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WO2006064739A1 - Procédé consistant à traiter un échantillon biologique pour effectuer une amplification d'un acide nucléique - Google Patents

Procédé consistant à traiter un échantillon biologique pour effectuer une amplification d'un acide nucléique Download PDF

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Publication number
WO2006064739A1
WO2006064739A1 PCT/JP2005/022676 JP2005022676W WO2006064739A1 WO 2006064739 A1 WO2006064739 A1 WO 2006064739A1 JP 2005022676 W JP2005022676 W JP 2005022676W WO 2006064739 A1 WO2006064739 A1 WO 2006064739A1
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Prior art keywords
nucleic acid
biological sample
sample
solution
gene
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PCT/JP2005/022676
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English (en)
Japanese (ja)
Inventor
Takeshi Nagasaka
Nagahide Matsubara
Noriaki Tanaka
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National University Corporation Okayama University
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Application filed by National University Corporation Okayama University filed Critical National University Corporation Okayama University
Publication of WO2006064739A1 publication Critical patent/WO2006064739A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention provides a nucleic acid derived from a target gene without separating and purifying the nucleic acid from the sample by a complicated process as in the prior art when amplifying a nucleic acid that may be contained in a biological sample. It is related with the processing method for extracting easily from a sample. Furthermore, the present invention relates to a method for amplifying a nucleic acid using the treatment method.
  • Intestinal flora and pathogenic bacteria, viruses, or other gene fragments present in mammalian digestive tract mucosal cells, malignant neoplastic cells, neoplastic cells, and secretions It is very useful for understanding the health status of individuals and for early detection of malignant neoplasms.
  • the current technology for separating and purifying genes in stool is cumbersome and laborious.
  • Nucleic acids isolated from stool force are generally obtained by dissolving stool in an alkaline lysate, centrifuging, and destroying alcohol.
  • nucleic acids from microorganisms are isolated by washing the sample with a phosphate buffer (PBS) or the like, crushing the cells by the salt benzil method, denaturing the protein, and precipitating with alcohol. It was broken. However, it has been pointed out that this method hardly recovers nucleic acid from some bacteria such as Gram-positive cocci.
  • PBS phosphate buffer
  • Non-patent Document 2 a method of crushing bacterial cells by collecting beads with a diameter of about 0.1 mm and shaking vigorously in the presence of phenol. According to this method, nucleic acids can be efficiently isolated from a wide variety of species.
  • MagExtractor manufactured by Toyobo
  • QIAamp Stool DNA Isolation Kit manufactured by QIAGEN
  • the former has a problem that the yield of nucleic acid is poor because the nucleic acid adsorbed on the beads is recovered with magnetic beads after the cells are crushed with beads.
  • the latter is a method in which cell membrane proteins are denatured by heating and nucleic acids are isolated, but this method also has a problem in yield.
  • Patent Document 1 There is also a method for extracting nucleic acids by using beads and gel filtration (Patent Document 1). Force The complexity of the work process and the direct workability cannot be avoided.
  • Patent Document 2 a method for extracting fecal force and DNA using a piece of dry filter paper.
  • the filter paper described in this document contains a stool sample that is not directly related to DNA extraction, and can be easily collected for handling such as transportation and storage by enclosing it in a plastic bag. Is. In order to extract DNA from stool samples contained in filter paper, the following steps (1) to (4) are required.
  • Non-Patent Literature 1 Sidransky D. et. Al. 1992.Identincation of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science. Apr. 3; 256 (5053): 102
  • Non-Patent Document 2 Wilson, K. ⁇ . And R. ⁇ . Blithington. 1996. Human colonic biota studied by ribosomal DNA sequence analysis. Appl. Environ. Micro Diol. 62: 2273-2278 — Publication No. 306175
  • Patent Document 2 JP 2001-221721 A
  • the problem to be solved by the present invention is to separate and purify nucleic acid such as sample force DNA by a complicated process when amplifying a nucleic acid derived from a target gene that may be contained in a biological sample. It is to establish and provide a treatment method for extracting nucleic acids easily.
