Method for extracting nucleic acid from formalin fixative

Abstract

To provide a method for extraction of nucleic acid from formalin in which a sample has been immersed.SOLUTION: A method for extracting nucleic acid, the method including a step (1) of adding a formalin neutralizing agent to formalin in which a sample has been immersed, and a step (2) of extracting nucleic acid from a mixture obtained in the step (1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for extracting nucleic acids from formalin after a sample has been soaked in the formalin. The nucleic acid extraction method of the present invention is characterized by including a step of adding a formalin neutralizer to the formalin after the sample has been soaked in the formalin. In one example, the formalin neutralizer includes one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid. The present invention also relates to a nucleic acid amplification method including a step of amplifying a nucleic acid of interest in a solution containing the nucleic acid obtained by the extraction method, and a sequencing method including a step of analyzing the sequence of the nucleic acid amplified by the amplification method. [Background technology]

[0002] In recent pathological diagnoses, formalin-fixed paraffin-embedded (FFPE) tissues have been widely used as samples for genetic testing using PCR, next-generation sequencing (NGS), etc., and the test results are used for the diagnosis and companion diagnosis of various diseases (Non-Patent Document 1). Furthermore, genetic research using such samples is useful for the discovery of new therapeutic drugs (Non-Patent Document 1).

[0003] However, in the case of very small specimens such as biopsy specimens, it has been reported that the quality and quantity of DNA and RNA recovered from FFPE samples is low, making it difficult to perform genetic tests such as PCR tests (Non-Patent Documents 2-4).

[0004] In forensic medical examinations, tests are sometimes performed using samples that have been preserved in formalin for a long period of time, but there is a problem that genetic testing cannot be performed due to factors such as low yields of DNA and RNA. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yasushi Y et al., Pathology International. 2020;20 (12) 921-931 [Non-patent document 2] Wang F et al., J Mol Diagn. 2007; 9(4):441-451 [Non-patent document 3] Srinivasan M et al., Am J Pathol. 2002; 161(6):1961-1971 [Non-patent document 4] Utako Oba et al., Biology Methods and Protocols 2022; Jul 26;7(1) Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an objective of the present invention is to provide a method for extracting nucleic acids from formalin after a sample has been soaked in the formalin. In particular, the objective of the present invention is to provide a nucleic acid extraction method characterized by including a step of adding a formalin neutralizer to the formalin after the sample has been soaked in the formalin. In one example, the neutralizer includes one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid. Another objective of the present invention is to provide a nucleic acid amplification method including a step of amplifying a nucleic acid of interest in a solution containing the nucleic acid obtained by the extraction method, and a sequencing method including a step of analyzing the sequence of the nucleic acid amplified by the amplification method.

[0007] The inventors have considered the fact that in the case of very small specimens such as biopsy specimens, the quality and quantity of DNA and RNA recovered from FFPE samples is low, making it difficult to perform genetic tests such as PCR tests or forensic tests, and that conventional specimen preparation only produces a cross-sectional specimen, which means that there is a possibility that a disease (e.g., cancer) may be overlooked. In light of this, they have attempted to obtain nucleic acids of a quality and quantity suitable for these tests from sources other than FFPE samples.

[0008] Therefore, the present inventors focused on formalin used to fix samples such as surgical specimens and biopsy specimens, and came up with the idea of ​​extracting nucleic acids from the formalin. After extensive investigation, they surprisingly found that by immersing a sample such as a surgical specimen or biopsy specimen in formalin and then adding hydrogen peroxide, or urea, citric acid, and calcium chloride to the formalin, nucleic acids can be efficiently extracted with minimal denaturation. Based on these findings, the present inventors conducted further research, and as a result, they have completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for extracting nucleic acids, comprising: (1) adding a formalin neutralizer to the formalin after the sample has been immersed; and (2) extracting nucleic acids from the mixture obtained in step (1); A method comprising: [2] The method according to [1], wherein the formalin neutralizer comprises one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid. [3] The method according to [1] or [2], wherein the formalin neutralizer comprises one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water. [4] The method according to [2] or [3], wherein the one or more compounds is hydrogen peroxide. [5] The method according to [1] or [2], wherein the formalin neutralizer comprises urea and acid. [6] The method according to [2] or [5], wherein the acid is an aqueous solution of citric acid. [7] The method according to [5] or [6], wherein the formalin neutralizer further contains calcium chloride. [8] The method according to any one of [1] to [7], further comprising adding a surfactant in step (1). [9] The method according to [8], wherein at least one of the surfactants is an ionic surfactant.

[10] The method according to [9], wherein at least one of the ionic surfactants is an anionic surfactant.

[11] The method according to

[10] , wherein the anionic surfactant is a polyoxyethylene alkyl ether sulfate or an alkyl ether sulfate.

[12] The method according to

[10] or

[11] , wherein at least one of the ionic surfactants is a cationic surfactant.

[13] The method according to

[12] , wherein the cationic surfactant is a quaternary ammonium salt.

[14] A method for amplifying nucleic acid, comprising the step of amplifying a target nucleic acid in a solution containing nucleic acid extracted by the method according to any one of [1] to

[13] .

