthermore, this nucleic acid extraction kit can contain cell wall degrading enzyme and Z or proteolytic enzyme, and can further contain magnesium salt and buffer. For example, they can be supplied as a stock solution or concentrated solution of a liquid (solution) dissolved and sealed together with a reagent such as a buffer solution required for each step.

Example

[0048] Hereinafter, the nucleic acid extraction method of the present invention will be specifically described with reference to examples.

[0049] [Example 1] Nucleic acid extraction of Legionella force

DNA was extracted from Legionella pneumophila (L. pneumophila) SG3 strain of Legionella genus and cells of Legionella genus bacteria (Legionella sp.) Of a different species by the following procedure, and its presence was confirmed.

[0050] Preparation of chromosomal DNA

Legionella cells were suspended in a buffer solution to prepare a 5 L test sample. In this test sample 2 L of IB solution (10 mM deoxycholic acid, 0.35 mM glycolic acid, lOOmM EDTA-2Na) was added and incubated at 37 ° C for 15 minutes. Next, add 2 L of the liquid (O.lmAU proteinase K (QIAGEN)), incubate at 50 ° C for 10 minutes, and then incubate at 80 ° C for 5 minutes. . Next, 2 L of sag (25 mM MgCl, 0.5 mM TA

2

PS Buffer (pH 9.5)) was added to prepare a nucleic acid sample (Example) containing the chromosomal DNA extracted from Legionella cell force. A nucleic acid sample (control) treated in the same manner as described above was prepared, except that the same volume of sterilized water was added instead of the above reaction solutions ΙΒ to ΠΙΒ.

[0051] Amplification of DNA

The nucleic acid samples prepared above were cooled to room temperature, and PCR was performed using each of the samples as cages to obtain amplified fragments containing the gene of Legionella. For the PCR reaction, the forward primer Legll 3F (5, -GCACGTGTGTAGCCCTACCC-3 ′; sequence identification number 1) and the reverse primer Lrgl 115R (5′-ACTGGAACTGAGACACGGTC-3 ′; sequence identification number 2) were used as the primer set. The PCR reaction solution is 5 μL of X 10 Reaction Buffer (Ex Taq Buffer (+ Mg ²⁺ ), manufactured by Takara Bio Inc.), 5 μL of 2.5 mM dNTPs, 1 μL each of ΜρΜ / μL primer, 5 U I μL Ex Taq (manufactured by Takara Bio Inc.) was prepared in 0.5 μL, the nucleic acid sample prepared above was prepared in 12.5 μL, and sterilized water was added in 25 L to make a total volume of 50 L. The reaction was performed for 40 cycles, with denaturation at 94 ° C for 60 seconds, denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. Thereafter, the reaction was terminated by a final extension reaction at 72 ° C for 240 seconds. Next, each amplification product obtained by the PCR reaction was subjected to electrophoresis based on a conventional method to visualize the nucleic acid fragment.

[0052] FIG. 1 shows the results of visualizing the amount of DNA extracted by gel electrophoresis. For each of pneumophi la SG3 and Legionella sp., It corresponds to 3. OX 10 °, 3. OX 10 ¹ , 3. OX 10 0 ² , 3.0 X 10 ³ , 3.0 X 10 ⁴ cells Nucleic acid samples to be loaded were loaded into each lane. The control nucleic acid sample was similarly loaded. In the figure, gels 1 and 2 show the results of the examples treated with the IB solution to the filtrate, which are the nucleic acid extraction methods of the present invention, gel 1 shows the results of pneumophila SG3 strain, and gel 2 shows the results of Legionella sp. Gels 3 and 4 show the results of the control, gel 3 shows the results of L. pneumophila SG3 strain, and gel 4 shows the results of Legionella sp. In the figure, M represents a molecular weight marker of 100 bp ladder. Nucleic acid of the present invention In the example where nucleic acid extraction was performed by the extraction method, gene amplification occurred even with a small cell force for each of L. pneumophila SG3 strain and Legione Hasp. The amplified DNA band became thicker as the number of cells increased. As a result, quantitative amplification occurred depending on the number of cells (gels 1 and 2). From this, it was found that the nucleic acid extraction method of the present invention enables stable nucleic acid extraction and amplification. Furthermore, it can be seen that the nucleic acid extraction method of the present invention can be applied as a quantitative PCR method to detect and quantify Legionella. On the other hand, in the control, the PCR product has a strong correlation between the number of generated cells and the thickness of the amplified DNA band. There is a possibility that the DNA extraction or PCR reaction may be inhibited (gels 3 and 4). ).

