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WO2000008136A1 - Amplification enzymatique d'acide nucleique - Google Patents

Amplification enzymatique d'acide nucleique Download PDF

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Publication number
WO2000008136A1
WO2000008136A1 PCT/JP1999/004189 JP9904189W WO0008136A1 WO 2000008136 A1 WO2000008136 A1 WO 2000008136A1 JP 9904189 W JP9904189 W JP 9904189W WO 0008136 A1 WO0008136 A1 WO 0008136A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
organic solvent
enzymatic amplification
amplification
pcr
Prior art date
Application number
PCT/JP1999/004189
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English (en)
Japanese (ja)
Inventor
Isao Karube
Sadayori Hoshina
Kazunori Ikebukuro
Eriko KAI
Original Assignee
Center For Advanced Science And Technology Incubation, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Center For Advanced Science And Technology Incubation, Ltd. filed Critical Center For Advanced Science And Technology Incubation, Ltd.
Priority to AU49340/99A priority Critical patent/AU4934099A/en
Publication of WO2000008136A1 publication Critical patent/WO2000008136A1/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
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to an enzymatic amplification reaction of a nucleic acid.
  • the present invention relates to a method for performing a nucleic acid amplification reaction without being affected by an inhibitory factor, when the inhibitory factor coexists in a sample.
  • Amplification reactions based on enzymatic synthesis of nucleic acids are widely implemented as gene detection techniques. Particularly in the detection and identification of pathogenic microorganisms, nucleic acid amplification reaction is an indispensable analytical technique.
  • the most widespread nucleic acid amplification reaction is the polymerase chain reaction (PCR).
  • PCR is an application of an enzymatic reaction in which a MA-dependent MA polymerase such as Taq polymerase synthesizes a complementary strand starting from the 3 ′ end of a primer hybridized to a complementary strand, for amplification of nucleic acids.
  • nucleic acids can be amplified exponentially.
  • PCR reaction is excellent in terms of specificity and amplification efficiency, it has a problem that it cannot actually avoid the influence of coexisting components.
  • various components coexisting in the biological sample from which the type III nucleic acid is derived have an inhibitory effect on PCR.
  • Extraction and purification of nucleic acids include, for example, protein denaturation by protease phenol and alcohol precipitation to separate nucleic acids from aqueous solution after their treatment. Has been implemented. These treatments are unsuitable for processing large amounts of samples, and have the potential to break long-chain DNA during the treatment. (Shinsei Kagaku Experimental Course • Nucleic Acid I “Separation and Purification”, edited by The Biochemical Society of Japan, P13-15).
  • a special DNA polymerase ("TaKaRa ExTaq", a registered trademark of Takara Shuzo Co., Ltd.), which is improved so as not to be affected by co-existing components, is commercially available.
  • This DNA polymerase enables PCR in the presence of blood and medium components, which are difficult to amplify without sample pretreatment with ordinary DNA polymerase.
  • AmpDirect (Registered trademark, manufactured by Shimadzu Corporation) contains a component that can block the effect of PCR inhibition by protein sugar present in a blood sample. It is possible to perform PCR on blood samples simply by treating with AmpDirect prior to PCR. However, these measures alone may not be effective enough, and PCR is usually performed in combination. In addition, AmpDirect is effective in amplifying DNA, but it cannot be used in amplification reaction with RNA polymerase because interference with the enzyme reaction is observed even after AmpDirect treatment.
  • An object of the present invention is to provide a novel method for enzymatically amplifying a nucleic acid using a microorganism present in a biological sample such as feces as a sample, which can omit a complicated extraction and purification operation of the nucleic acid. More specifically, an object of the present invention is to provide a method for enzymatically amplifying a nucleic acid, which can surely remove a factor that inhibits an enzymatic synthesis reaction and can be performed with a simpler operation.
  • the present inventors have studied a method for reliably removing the influence of a factor that inhibits an enzymatic synthesis reaction. As a result, they have found that washing a nucleic acid-containing sample with a specific organic solvent effectively removes the influence of factors that inhibit the enzymatic synthesis reaction of nucleic acids, and completed the present invention. That is, the present invention relates to the following enzymatic amplification methods for nucleic acids and their applications.
  • (I) a method comprising washing a test sample with an organic solvent to remove a substance that inhibits an enzymatic amplification reaction of a nucleic acid, and enzymatically amplifying a nucleic acid of a cell contained in the test sample.