[15] A step of analyzing the sequence of the nucleic acid amplified by the method according to

[14] . A sequencing method comprising:

[16] A kit for extracting nucleic acids from formalin after immersion of a sample, comprising one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid. [Effects of the Invention]

[0010] The present invention makes it possible to obtain sufficient amounts of nucleic acids of a quality suitable for genetic testing in pathological diagnosis from the formalin used to prepare such samples, without relying on conventional samples such as FFPE samples. Therefore, it is possible to obtain sufficient amounts of nucleic acids of a quality suitable for genetic testing in pathological diagnosis, even from minute specimens such as biopsy specimens. Furthermore, for samples that were previously untestable, such as long-term stored FFPE samples, genetic testing using PCR, next-generation sequencing (NGS), and the like becomes possible, as long as the formalin used to prepare the sample is preserved. Furthermore, even for samples such as biological tissues and cells preserved immersed in a solution containing formalin (e.g., liquid-immersion specimens (so-called formalin-preserved specimens), specimens for liquid-based cytology, etc.), the present invention makes it possible to extract nucleic acids from the formalin in which the sample is preserved and ultimately analyze the sequence of the nucleic acid, thereby enabling various searches in basic medicine, forensic medicine, and biology (e.g., the search and identification of novel diagnostic markers and disease-causing genes, etc.). [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 relates to the DIN values ​​of nucleic acids (DNA) obtained by the nucleic acid extraction method of the present invention. [Figure 2] FIG. 2 shows the results of housekeeping gene amplification using nucleic acids (DNA and RNA) obtained by the nucleic acid extraction method of the present invention (FIG. 2, left (DNA, GAPDH); FIG. 2, right (RNA, RPS18)). DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Nucleic acid extraction method of the present invention The present invention provides a method for extracting nucleic acids from formalin after immersion of a sample. Specifically, the method comprises the steps of: (1) adding a formalin neutralizer to the formalin after the sample has been immersed; and (2) extracting nucleic acids from the mixture obtained in step (1); (hereinafter, this may be referred to as the "nucleic acid extraction method of the present invention").

[0013] As used herein, "formalin" refers to an aqueous solution containing formaldehyde, typically further containing methanol. The concentration of formaldehyde in formalin is typically 35 to 38% (w / v) according to the Japanese Pharmacopoeia. As used herein, for example, 20% formalin contains 1 / 5 of the volume of undiluted formalin (e.g., Japanese Pharmacopoeia formalin), and the same applies to other percentages of formalin.

[0014] As used herein, "formalin after sample immersion" refers to a solution (hereinafter sometimes referred to as "fixative") obtained by diluting undiluted formalin (e.g., Japanese Pharmacopoeia formalin) in which at least a portion of a sample such as a (biological) tissue fragment has been immersed. Therefore, the formalin after sample immersion may or may not contain a sample such as a (biological) tissue fragment.

[0015] In one embodiment, the formalin after the sample immersion may be formalin in which a sample such as a (biological) tissue slice is being fixed, or may be formalin after the sample has been fixed, etc. In addition, in either case of the formalin, a sample such as a (biological) tissue slice may or may not be present therein.

[0016] In another embodiment, the formalin after immersion of the sample may be formalin in a liquid-immersion preparation of the sample, such as a (biological) tissue slice, etc. The sample, such as a (biological) tissue slice, may or may not be present in the formalin.

[0017] In step (1) of the present invention, the formalin to which the formalin neutralizer is added may be the entire amount of formalin after immersion of a sample such as a (biological) tissue piece, or a part thereof. When the compound is added to a part of the formalin after immersion of the sample, the compound may be added to a part of the formalin after immersion of the sample.

[0018] The dilution ratio of formalin stock solution is typically 10% to 20% (v / v), and dilution can be performed using, for example, distilled water or various buffer solutions. In this specification, formalin stock solution diluted with various buffer solutions is referred to as buffered formalin (e.g., 10% neutral buffered formalin, etc.).

[0019] The time for which a sample such as a biological tissue fragment is immersed in formalin is not particularly limited, as long as nucleic acids of the desired quality and quantity can be extracted in step (2) of the nucleic acid extraction method of the present invention. In one embodiment, when a (biological) tissue fragment is fixed with the intention of producing an FFPE sample, the fixation time may vary depending on the fixative, the size of the biological tissue fragment, etc. For example, when a 10% neutral buffered formalin solution is used as the fixative, the immersion time is 6 to 72 hours, preferably 24 to 48 hours.

[0020] The origin of the sample to be immersed in formalin is not particularly limited and may be either an animal or a plant. Examples of animals include, but are not limited to, fish, amphibians, reptiles, birds, and mammals, with mammals being preferred. Examples of mammals include, but are not limited to, rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, with humans being preferred.

[0021] The sample to be immersed in formalin may be either plant tissue or animal tissue. Examples of animal tissue include, but are not limited to, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, eye, and brain. Examples of plant tissue include, but are not limited to, meristematic tissue and permanent tissue.

[0022] The formalin used in the nucleic acid extraction method of the present invention after sample immersion may be formalin used immediately after at least a portion of a sample such as a (biological) tissue slice has been immersed in the fixative, or may be formalin used several months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months), one year, or multiple years (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 years) or more. Furthermore, it may be formalin used at any point during fixation of a (biological) tissue slice in preparation for an FFPE sample, etc. It may also be formalin used after fixation of a (biological) tissue slice in preparation for an FFPE sample, etc. The formalin after fixation may be that used immediately after fixation, or may be that stored for several months (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months), one year, or multiple years (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 years) or more for disposal, etc. Alternatively, the formalin used after sample immersion may be that used in a liquid-immersed specimen.

[0023] As used herein, the term "formalin neutralizer" refers to a substance that can accelerate the decomposition of formaldehyde, reduce the toxicity of formaldehyde, or at least inhibit the formation of methylene bridges between formaldehyde and nucleic acids. Specific examples of formalin neutralizers include one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and acid.

[0024] Peroxides that generate hydrogen peroxide in water and are used in step (1) of the nucleic acid extraction method of the present invention include, but are not limited to, inorganic peroxides and hydrogen peroxide adducts. Preferred are percarbonate, tripolyphosphate-hydrogen peroxide adduct, pyrophosphate-hydrogen peroxide adduct, urea-hydrogen peroxide adduct, sulfate-hydrogen peroxide adduct, perborate, persilicate, and peroxide salts. More preferred are sodium percarbonate, sodium tripolyphosphate-hydrogen peroxide adduct, sodium pyrophosphate-hydrogen peroxide adduct, urea-hydrogen peroxide adduct, 4NaSO·2HO, sodium perborate monohydrate, sodium perborate tetrahydrate, sodium persilicate, sodium peroxide, and calcium peroxide. Even more preferred are sodium percarbonate and / or sodium perborate. In one embodiment of the present invention, the one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water include at least hydrogen peroxide.