  • the present inventors have conducted extensive research, and as a result, dissolved a biological sample in a lysis solution, and contained the lysis solution in a carrier for development, thereby inhibiting the reaction. And the nucleic acid contained in the sample is separated and adsorbed on the development carrier, the development carrier can be directly used for the nucleic acid amplification reaction, and it may be contained in a biological sample.
  • the method of the present invention was completed by successfully amplifying a nucleic acid derived from a target gene simply and efficiently.
  • the present invention comprises the following.
  • a method of processing a biological sample to amplify nucleic acid derived from a gene that may be contained in the biological sample including the following steps:
  • a method for analyzing a gene present in a biological sample wherein a target gene is identified from the nucleic acid amplification product obtained by the amplification method described in item 4 above.
  • a nucleic acid derived from a target gene can be easily extracted from a biological sample without being separated and purified by a complicated process as in the past, and a nucleic acid amplification reaction can be performed. It becomes.
  • FIG. 1 is an explanatory diagram for preparing a stool solution by adding stool to a sample dissolution buffer.
  • FIG. 2 is a view showing a state where a fecal lysate is centrifuged and separated into a supernatant and a precipitate.
  • FIG. 3 is a view showing a state in which a developing carrier is immersed in a supernatant portion of a stool solution, and the developing carrier including the supernatant portion is dried.
  • FIG. 4 is a view showing a state in which a nucleic acid is extracted by immersing a development carrier including a supernatant portion in a PCR buffer solution.
  • FIG. 5 is a diagram showing an example of a sample solution when stool is used as a sample. (Example 1)
  • FIG. 6 is a diagram showing an example of a sample solution adsorbed and dried using a developing carrier.
  • FIG. 7 shows the results of amplification of the KRAS gene.
  • FIG. 8 shows the results of BRAF gene amplification.
  • FIG. 9 shows the results of amplification of ThrA gene (derived from E. coli). (Example 2)
  • FIG. 10 shows the results of simultaneous amplification of KRAS gene and BRAF gene. (Example 2) Explanation of symbols
  • biological sample refers to tissues, blood, serum, feces, urine, semen, sputum, saliva, nasal discharge, cerebrospinal fluid, tears, and other body fluids collected from mammals. Particularly preferred is feces. Feces are excrement obtained from mammals. Feces include intestinal mucosa cells, blood cells, intestinal cell flora, pathogenic microorganisms (eg, bacteria and viruses), and intestinal neoplasia (including colon cancer and polyps).
  • genes that may be contained in a biological sample refers to various disease-related genes and microorganism-related genes that may be contained in a biological sample. Examples include genes causing genetic diseases, genes derived from colon cancer tissue, genes derived from resident bacteria (microbes, viruses, etc.), and the like.
  • a “gene-derived nucleic acid” is almost synonymous with a gene, but a DNA fragment or an RNA fragment containing a desired position necessary for analysis is not necessary, rather than a complete gene sequence. .
  • the biological sample processing method of the present invention is based on a method including at least the following steps.
  • Step (a) lysing the biological sample with a lysis buffer
  • Step (b) A step of including a sample solution in which a biological sample is dissolved in a developing carrier;
  • Step (c) A step of bringing the obtained carrier for development directly into contact with a nucleic acid amplification reaction solution containing a target nucleic acid-specific primer.
  • the biological sample in order to dissolve the solid matter contained in the sample, the biological sample is first dissolved.
  • lysis solution a known solution can be used.
  • Tris-EDTA buffer or 8.4% (1M) sodium bicarbonate solution can be used as a stool sample solution, and a solution having a pH value of about 9 containing Tris-EDTA buffer is preferable.