[0053] Legionella is an intracellularly proliferating Gram-negative rod. In protozoa, algae, and the human body, it grows in neutrophils and macrophages, causing pneumonia (legionellosis). Inhabiting a moist environment, it grows in natural water systems such as rivers, as well as in cooling towers and recirculating tubs, and human outbreaks of pneumonia may occur. Under such circumstances, in order to detect Legionella rapidly and accurately, it is very useful from the viewpoint of epidemiological viewpoint to extract the nucleic acid using the nucleic acid extraction method of the present invention and provide it for prayer. In particular, legionellosis effectively suppresses Legionella infection in humans and the associated pneumonia by applying this method to nucleic acid extraction of species that are often caused by L. p neumophila. can do.

[0054] [Example 2] Nucleic acid extraction from cultured human cells

DNA was extracted from human cultured cells (Hela S3) according to the following procedure, and its presence was confirmed.

[0055] Preparation of chromosomal DNA

Hela S3 cells were suspended in a buffer to prepare a 5 L test sample. To this test sample, add two 18 solutions (1 (^ \ 1 deoxycholic acid, 0.35 mM glycolic acid, lOOmM EDTA'2Na) and incubate for 15 minutes at 37 ° C. The solution (O.lmAU proteinase K (QIAGEN)) was added and incubated at 50 ° C for 10 minutes, followed by further incubation at 80 ° C for 5 minutes, then 2 L Nose solution (25mM MgCl, 0.5mM TAPS Bu

2

ffer (pH 9.5)) was added to prepare a nucleic acid sample (Example) containing chromosomal DNA extracted from HelaS3 cells. A nucleic acid sample (control) was prepared in the same manner as described above except that the same volume of sterilized water was added instead of the reaction solutions (1) to (8). [0056] DNA amplification

The nucleic acid sample prepared above was cooled to room temperature, and PCR was performed using primers designed based on the human globin gene, each in a saddle shape, to obtain an amplified fragment containing the gene for Hela S3 cells. For the PCR reaction, forward primer KM29 (5, -GGTTGGCCAA TCTACTCCCAGG-3 '; sequence identification number 3) and reverse primer PC04 (5'-CAACTT CATCCACGTTCACC-3'; sequence identification number 4) were used as a primer set. PCR reaction solution is 5 μL of X 10 Reaction Buffer (Ex Taq Buffer (+ Mg ²⁺ ), manufactured by Takarabio), 5 μL of 2.5 mM dNTPs, 1 μL of lOpmol / μL primer, Prepare 5 U / μL Ex Taq (manufactured by Takara Bio Inc.) 0.5 μL, 12.5 μL of the nucleic acid sample prepared above, and 25 μL of sterilized water for a total volume of 50 L. The reaction consists of denaturation at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds for one cycle. Thereafter, the reaction was terminated by a final extension reaction at 72 ° C for 240 seconds. Next, each amplification product obtained by the PCR reaction was subjected to electrophoresis based on a conventional method to visualize nucleic acid fragments.

[0057] FIG. 2 shows the results of visualizing the amount of DNA extracted by gel electrophoresis. Nucleic acid samples corresponding to 1 × 10 °, 1 × 10 ¹ , 1 × 10 ² , 1 × 10 ³ cells were loaded into each lane. The control console was loaded in the same way. In the figure, gel A shows the results of the examples treated with the IB solution to the filtrate as the nucleic acid extraction method of the present invention, and gel B shows the results of the control. In the figure, M represents a molecular weight marker of lOObp ladder. In the case of the example in which nucleic acid extraction was performed by the nucleic acid extraction method of the present invention, it can be seen that even a small amount of cells had amplified the gene (gel A). From this, it was found that the nucleic acid extraction method of the present invention enables stable nucleic acid extraction and amplification. On the other hand, a PCR product was produced in the control, but the signal was much weaker than in the Examples, indicating that the DNA extraction efficiency was poor (Gel B).