[0025] The acid used in step (1) of the nucleic acid extraction method of the present invention is not particularly limited as long as it is a substance that generates hydrogen ions when dissolved in water, but is preferably an acid with a pH of 6.0 or less, more preferably an acid with a pH of 3.0 or less. Specific examples include citric acid, hydrochloric acid, nitric acid, acetic acid, phosphoric acid, and oxalic acid, with citric acid being preferred.

[0026] When the formalin neutralizer used in step (1) of the nucleic acid extraction method of the present invention contains urea and an acid, it may further contain calcium chloride.

[0027] In step (1) of the nucleic acid extraction method of the present invention, a surfactant may be added to the formalin after immersion of the sample in addition to a formalin neutralizer (e.g., a neutralizer containing one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, a neutralizer containing urea and an acid, etc.). The surfactant may be an ionic surfactant, a nonionic surfactant, or a combination thereof, but it is preferable that at least one of the surfactants is an ionic surfactant. The ionic surfactant may be a cationic surfactant, an anionic surfactant, an amphoteric surfactant, or a combination thereof, but it is preferable that at least one of the surfactants is an anionic surfactant. It is more preferable that at least one of the ionic surfactants is an anionic surfactant and at least one of the ionic surfactants is a cationic surfactant.

[0028] Examples of anionic surfactants include, but are not limited to, alkylbenzenesulfonates, preferably alkylbenzenesulfonates having an alkyl group with 10 to 20 carbon atoms, more preferably 10 to 15 carbon atoms; alkyl sulfates, preferably alkyl sulfates having an alkyl group with 8 to 18 carbon atoms, more preferably 10 to 14 carbon atoms; and polyoxyethylene alkyl ether sulfates, preferably polyoxyethylene alkyl ether sulfates having an alkyl group with 8 to 18 carbon atoms, more preferably 10 to 14 carbon atoms. Among these, polyoxyethylene alkyl ether sulfates are preferred, more preferably polyoxyethylene alkyl ether sulfates having an alkyl group with 8 to 18 carbon atoms, even more preferably 10 to 14 carbon atoms. The average number of EO moles added of polyoxyethylene alkyl ether sulfates is preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5. Alkali metals such as sodium and potassium are preferred as counterions to these anionic surfactants.

[0029] Examples of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants other than those mentioned above include anionic surfactants such as olefin sulfonates, alkanesulfonates, fatty acid salts, alkyl or alkenyl ether carboxylates, α-sulfofatty acid salts, and α-sulfofatty acid esters; nonionic surfactants such as polyoxyethylene or polyoxypropylene or copolymers thereof, polyoxyethylene alkyl or alkenyl ethers, polyoxyethylene alkylphenyl ethers, higher fatty acid alkanolamides or alkylene oxide adducts thereof, sucrose fatty acid esters, and alkylglycosides; amphoteric surfactants such as amine oxides, sulfobetaines, and carbobetaines; and cationic surfactants such as quaternary ammonium salts. In one embodiment, it is preferable to blend a polyoxyethylene alkyl or alkenyl ether having an alkyl or alkenyl group having 10 to 20 carbon atoms, preferably 10 to 18 carbon atoms, and a polyoxyethylene chain having an average condensation degree of 4 to 20, preferably 4 to 15, in the molecule, or a fatty acid having an alkyl group having 8 to 18 carbon atoms, preferably 10 to 18 carbon atoms, or an alkyl group composition derived from coconut oil or beef tallow, or a sodium or potassium salt thereof.

[0030] Further, examples of anionic surfactants other than those mentioned above include linear alkylbenzenesulfonic acid or its salts; α-olefin sulfonates; linear or branched alkyl sulfate ester salts; alkyl ether sulfate ester salts; alkenyl ether sulfate ester salts; alkanesulfonates; α-sulfofatty acid ester salts; carboxylic acid type anionic surfactants such as fatty acid salts, alkyl ether carboxylate salts, polyoxyalkylene ether carboxylate salts, alkylamide ether carboxylate salts, alkenylamide ether carboxylate salts, and acylaminocarboxylate salts; and phosphate ester type anionic surfactants such as alkyl phosphate ester salts, polyoxyalkylene alkyl phosphate ester salts, polyoxyalkylene alkylphenyl phosphate ester salts, and glycerin fatty acid ester monophosphate ester salts.

[0031] Examples of salt forms include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium; and alkanolamine salts such as monoethanolamine salts and diethanolamine salts. Among the above, in one embodiment, the linear alkylbenzenesulfonic acid or salt thereof is preferably one in which the linear alkyl group has 8 to 16 carbon atoms, and particularly preferably one in which the linear alkyl group has 10 to 14 carbon atoms. In one embodiment, the α-olefin sulfonate preferably has 10 to 20 carbon atoms. In one embodiment, alkyl sulfates having 10 to 20 carbon atoms are preferred. In one embodiment, the alkyl ether sulfate or alkenyl ether sulfate is preferably a polyoxyethylene alkyl ether sulfate or polyoxyethylene alkenyl ether sulfate having a linear or branched alkyl group or alkenyl group having 10 to 20 carbon atoms and having an average of 1 to 10 moles of ethylene oxide added thereto. In one embodiment, the alkanesulfonate is a secondary alkanesulfonate having an alkyl group preferably having 10 to 20 carbon atoms, more preferably having 14 to 17 carbon atoms. In one embodiment, the α-sulfofatty acid ester salt is preferably one having 10 to 20 carbon atoms. In one embodiment, the fatty acid salt preferably has 10 to 20 carbon atoms. In one embodiment, among the above, at least one selected from the group consisting of linear alkylbenzenesulfonic acid or a salt thereof, an alkane sulfonate, an α-olefin sulfonate, an alkyl ether sulfate, and an alkenyl ether sulfate is preferred. Also, in one embodiment, it is preferred to use linear alkylbenzenesulfonic acid or a salt thereof, and an alkane sulfonate in combination.