  • the amount of lysate added to the biological sample can be determined in relation to the amount of sample. For example, a solution of about 0.1 to 100, preferably 1 to 30 and more preferably about 3 to 7 liters per lmg of sample can be used. The obtained solution is called a sample solution.
  • the sample solution is preferably further heated as desired.
  • the heating is, for example, 60 to 95. C, can be performed for 10 minutes to 10 hours.
  • the sample solution that has been or has not been subjected to the heat treatment can be centrifuged, and the supernatant can be collected and subjected to the next step. Centrifugation may be appropriately performed at a rotational speed of about 3,000 to 8,000 rpm, preferably 4,000 to 6, OOO rpm, more preferably about 5,000 rpm for 1 to 5 minutes.
  • the sample solution obtained by the above step (a) or the supernatant of the sample solution obtained by centrifugation is contained in the developing carrier.
  • the “developing carrier” is not particularly limited as long as it can separate a component necessary for analysis and an unnecessary component by contacting a sample solution or a supernatant.
  • the developing carrier having such a function is desirably one having a retention particle diameter of 1 to 7 m.
  • the retained particle size here refers to the value obtained from the leaked particle size when barium sulfate or the like specified in JIS P 3801 [filter paper (for chemical analysis)] is naturally filtered.
  • An example of such a developing carrier is filter paper (retained particle diameter of 1 to 7 m).
  • the filter paper is not limited to paper, and examples thereof include liquid separation filter paper, chromatographic filter paper, glass fiber filter paper, composite fiber filter paper, silica fiber filter paper, polyflon filter, and cylindrical filter paper. Further, a filter paper having anion exchange ability may be used. Examples of commercially available products include ADVANTEC standard filter papers No. 2 and No. 26, and high purity filter paper No. 5B.
  • the size of the developing carrier depends on the amount of the sample solution. In general, a strip of 10 to 200 mm in length and 1 to 10 mm in width is preferably used.
  • the thickness of the development carrier is generally 0.2 to 0.8 mm fc.
  • step (a) "Including the sample solution in the developing carrier” means immersing 2 to 10 mm of the tip of the developing carrier in the sample solution or supernatant obtained in the above step (a) for 0.5 to 3 seconds. It means to be immersed in.
  • the sample solution or supernatant soaked in the development carrier is naturally developed by the surface tension of the development carrier, and the nucleic acid contained in the sample solution is separated.
  • the nucleic acid can be separated from the amplification reaction inhibitory substance, such as rot acid, present in the sample solution with the use of the carrier for expansion.
  • a hanging process may be performed under suction conditions using, for example, a vacuum pump. Deployment is sufficient at room temperature, but humidity 40
  • the developing carrier containing the sample solution or the supernatant can be dried as necessary.
  • it can be dried under the above suction conditions, or it can be dried by air drying.
  • the development carrier obtained by the above step (b) is used as a nucleus containing a target nucleic acid-specific primer.
  • Contact the acid amplification reaction solution For example, immerse the carrier for development containing the supernatant of the sample solution in the nucleic acid amplification reaction solution in step (c)!
  • a development carrier containing a sample solution for example, a stool solution
  • the portion of the development carrier colored by contact with the stool solution is lightly colored. What is necessary is just to immerse in the said nucleic acid amplification reaction solution.
  • the "nucleic acid amplification reaction solution” may be a reaction solution used in a desired nucleic acid amplification method.
  • a currently known method can be applied as the nucleic acid amplification method.
  • PCR method Science, 230: 1350-1354, 1985
  • NASBA method Nucleic Acid Sequence Based Amplification method, Nature, 350, 91-92, 1991
  • LAMP method Japanese Patent Laid-Open No. 2001-242 169.
  • the PCR method can be preferably applied.
  • the PCR method will be described as an example.
  • the desired nucleic acid can be extracted into the nucleic acid reaction solution.
  • the extracted nucleic acid can be subjected to the nucleic acid amplification treatment by either of the following methods (i) or (ii). You can choose which method to use depending on the situation.