[Example 3] Nucleic acid extraction from rice seeds

Chromosomal DNA preparation

As rice seeds, one commercially available rice koshihikari rice was dissolved in 50 μL of TE Buffer, and then heated at 95 ° C for 5 minutes in a 0.2 mL tube to prepare a test sample. Of this, 5 L was dispensed, and the second 18 liquids (1 (^ \ 1 deoxycholic acid, 0.35 mM glycolic acid, lOOmM EDTA-2Na ) Was added and incubated at 37 ° C for 15 minutes. Next, add 2 μL of the broth (O.lmAU proteinase K (QIAGEN)), incubate at 50 ° C for 10 minutes, and then incubate at 80 ° C for 5 minutes. did. Next, 2 L of sag (25 mM MgCl, 0.5 mM TA

Was added PS Buffer (pH 9. ^5)), nucleic acid specimen containing chromosomal DNA extracted from rice seeds (Example) was prepared. In addition, a nucleic acid sample (control) treated in the same manner as described above was prepared, except that the same volume of sterilized water was added instead of the reaction solutions (1) to (8).

[0059] DNA amplification

The nucleic acid samples prepared above were cooled to room temperature, and PCR was carried out using each as a saddle type to obtain an amplified fragment containing a gene derived from rice seeds. For the PCR reaction, forward primer IR F170 (5′-GTAAATGCCCTTTTTTCCCC-3 ′; SEQ ID NO: 5) and reverse primer IRF170 (5′-CGAACCCTCGGTAAACAAAA-3 ′; SEQ ID NO: 6) were used as a primer set. PCR reaction solution is 5 μL of X 10 Reaction Buffer (Ex Taq Buffer (+ Mg ²⁺ ), manufactured by Takara Bio Inc.), 5 μL of 2.5 mM dNTPs, and 1 μL each of lOpmol / μL primer. 5 U / μL Ex Taq (manufactured by Takara Bio Inc.) was added at 0.5 μL, the nucleic acid sample prepared above was added at 12.5 L, and sterilized water was added at 25 μL to prepare a total volume of 50 μL. The reaction was carried out for 40 cycles, with denaturation at 94 ° C for 60 seconds, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. Later, the reaction was terminated by a final extension reaction at 72 ° C for 240 seconds. Next, each amplification product obtained by the PCR reaction was subjected to electrophoresis based on a conventional method to visualize nucleic acid fragments.

[0060] FIG. 3 shows the results of visualizing the amount of DNA extracted by gel electrophoresis. The thus obtained nucleic acid sample to the operation, without dilution, or, respectively, 1.0 X 10- 1.0 X 10, a diluted to 1.0 X 10- ³ were loaded in each lane. The controls were loaded in the same way. In the figure, lanes 1 to 5 show the results of Examples treated with the IB solution to the filtrate, which are the nucleic acid extraction methods of the present invention, and lanes 6 to 9 show the results of controls. In the figure, M represents a molecular weight marker of 10 Obp ladder. In the example in which nucleic acid extraction was performed based on the nucleic acid extraction method of the present invention, gene amplification occurred even from a low concentration sample, and the amplified DNA band became thicker as the concentration increased. Quantitative amplification depending on the concentration occurred. Therefore, the nucleic acid extraction method of the present invention is a quantitative PCR method, It can be seen that the method can be applied to the detection and quantification of nucleic acids contained in plant seed-derived samples such as milled rice (lanes 2-5). On the other hand, in the control, the PCR product may interfere with nucleic acid extraction or PCR reaction in which there is no correlation between the concentration of the generated test sample and the thickness of the amplified DNA band (lanes 6 to 6). 9).