[0032] Examples of cationic surfactants other than those mentioned above include alkyltrimethylammonium salts such as tetradecyltrimethylammonium chloride, dialkyldimethylammonium salts, benzalkonium salts, benzethonium salts, pyridinium salts, imidazolium salts, etc. Counter ions of these salts include halogen ions, etc.

[0033] Examples of amphoteric surfactants other than those mentioned above include alkylcarboxybetaine, alkylsulfobetaine, alkylhydroxysulfobetaine, alkylamidobetaine, and imidazolinium betaine.

[0034] The amount of formalin neutralizer (e.g., one containing one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, one containing urea and an acid, etc.) added to the formalin after sample immersion is not particularly limited as long as it allows extraction of the desired amount of nucleic acid. The amount of hydrogen peroxide and peroxides that generate hydrogen peroxide in water added to the formalin after sample immersion is typically such that the volume ratio of hydrogen peroxide (10% to 34% (w / w)) to the formalin after sample immersion (10% (w / w) to 40% (w / w)) is 10:1 to 1:10, more typically 5:1 to 1:1, and even more typically 4:1 to 3:1.

[0035] Furthermore, the amount of urea to be added to the formalin after the sample has been immersed is typically 1.0% to 20% (v / v), more typically 5.0% to 10% (w / v), relative to the formalin after the sample has been immersed (10% (w / w) to 40% (w / w)).

[0036] The amount of acid added to the formalin after immersion of the sample is typically 0.1% to 20% (v / v), more typically 1.0% to 5.0% (w / v), relative to the formalin after immersion of the sample (10% (w / w) to 40% (w / w)). For example, when citric acid is used, the concentration after addition is 1.0% (w / v).

[0037] The amount of calcium chloride to be added to the formalin after the sample has been immersed is typically 0.1 to 20% (v / v), more typically 1.0% to 5.0% (w / v), relative to the formalin after the sample has been immersed (10% (w / w) to 40% (w / w)).

[0038] The amount of surfactant added to the formalin after sample immersion is not particularly limited, as long as it can achieve the desired nucleic acid extraction. Typically, the surfactant is added to the formalin (10% (w / w) to 40% (w / w)) after sample immersion at a concentration of 0.1% to 10% (w / v), more typically 1.0% to 5.0% (w / v) after addition. For example, when sodium dodecyl sulfate (SDS) is used as the surfactant, the concentration after addition is 3.3% (w / v). The surfactant may be added to the formalin after sample immersion at any time and in any form, as long as it is before performing step (2) of the nucleic acid extraction method of the present invention.

[0039] In step (1) of the present invention, one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water may be added to the formalin after the sample has been immersed, followed by stirring and mixing using a vortex mixer or the like. Alternatively, step (2) may be performed after removing the formalin and the like from the mixture. The method for removing the formalin and the like is not particularly limited as long as it allows the desired quality and quantity of nucleic acid to be extracted in step (2). For example, the mixture may be separated into a supernatant and a precipitate by centrifugation or the like, the supernatant removed, and distilled water (Milli-Q water) added to the precipitate. The mixture may then be stirred and mixed, and further separated into a supernatant and a precipitate by centrifugation. The precipitate obtained by removing the supernatant may be used in step (2).

[0040] The extraction of nucleic acids in step (2) of the present invention can be carried out by a method known per se (e.g., Agnieszka K Sarnecka et al., Contemp Oncol (Pozn). 2019;23(1):52-58. doi: 10.5114 / wo.2019.83875. Epub 2019 Mar 26., Samantha J McDonough et al., PLoS One. 2019 Apr 11;14(4):e0211400. doi: 10.1371 / journal.pone.0211400. eCollection 2019., etc.). Specific examples include DNA extraction methods using phenol / chloroform, ethanol, sodium hydroxide, CTAB, etc. Other examples include RNA extraction methods using the AGPC (Acid Guanidium-Phenol-Chloroform) method. Nucleic acids can be extracted using commercially available DNA or RNA extraction kits (e.g., QIAamp DNA FFPE Tissue Kit (QIAGEN), NucleoSpin (registered trademark) DNA FFPE XS (Takara Bio), Maxwell (registered trademark) FFPE Plus DNA Kit (Promega), WaxFree TM Paraffin Sample DNA Extraction Kit (TrimGen Corporation), RELIAPREP TM FFPE gDNA Miniprep System (Promega), NucleoSpin® RNA (Takara Bio), ReliaPrep TM This may also be performed using FFPE Total RNA Miniprep System (Promega), Maxwell (registered trademark) RSC RNA FFPE Kit (Promega), etc.

[0041] In one embodiment, fragmented nucleic acids (e.g., DNA of 500 bp or less) and other contaminants may be removed from the solution containing the nucleic acids extracted in step (2) of the present invention by, for example, a method using a silica-based solid phase material (e.g., silica membrane, silica monolith, etc.), a method using an ion exchange carrier or a gel filtration carrier, a method using magnetic beads, or the like.