  • a PCR buffer can be used as the nucleic acid amplification reaction solution.
  • Ampdirect crude DNA buffer (Shimadzu Corporation) is preferably used, but not limited to this, widely known PCR buffers can be used.
  • step (b) Method of performing nucleic acid amplification treatment by immersing the development carrier obtained in step (b) in the nucleic acid amplification reaction solution for 0 seconds, and then removing the development carrier.
  • step (ii) A method in which a necessary portion of the development carrier obtained in step (b) is cut out and nucleic acid amplification treatment is performed while the cut out development carrier is immersed in a nucleic acid amplification reaction solution.
  • the reaction solution soaked with the above-described carrier for development contains a target nucleic acid-specific primer, the reaction solution is directly applied to a nucleic acid amplification processing apparatus to cause a nucleic acid amplification reaction. Can do.
  • the present invention also includes detecting and identifying the target gene using the nucleic acid amplification product obtained by the above method. For example, by performing hybridization using a DNA chip, a clone library method, denatured radish endogel electrophoresis (DGGE method), temperature dalaziendo gel electrophoresis (TGGE) method, SSCP method, for example, Various genetic diseases and tumors It is possible to detect and identify a disease or a specific microorganism that constitutes a microflora in feces.
  • DGGE method denatured radish endogel electrophoresis
  • TGGE temperature dalaziendo gel electrophoresis
  • SSCP method for example, Various genetic diseases and tumors It is possible to detect and identify a disease or a specific microorganism that constitutes a microflora in feces.
  • the biological sample is feces
  • various disease-related gene markers such as genes present in the feces, tumor markers in the feces, and microorganism-related genes such as E. coli. .
  • the present invention extends to a reagent kit including a biological sample dissolving solution and a nucleic acid separation developing carrier that can be used in the nucleic acid amplification method.
  • sample lysis buffer 500 mmol / L Tris-HC1, 16 mmol / L EDTA, lOmmol / L NaCl, pH 9.0 (sample lysis buffer) was prepared.
  • LOOmg of stool was taken in a 1.5 ml tube and 500 L of sample dissolution buffer was added to dissolve the stool.
  • About 20 liters of sample dissolution buffer is desirable for 20 mg of stool to be dissolved.
  • a sample solution obtained by dissolving stool was heat-treated at 95 ° C for 10 minutes. After further centrifugation at 5,000 rpm for 5 minutes, the developing carrier was immersed in the supernatant of the sample solution.
  • the KRAS and BRAF genes present in feces and the thrA gene derived from E. coli were amplified. Rinse the fecal solution supernatant with the spreading carrier part immersed in 50 L of Ampdirect crude DNA buffer (Shimadzu Corporation), which is a buffer solution for each PCR containing the following primers (A) to (D): DNA was extracted.
  • Ampdirect crude DNA buffer Shiadzu Corporation
  • FK-F 5'-TGACTGAATATAAACTTGTGGTAG (SEQ ID NO: 1)
  • FK-106R 5'- ATTGTTGGATCATATTCGTC (SEQ ID NO: 2)
  • FB-F 5'- TAGGTGATTTTGGTCTAGCT (SEQ ID NO: 3)
  • FB-115R 5 -ATCAGTGGAAAAATAGCCTC (SEQ ID NO: 4)
  • thrA-F 5 CCCCGCCAAAATCACCAACCATCT (SEQ ID NO: 5)
  • thrA-101R 5'- CGGCAAAAATACGTTCGGCATCG (SEQ ID NO: 6)
  • nucleic acid amplification method of the present invention it is possible to quickly and sensitively perform tests necessary for genetic diagnosis, early cancer diagnosis, infectious disease diagnosis and the like.
  • the method of the present invention genetic diagnosis of various biological samples, particularly fecal force, can be performed very easily and reliably.