[0061] [Example 4, Comparative Examples 1 and 2]

Comparison of nucleic acid extraction method of the present invention and conventional method for DNA extraction from cryptosporidium oocysts

The effectiveness of the nucleic acid extraction method of the present invention will be further described below by taking as an example DNA extraction from Cryptosporidium oocysts and a series of analyzes using the extracted DNA. At the same time, a comparison was made with the previous method of the present inventors. The conventional nucleic acid extraction method is the nucleic acid extraction method disclosed in the above-mentioned “use water and wastewater”.

[0062] [Example 4] Extraction by nucleic acid extraction method of the present invention

Using the nucleic acid extraction method of the present invention, DNA was extracted from Cryptosporidium oocysts and PCR amplification was performed on the extracted DNA.

[0063] Chromosomal DNA extraction

A sample of 5 μL was prepared by suspending ³ × 10 ³ oocysts of Cryptosporidium in a buffer solution. To this test sample, 2 L of IB solution (10 mM deoxycholic acid, 0.35 mM glycolic acid, lOOmM EDTA'2Na) was added and incubated at 37 ° C for 15 minutes. Subsequently, 2 μL of a filtrate (O.lmAU proteinase K (manufactured by QIAGEN)) was added and incubated at 50 ° C. for 10 minutes. Thereafter, proteinase K was inactivated by further incubation at 80 ° C. for 5 minutes. Next, the 2 1 solution (25 mM MgCl

2. A nucleic acid sample was prepared by adding 0.5 mM TAPS Buffer (pH 9.5)).

[0064] Amplification of DNA

The nucleic acid sample prepared above was cooled to room temperature, PCR was performed using this as a cage, and an amplified fragment containing a gene derived from Talibutosporidium was obtained. For the PCR reaction, forward primer 18SF (5, -GGAACCTGGTTGATCCTGCCAG-3 '; SEQ ID NO: 7) and reverse primer 18SR (5, -GGTCGATCCCCTAACTTTCGTT-3'; SEQ ID NO: 8) were used as a set of primers. PCR reaction solution is X 10 Reaction Buffer (buffer for Blend Taq (+ M g) 5 μL of Toyobo Co., Ltd.), 5 μL of 2 mM dNTPs, and lOpmol / μL primer: LL, 0.5 U / μL of Blend Taq (Toyobo Co., Ltd.) 0.5 μL Then, 12.5 μL of the nucleic acid sample prepared above and 25 μL of sterilized water were added to prepare a total volume of 50 μL. The reaction was carried out for 40 cycles with denaturation at 94 ° C for 60 seconds, denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 60 seconds. Later, the reaction was terminated by a final extension reaction at 72 ° C for 240 seconds.

Next, each amplification product obtained by the PCR reaction was subjected to electrophoresis based on a conventional method to visualize nucleic acid fragments.

[0065] [Comparative Example 1] Extraction by conventional method 1

Using the conventional method of the present inventors, which was the basis for the improvement of the nucleic acid extraction method of the present invention, DNA was extracted from cryptosporidium oocysts, and PCR amplification was performed on the extracted DNA. First, a test sample of Cryptosporidium oocyst was prepared in the same manner as in Example 4 above. To this test sample, 2 L of reaction solution IA (3.5 mM deoxycholic acid (final concentration lmM), 35 mM glycocholic acid (final concentration 10 mM)) was added and incubated at 37 ° C for 15 minutes. Subsequently, 4 L of reaction solution IIA (17 mAU proteinase K (600 mAU / mL, manufactured by QIAG EN), 9.7 mM EDTA ′ 2Na) was added and incubated at 70 ° C. for 10 minutes. Thereafter, the proteinase K was inactivated by further incubation at 94 ° C. for 10 minutes. The nucleic acid sample thus prepared was subjected to PCR and electrophoresis in the same manner as in Example 4 above.

[0066] [Comparative Example 2] Extraction by conventional method 2

The nucleic acid sample obtained in Comparative Example 1 was subjected to electrophoresis in the same manner except that the nucleic acid sample was diluted to 1/10 with a buffer solution.