[0042] The silica-based solid phase material is preferably a silica monolith or a solid phase material having a porous silica membrane. From the viewpoint of facilitating the removal of fragmented nucleic acids and other contaminants, or the washing and elution of nucleic acids, the solid phase material is preferably in the form of a spin column, a column-equipped pipette tube, or a column cartridge that can be attached to a centrifuge tube or pipette tube. Furthermore, from the viewpoints of contamination prevention and operational efficiency, a column cartridge that can be attached to a spin column or centrifuge tube is more preferable. Furthermore, considering that the amount of nucleic acid recovered is relatively small, the solid phase material is preferably in a form that can purify small amounts of nucleic acid, such as a miniprep column. Examples of the solid phase material include commercially available columns for nucleic acid purification and concentration, such as NucleoSpin® gDNA Clean-up XS (Machley-Nagel), Wizard® SV Gel and PCR Clean-Up System (Promega), and minElute PCR Purification Kit (QIAGEN).

[0043] A washing solution used to remove or wash fragmented nucleic acids and other contaminants from a silica-based solid phase material typically includes a solution (e.g., an aqueous solution) containing at least one water-soluble organic solvent and a water-soluble salt. The water-soluble organic solvent contained in the washing solution typically includes a water-soluble alcohol. Examples of water-soluble alcohols include methanol, ethanol, propanol, and butanol. Propanol may be either isopropanol or n-propanol, and butanol may be either linear or branched. These water-soluble alcohols may also be used in combination. Examples of water-soluble salts contained in the washing solution include halide salts, more specifically, chloride salts. Examples of water-soluble salts include monovalent or divalent cation salts, specifically, alkali metal salts, alkaline earth metal salts, and more specifically, sodium salts, potassium salts, and the like.

[0044] Desorption of nucleic acids from the solid-phase material can typically be achieved by passing an elution solution through the solid-phase material to elute the nucleic acids. Examples of the elution solution used for eluting nucleic acids include distilled water and Tris-EDTA (TE) buffer. The washing and elution solutions provided with commercially available nucleic acid purification or concentration columns may be used according to the manufacturer's instructions. Washing and elution of the solid-phase material can be achieved by known procedures. Typically, for example, when the solid-phase material is attached to a spin column, a solution containing nucleic acids is passed through the column, a washing solution is added to the column to which the nucleic acids have been adsorbed, and the column is centrifuged to wash away fragmented nucleic acids and other contaminants along with the washing solution. Subsequently, an elution solution is added to the column, and the column is centrifuged to elute the nucleic acids from the column.

[0045] The quality of the nucleic acid extracted in step (2) of the present invention may be confirmed. As an indicator of DNA quality, a known indicator may be used, such as a ΔCt value or a DIN (DNA Integrity Number) value. A DIN value of 2.0 or higher is typically preferred. As an indicator of RNA quality, a known indicator may be used, such as a RIN (RNA Integrity Number) value.

[0046] 2. Nucleic Acid Amplification Method of the Present Invention The present invention provides a method for amplifying nucleic acids (hereinafter, sometimes referred to as the "nucleic acid amplification method of the present invention"), which comprises the step of amplifying a target nucleic acid in a solution containing nucleic acids extracted by the above-mentioned nucleic acid extraction method of the present invention. The nucleic acid may be DNA or RNA.

[0047] The method for amplifying a target nucleic acid in the nucleic acid amplification method of the present invention is not particularly limited as long as it amplifies the target nucleic acid, and can be performed using methods such as polymerase chain reaction (PCR), ligase chain reaction, MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), MDA (Multiple Displacement Amplification), strand displacement amplification (SDA), rolling circle amplification (RCA), cross priming amplification, loop-mediated isothermal amplification (LAMP), ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acids), T7 RNA amplification, and transcription-mediated amplification (TMA). These methods are known to those skilled in the art and can be performed using conventional techniques. In one embodiment, the target DNA is amplified by PCR in the nucleic acid amplification method of the present invention.

[0048] In the nucleic acid amplification method of the present invention, the nucleic acid (e.g., DNA (gene), RNA) to be amplified is not particularly limited, and examples include genes that are the subject of testing in genetic testing for specific diseases. More specifically, examples include immune-related gene rearrangement (rearrangement) tests for malignant lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, etc., and examples of genes that are the subject of such testing include the IgH (immunoglobulin heavy chain) gene, IgK (immunoglobulin light chain kappa) gene, IgL (immunoglobulin light chain lambda) gene, TRB (T cell receptor beta chain) gene, TRG (T cell receptor gamma chain) gene, TRD (T cell receptor delta chain) gene, etc. In addition, for example, breast cancer HER2 protein (IHC) 4B5 testing, epidermal growth factor receptor (EGFR) gene mutation testing in lung cancer patients, detection of Mycobacterium tuberculosis DNA, testing using the AmoyDx (registered trademark) lung cancer multi-gene PCR panel (Riken Genesis Co., Ltd.), HRMA (high resolution melting curve analysis) testing, testing using the my Choice (registered trademark) diagnostic system (Myriad Genetics, Inc.), KRAS G12C mutation analysis, EZH2 gene mutation analysis, malignant melanoma BRAF mutation analysis, MSI testing, MEBGEN RASKET TM Examples of such genes include those that are the subject of testing using the -B kit (Medical and Biological Laboratories Co., Ltd.).

[0049] The quality of nucleic acids amplified by the nucleic acid amplification method of the present invention may be confirmed. As an index of DNA quality, a known index may be used, such as a ΔCt value or a DIN (DNA Integrity Number) value. A DIN value of 2.0 or higher is typically preferred. As an index of RNA quality, a known index may be used, such as a RIN (RNA Integrity Number) value.

[0050] For matters other than those mentioned above regarding the nucleic acid amplification method of the present invention, the contents described above in "1. Nucleic acid extraction method of the present invention" are all applicable.

[0051] 3. Sequencing Method of the Present Invention The present invention provides a sequencing method comprising a step of analyzing the sequence of nucleic acid amplified by the nucleic acid amplification method of the present invention. The sequencing method may be performed by conventional techniques such as the Sanger method, or by massively parallel sequencing. Massively parallel sequencing includes, for example, next-generation sequencing (NGS).