  • feces with unique information which is unique to other biological samples, can be used practically and simply as a non-invasive DNA material is an operation that is useful for the diagnosis of various diseases. Enables the processing of large numbers of specimens. Therefore, it can be applied to various types of screening for colorectal cancer and the like in the normal population, and it is also possible to quickly detect causes such as mass-onset infections.
  • the method of the present invention has contributed to preventive medicine, which has become increasingly important in recent years, and has led to mass outbreaks. Application to rapid diagnosis of infectious diseases that occur is expected.

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Abstract

L'invention a pour objet un procédé de traitement pour extraire commodément un acide nucléique lorsqu'on amplifie un acide nucléique provenant d'un gène cible susceptible d'être contenu dans un échantillon biologique, sans séparer ni purifier un acide nucléique, tel qu'un ADN, de l'échantillon en utilisant des procédures compliquées comme dans les procédés existants. L'invention concerne donc un procédé lequel comprend : (a) l'étape consistant à dissoudre un échantillon biologique dans une solution tampon pour dissoudre l'échantillon ; (b) l'étape consistant à imprégner un support de développement de la solution résultante ; et (c) l'étape consistant à mettre le support de développement en contact avec une solution d'amplification d'acide nucléique. Selon ce procédé, on peut amplifier commodément et efficacement un acide nucléique provenant d'un gène cible dans un échantillon biologique sans avoir recours à des procédures compliquées pour séparer et purifier l'acide nucléique.
PCT/JP2005/022676 2004-12-13 2005-12-09 Procédé consistant à traiter un échantillon biologique pour effectuer une amplification d'un acide nucléique WO2006064739A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011019505A (ja) * 2009-01-29 2011-02-03 Mukogawa Gakuin 特定遺伝子を検出する方法
JP2012157295A (ja) * 2011-02-01 2012-08-23 Mukogawa Gakuin 遺伝子の増幅方法、特定遺伝子の検出方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6493209B2 (ja) * 2013-08-06 2019-04-03 東洋紡株式会社 核酸増幅法
JP7403948B2 (ja) * 2018-10-31 2023-12-25 公益財団法人筑波メディカルセンター 試料の前処理方法

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US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
JPH08504081A (ja) * 1992-04-01 1996-05-07 ザ ジョーンズ ホプキンズ ユニバーシティー スクール オブ メディシン 糞便試料から単離した哺乳類の核酸を検出する方法、およびその検出用試薬
JP2001221721A (ja) * 2000-02-09 2001-08-17 Sapporo Imuno Diagnostic Laboratory:Kk 乾燥濾紙便による遺伝子診断
JP2002306175A (ja) * 2001-04-11 2002-10-22 Yakult Honsha Co Ltd 核酸の単離方法

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NL8900725A (nl) * 1989-03-23 1990-10-16 Az Univ Amsterdam Werkwijze en combinatie van middelen voor het isoleren van nucleinezuur.
JP2004201607A (ja) * 2002-12-26 2004-07-22 Asahi Kasei Corp 核酸吸着固相体上でのｌａｍｐ反応

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material
JPH08504081A (ja) * 1992-04-01 1996-05-07 ザ ジョーンズ ホプキンズ ユニバーシティー スクール オブ メディシン 糞便試料から単離した哺乳類の核酸を検出する方法、およびその検出用試薬
JP2001221721A (ja) * 2000-02-09 2001-08-17 Sapporo Imuno Diagnostic Laboratory:Kk 乾燥濾紙便による遺伝子診断
JP2002306175A (ja) * 2001-04-11 2002-10-22 Yakult Honsha Co Ltd 核酸の単離方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011019505A (ja) * 2009-01-29 2011-02-03 Mukogawa Gakuin 特定遺伝子を検出する方法
JP2012157295A (ja) * 2011-02-01 2012-08-23 Mukogawa Gakuin 遺伝子の増幅方法、特定遺伝子の検出方法

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