[0067] FIG. 4 shows the results of visualizing the amount of DNA extracted by gel electrophoresis. In the figure, Lane 1 shows the result of Comparative Example 1, Lane 2 shows the result of Comparative Example 2, and Lane 3 shows the result of Example 4. In the figure, M represents a molecular weight marker of lOObp ladder. When nucleic acid extraction was performed based on the nucleic acid extraction method of the present invention, gene amplification occurred even from a low concentration sample, and the amplified product could be detected with high sensitivity (lane 3). On the other hand, when the nucleic acid sample extracted by the conventional method of the present inventors is directly subjected to an amplification reaction, most of the amplification product is removed. I couldn't confirm it (lane 1). Therefore, in the conventional method, the reaction may be inhibited by any of the reagent groups added during the nucleic acid extraction at either the nucleic acid extraction or the PCR reaction stage. Therefore, in order to avoid such reaction inhibition, the conventional method requires the dilution of the nucleic acid extract during PCR. Even when the extracted nucleic acid sample is diluted to 1/10 concentration and used for the amplification reaction, Almost no confirmation was possible (lane 2). In other words, it can be seen that the analysis sensitivity decreased due to dilution.

[0068] Therefore, the nucleic acid extraction method of the present invention does not require any means such as dilution, and the nucleic acid extract can be directly applied to the nucleic acid amplification method and is contained in the test sample with high sensitivity. It can be seen that nucleic acids can be detected and quantified. It is particularly useful for analysis of trace samples because of its high detection sensitivity. On the other hand, it has been found that the conventional method has high utility value as a nucleic acid extraction method. It has been found that there are some problems in application to nucleic acid amplification methods, especially to trace samples. Further, the nucleic acid extraction method of the present invention, which has improved problems, has a wider utility value.

Industrial applicability

[0069] Since the present invention can efficiently extract nucleic acids regardless of the type of organism, medical fields such as diagnosis of diseases, inspection of environmental pollution, and identification of plant varieties and brands, etc. Can be used.

Brief Description of Drawings

[0070] [Fig. 1] Electrophoretic diagram showing the results of extraction of nucleic acid by the nucleic acid extraction method of the present invention using Legionella cells as a test sample.

[Fig. 2] Electrophoretic diagram showing the results of nucleic acid extraction by using the nucleic acid extraction method of the present invention using human cultured cells as a test sample.

[Fig. 3] Electrophoretic diagram showing the results of nucleic acid extraction by the nucleic acid extraction method of the present invention using Koshihikari rice mill as a test sample.

[Fig. 4] Electrophoretic diagram showing the results of comparing the nucleic acid extraction method of the present invention with the conventional nucleic acid extraction method using cryptosporidium oocysts as test samples.

Claims

The scope of the claims

[I] A nucleic acid extraction method comprising a step of bringing a test sample into contact with a chelating agent of deoxycholic acid, glycolic acid, and divalent cation to liberate nucleic acid.

[2] The nucleic acid extraction method according to [1], wherein the concentration of deoxycholic acid is 0.01 to 10 mM.

[3] The nucleic acid extraction method according to claim 1 or 2, wherein the concentration of the dalicholic acid is 0.1 to 100 mM.

[4] The method for extracting nucleic acid according to any one of claims 1 to 3, further comprising a step of bringing a protease into contact with the test sample to release the nucleic acid.

[5] The nucleic acid extraction method according to any one of claims 1 to 4, wherein the heat treatment is performed at 25 to 95 ° C for 5 to 120 minutes.

[6] Following the nucleic acid release step! The nucleic acid extraction method according to any one of claims 1 to 5, further comprising a step of adding a magnesium salt and a buffer solution.

7. The nucleic acid extraction method according to any one of claims 1 to 6, wherein the test sample is a sample containing a microorganism.

[8] Nucleic acid extraction kit containing deoxycholic acid, glycolic acid, and divalent cation chelating agent.

[9] Claims containing deoxycholic acid so that the final concentration is 0.01 to 10 mM when in use.

8. The nucleic acid extraction kit according to 8.

10. The nucleic acid extraction kit according to claim 8 or 9, comprising glycolic acid so that the final concentration is 0.1 to 100 mM when in use.

[II] The nucleic acid extraction kit according to any one of claims 8 to 10, comprising a proteolytic enzyme.

[12] The nucleic acid extraction kit according to any one of claims 8 to 8, comprising a magnesium salt and a buffer.