[0052] Next-generation sequencing is a method for obtaining sequence information using a next-generation sequencer. Compared to the Sanger sequencing method, next-generation sequencing allows for a much larger number of sequencing reactions to be performed simultaneously (see, for example, Rick Kamps et al., Int. J. Mol. Sci., 2017, 18(2), p. 308 and Int. Neurourol. J., 2016, 20(Suppl. 2), S76-83). Various systems for next-generation sequencing are available, including, but not limited to, the Roche Genome Sequencer (GS) FLX System, Illumina HiSeq or Genome Analyzer (GA), Life Technologies Support Oligonucleotide Ligation Detection (SOLiD) System, Polonator G.007 System, and Helicos BioSciences HeliScope Gene Sequencing System.

[0053] The general steps of next-generation sequencing are as follows, but are not limited to these. Next-generation sequencing begins with sample preparation. In this step, the DNA to be analyzed is enzymatically or mechanically fragmented to match the read length of the next-generation sequencer. Subsequently, in many cases, an adapter sequence required for the subsequent sequencing step is added. Furthermore, to analyze a specific gene region, the specific gene region may be enriched by PCR or the like, or a region having a specific sequence may be concentrated using a probe or the like. Enrichment of a gene region can be performed, for example, by a 4- to 12-cycle amplification step, and enrichment using a probe can be performed by utilizing a label (e.g., biotin) attached to the probe.

[0054] Next, sequencing is performed. The details of this process vary depending on the type of next-generation sequencer, but typically, the DNA is linked to the substrate via an adapter sequence, and the sequencing reaction is performed using the adapter sequence as a priming site. For details of the sequencing reaction, see, for example, Rick Kamps et al., Int. J. Mol. Sci., 2017, 18(2), p. 308 and Int. Neurourol. J., 2016, 20(Suppl. 2), S76-83. Finally, data output is performed. In this process, a collection of sequence information (reads) obtained by the sequencing reaction is obtained.

[0055] For matters other than those mentioned above regarding the sequencing method of the present invention, the contents described above in "1. Nucleic acid extraction method of the present invention" and "2. Nucleic acid amplification method of the present invention" are all applicable.

[0056] 4. Nucleic acid extraction kit of the present invention The present invention provides a kit for extracting nucleic acids from formalin after immersion of a sample, comprising one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid.

[0057] Peroxides that generate hydrogen peroxide in water and can be included in the nucleic acid extraction kit of the present invention include, but are not limited to, inorganic peroxides and hydrogen peroxide adducts. Preferred are percarbonate, tripolyphosphate-hydrogen peroxide adduct, pyrophosphate-hydrogen peroxide adduct, urea-hydrogen peroxide adduct, sulfate-hydrogen peroxide adduct, perborate, persilicate, and peroxide salts. More preferred are sodium percarbonate, sodium tripolyphosphate-hydrogen peroxide adduct, sodium pyrophosphate-hydrogen peroxide adduct, urea-hydrogen peroxide adduct, 4NaSO·2HO, sodium perborate monohydrate, sodium perborate tetrahydrate, sodium persilicate, sodium peroxide, and calcium peroxide. Even more preferred are sodium percarbonate and / or sodium perborate. In one embodiment of the present invention, the one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water include at least hydrogen peroxide.

[0058] The acid that can be contained in the nucleic acid extraction kit of the present invention is not particularly limited as long as it is a substance that generates hydrogen ions when dissolved in water, but is preferably an acid with a pH of 6.0 or less, more preferably an acid with a pH of 3.0 or less. Specific examples include citric acid, hydrochloric acid, nitric acid, acetic acid, phosphoric acid, and oxalic acid, with citric acid being preferred.

[0059] When the nucleic acid extraction kit of the present invention contains urea and an acid, it may further contain calcium chloride.

[0060] The nucleic acid extraction kit of the present invention may further contain a surfactant. The surfactant may be an ionic surfactant, a nonionic surfactant, or a combination thereof, but it is preferable that at least one of the surfactants is an ionic surfactant. The ionic surfactant may be a cationic surfactant, an anionic surfactant, an amphoteric surfactant, or a combination thereof, but it is preferable that at least one of the ionic surfactants is an anionic surfactant. It is more preferable that at least one of the ionic surfactants is an anionic surfactant and at least one of the ionic surfactants is a cationic surfactant.

[0061] For matters other than those described above regarding the nucleic acid extraction kit of the present invention, the contents described above in "1. Nucleic acid extraction method of the present invention," "2. Nucleic acid amplification method of the present invention," and "3. Sequencing method of the present invention" are all applicable.

[0062] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way. [Example]

[0063] Example 1: Nucleic acid extraction from formalin solution after sample immersion (hydrogen peroxide system) Specimen: Colon cancer immersed in formalin solution (stored for 1 week) (0.75 ml) Substances to be added to the sample (0.25 ml each) (i) Hydrogen peroxide 30% (w / w) (Fujifilm Wako Pure Chemical Industries, Ltd.) (ii) Wide Haiter (registered trademark) (Kao Corporation) (iii) Bright (registered trademark) (Lion Corporation) Wide Haiter (registered trademark) contains hydrogen peroxide, polyoxyethylene alkyl ether sulfate ester salt, and quaternary ammonium salt. Brite® contains hydrogen peroxide and alkyl ether sulfates.

[0064] Nucleic acid extraction method (control) The formalin solution after immersion (fixation) of the colon cancer cells was centrifuged at 11,000 × g for 5 minutes, and the supernatant was removed. 1,000 μl of distilled water (Milli-Q water) was added and vortexed. The mixture was then centrifuged at 11,000 × g for 5 minutes, and the supernatant was removed. After removing the supernatant, 1,000 μl of distilled water (Milli-Q water) was added and vortexed. The mixture was then centrifuged at 11,000 × g for 5 minutes, and the supernatant was removed. Nucleic acid extraction was performed using commercially available kits (DNA: RELIAPREP™ FFPE gDNA Miniprep System (Promega), RNA: ReliaPrep™ FFPE Total RNA Miniprep System (Promega)) according to the manufacturer's instructions. For example, the protocol for the commercially available kit (DNA: RELIAPREP™ FFPE gDNA Miniprep System (Promega)) is as follows: (Contains additives) Each of the above substances was added to the formalin solution after the colon cancer immersion (fixation) and vortexed. After leaving it to stand for 15 minutes, it was centrifuged at 11,000 x g for 5 minutes and the supernatant was removed. 1,000 μl of distilled water (Milli-Q water) was added and vortexed. Then, it was centrifuged at 11,000 x g for 5 minutes and the supernatant was removed. Nucleic acid was extracted using a commercially available kit (DNA:RELIAPREP TM FFPE gDNA Miniprep System (Promega), RNA:ReliaPrep TM The FFPE Total RNA Miniprep System (Promega) was used according to the manufacturer's instructions. TM The protocol for the FFPE gDNA Miniprep System (Promega) is as follows.

[0065] Sample dissolution Add 200 μl of Lysis Buffer Add 20 μl of Proteinase K and vortex. Lightly spin down Incubate at 56℃ (heat block) for 1 hour Incubate at 80℃ (heat block) for 4 hours After incubation, return to room temperature and spin down RNase treatment Add 10 μl of RNase A solution and pipette Incubate at room temperature (20-25°C) for 5 minutes (stand still). ·DNA binding Add 220 μl of BL Buffer Add 240 μl of ethanol (95-100%) and vortex. Centrifuge at 10,000 x g for 15 seconds (spin down) Place the ReliaPrep FFPE Binding Column in the Collection Tube. Transfer the sample (entire volume) to the column and centrifuge at 10,000 x g for 30 seconds. Discard the flow-through collected in the Collection Tube and place the column back in the Collection Tube. Column washing Add 500 μl of Wash Solution to the column and centrifuge at 10,000 × g for 30 seconds. Discard the flow-through, add 500 μl of Wash Solution to the column again, and centrifuge at 10,000 × g for 30 seconds. Discard the flow-through, open the column, and centrifuge at 16,000 x g for 3 minutes to dry the column. DNA elution Transfer the column to a new 1.5 ml centrifuge tube and add 20 μl of Elution Buffer. Centrifuge at 16,000 x g for 1 minute (DNA elution) Store at -20℃ (refrigerator)

[0066] The results are shown in Tables 1 and 2.

[0067] [Table 1]

[0068] [Table 2]

[0069] As can be seen from Table 1, compared to the control, the DNA concentration was 26.1 times higher for 30% hydrogen peroxide, 59.2 times higher for Wide Haiter (registered trademark), and 15.8 times higher for Bright (registered trademark). Therefore, it was found that adding a solution containing at least hydrogen peroxide to formalin after sample immersion (fixation) enabled the extraction of significantly larger amounts of nucleic acids than the control. Furthermore, based on the A260 / 280, an index of DNA purity, it is believed that the addition of 30% hydrogen peroxide, Wide Haiter®, or Bright® resulted in higher purity of extracted DNA than the control.

[0070] Regarding RNA, although the amount of extraction was lower than that of the control, the A260 / 280, an index of RNA purity, indicates that the addition of 30% hydrogen peroxide, Wide Haiter (registered trademark), and Bright (registered trademark) all resulted in higher purity of RNA after extraction compared to the control.

[0071] Example 2: Examination of the DIN value of extracted nucleic acids The DIN value of the nucleic acid extracted in Example 1 was measured using an Agilent Tape Station. The results are shown in Table 3 and Figure 1.

[0072] [Table 3]

[0073] A DIN value of 2.0 or higher is typically preferred, and the results in Table 3 and Figure 1 indicate that, compared to the control, the addition of 30% hydrogen peroxide, Wide Haiter (registered trademark), and Bright (registered trademark) all result in the extraction of nucleic acids of higher quality.

[0074] Example 3: Comparison of formalin solution and FFPE samples after sample immersion The nucleic acid (DNA) extracted from the formalin solution after immersion (fixation) of a colon cancer tissue sample was compared with the nucleic acid (DNA) extracted from the FFPE sample prepared from the immersion (fixed) sample. Extraction of nucleic acids from the formalin solution after immersion (fixation) of colon cancer tissue samples and extraction of nucleic acids (DNA) from FFPE samples prepared from the immersion (fixed) samples were performed in the same manner as in Example 1, and Wide Haiter (registered trademark) was used as the substance added to the specimens in both cases. The results are shown in Table 4.

[0075] [Table 4]

[0076] As can be seen from Table 4, the extraction method of the present invention made it possible to extract nucleic acids (DNA) at a concentration and purity equivalent to that of FFPE samples prepared from the soaked (fixed) sample, even when using the formalin solution after the sample had been soaked (fixed).

[0077] Example 4: Examination of nucleic acid extraction from samples derived from other organs Specimen: Formalin solution after immersion (fixation) of lung squamous cell carcinoma Nucleic acids (DNA) were extracted from the above specimens in the same manner as in Example 1. The results are shown in Table 5.

[0078] [Table 5]

[0079] As shown in Table 5, the extraction method of the present invention can be applied to tissues (organs) other than the intestine, and is therefore considered to be applicable to a wide range of tissues in the body.

[0080] Example 5: PCR amplification of DNA extracted with hydrogen peroxide The housekeeping gene (GAPDH) was amplified by PCR using the DNA extracted in Example 1 (Fig. 2, left). As shown in Fig. 2, left, the DNA extracted using the hydrogen peroxide system was also amplified by PCR. The results of Examples 1 to 5 above show that when nucleic acids (especially DNA) were extracted using a hydrogen peroxide system, 16 to 61 times the amount of DNA could be extracted compared to the control, and the DIN value was high, making it possible to obtain DNA that could be amplified by PCR.

[0081] Example 6: Nucleic acid extraction from formalin solution after sample immersion (urea system) Specimen: Colon cancer immersed in formalin solution (stored for 1 week) (1.0 ml) Neutralizer to be added to the sample (0.25 ml each) (i) Formalex Clear (Pharma Co., Ltd.) (ii) Neutralizer (urea-based) (composition: urea (25% (w / v)) (Fujifilm Wako Pure Chemical Industries, Ltd.), citric acid (5% (w / v)) (Nacalai Tesque, Inc.), calcium chloride (5% (w / v)) (Fujifilm Wako Pure Chemical Industries, Ltd.), and water)

[0082] Using the above, DNA and RNA were extracted in the same manner as the nucleic acid extraction method in Example 1. The results are shown in Tables 6 and 7.

[0083] [Table 6]

[0084] [Table 7]

[0085] As shown in Table 6, the amount of DNA extracted was significantly increased when a formalin neutralizer was used compared to the control. Furthermore, the amount of DNA extracted was significantly increased when a neutralizer (urea-based) was used compared to Formalex Clear, a commercially available formalin neutralizer.

[0086] As shown in Table 7, when the formalin neutralizer was used, the amount of RNA extracted was significantly increased compared to the control. Furthermore, when the neutralizer (urea-based) was used, the amount of RNA extracted was significantly increased compared to Formalex Clear, a commercially available formalin neutralizer.

[0087] Example 7: Nucleic acid extraction from formalin solution (urea-based) after immersion in long-term stored samples (connective tissue) RNA was extracted from the formalin solution after immersion of the samples stored for a long period of time (storage period: 4 years and 6 months) in the same manner as in Example 5. The results are shown in Table 8.

[0088] [Table 8]

[0089] As shown in Table 8, when a neutralizing agent (urea-based) was used, the amount of extracted RNA increased significantly (about 15-fold) compared to the control.

[0090] Example 8: PCR amplification of RNA extracted using a neutralizing agent (urea-based) The housekeeping gene (RPS18) was amplified by PCR using the RNA extracted in Example 6 (Figure 2, right). As shown in Figure 2, right, the RNA extracted using a neutralizing agent (urea-based) was also amplified by PCR.

[0091] As can be seen from the results of Examples 1 to 8 above, it was found that the present invention makes it possible to obtain sufficient amounts of nucleic acids of a quality suitable for genetic testing in pathological diagnosis from the formalin used to prepare conventional samples such as FFPE samples, without relying on these samples. [Industrial Applicability]

[0092] The present invention is useful because it enables the extraction of sufficient amounts of nucleic acids of a quality suitable for genetic testing in pathological diagnosis from the formalin used to prepare such samples, without relying on conventional samples such as FFPE samples. Therefore, it is possible to obtain sufficient amounts of nucleic acids of a quality suitable for genetic testing in pathological diagnosis, even from small specimens such as biopsy specimens. Furthermore, it is useful because it enables genetic testing using PCR, next-generation sequencing (NGS), and the like, for samples that were previously untestable, such as long-term stored FFPE samples, as long as the formalin used to prepare the sample is preserved. Furthermore, even for samples such as biological tissues and cells preserved immersed in a solution containing formalin (e.g., liquid-immersion specimens (so-called formalin-preserved specimens), specimens for liquid-based cytology, etc.), the present invention enables the extraction of nucleic acids from the formalin in which the sample is preserved, and ultimately allows the analysis of the nucleic acid sequence. This is useful because it enables various searches (e.g., the search and identification of new diagnostic markers and disease-causing genes) in the fields of basic medicine, forensic medicine, and biology.

Claims

1. 1. A method for extracting nucleic acids, comprising: (1) adding a formalin neutralizer to the formalin after the sample has been immersed; and (2) A step of extracting nucleic acids from the mixture obtained in step (1). A method comprising:

2. 10. The method of claim 1, wherein the formalin neutralizer comprises one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid.

3. 3. The method of claim 1 or 2, wherein the formalin neutralizer comprises one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water.

4. 4. The method of claim 2 or 3, wherein the one or more compounds is hydrogen peroxide.

5. 3. The method of claim 1 or 2, wherein the formalin neutralizer comprises urea and an acid.

6. 6. The method of claim 2 or 5, wherein the acid is citric acid.

7. 7. The method of claim 5 or 6, wherein the formalin neutralizer further comprises calcium chloride.

8. The method according to any one of claims 1 to 7, wherein a surfactant is further added in step (1).

9. The method of claim 8 , wherein at least one of the surfactants is an ionic surfactant.

10. 10. The method of claim 9, wherein at least one of the ionic surfactants is an anionic surfactant.

11. The method of claim 10, wherein the anionic surfactant is a polyoxyethylene alkyl ether sulfate or an alkyl ether sulfate.

12. 12. The method of claim 10 or 11, wherein at least one of the ionic surfactants is a cationic surfactant.

13. 13. The method of claim 12, wherein the cationic surfactant is a quaternary ammonium salt.

14. A method for amplifying a nucleic acid, comprising the step of amplifying a target nucleic acid in a solution containing the nucleic acid extracted by the method according to any one of claims 1 to 13.

15. A step of analyzing the sequence of the nucleic acid amplified by the method of claim 14. A sequencing method comprising:

16. A kit for extracting nucleic acids from formalin after soaking a sample, comprising one or more compounds selected from the group consisting of hydrogen peroxide and peroxides that generate hydrogen peroxide in water, or urea and an acid.

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