  • a method for enzymatic amplification of acids comprising washing a test sample with an organic solvent to remove a substance that inhibits an enzymatic amplification reaction of a nucleic acid, and enzymatically amplifying a nucleic acid of a cell contained in the test sample.
  • the organic solvent is selected from the group consisting of 70% ethanol aqueous solution, methanol, 2-propanol, acetone, acetonitrile, dimethyl sulfoxide, butanol, 2-butanol, and ethyl acetate. Enzymatic amplification method of the described nucleic acid
  • the test sample to be subjected to the present invention is excrement such as feces, urine, or sweat, body fluid such as blood, semen, saliva, gastric juice, or bile.
  • body fluid such as blood, semen, saliva, gastric juice, or bile.
  • body fluid such as blood, semen, saliva, gastric juice, or bile.
  • these biological samples may be further fractionated and a part thereof may be taken out.
  • feces contain many relatively large residues, such as undigested solids. Such solids hinder accurate pitting operations, so it is advisable to remove them by filtration or light centrifugation in advance.
  • the factor that inhibits the enzymatic synthesis reaction of nucleic acids whose effects should be removed by washing means an unspecified component that inhibits the enzymatic synthesis reaction described below by being mixed into an enzyme reaction system. I do. Although unspecified, the effect of removing substances that act as inhibitors can be easily confirmed by observing the results of enzymatic amplification reactions. For example, when feces are used as a sample, it has already been described that the inhibitory effect on PCR of a porphyrin compound such as bile salt is assumed. However, the components of stool are so diverse that it is difficult to explain the inhibitory effects of stool samples on PCR with these compounds alone.
  • washing of a biological sample using an organic solvent can be performed by various operations. For example, washing by centrifugation is a typical washing operation. Specific operation will be described by taking a case where the present invention is applied to a stool sample as an example. First, suspend the stool sample in a suitable buffer and remove it at 2000 rpm to remove large solids. Centrifuge for about 5 minutes and collect the supernatant. The obtained supernatant is centrifuged at 15000 rpm for 15 minutes, and the supernatant is discarded. A hydrophilic organic solvent is added to the remaining sediment, followed by centrifugation again at 15000 rpm for 10 minutes, and washing is performed. As a result, the fraction that can be recovered as a precipitate can be directly applied to PCR. If good results cannot be obtained, washing with an organic solvent can be repeated to remove the reaction inhibitor more reliably.
  • the washing operation according to the invention can also be carried out in combination with the filling process.
  • a washing operation based on the present invention is carried out using a filter that traps Escherichia coli cells and allows smaller molecules to pass through.
  • the E. coli cells captured on the filter may be recovered and used as a substrate for PCR. If the filter is clogged with a sample containing a large amount of insoluble solids such as feces, it is recommended that the sample be passed through a prefill for removing large solids and then trapped in a filter for washing. It is advantageous to configure.
  • a plurality of filters necessary for the washing operation of the present invention using a filter can be combined with a syringe for liquid feeding and an organic solvent for washing to form a kit. Washing by Fil Yuni has the advantage that it can be easily performed even in an environment without special equipment compared to centrifugation. Alternatively, a series of washing steps can be automated using a filter plate equipped with an ultrafiltration filter. A system for automating the extraction process of genomic / plasmid of microorganisms is known. The operating principle of this type of system can also be applied to the cleaning method according to the invention.
  • various organic solvents can be used. Of these, hydrophilic or amphiphilic organic solvents are preferred.
  • hydrophilic organic solvent for example, ethanol, methanol, 2-propanol, propanone (acetone), ether nitrile (acetonitrile), dimethyl sulfoxide (DMS0) and the like can be used.
  • amphiphilic organic solvent bushanol, 2-butanol, ethyl acetate and the like can be used.
  • the hydrophilicity of the solvent can be quantitatively compared using the relative dielectric constant (permittivity) as an index.
  • preferred organic solvents include those having a relative dielectric constant ⁇ in the range of 5 to 40 at the temperature at which washing is performed (20 to 25 ° C).
  • an organic solvent having a relative dielectric constant e in the range of 10 to 25 is particularly effective.
  • the relative permittivity is a ratio when the permittivity of vacuum is set to 1. In general, the dielectric constant often means the relative permittivity.
  • the magnitude of £ has a significant effect on the strength of the interaction between ions in solution.
  • the relative permittivity of water 25 ° C is 78, which means that the electrostatic force in water is 1/78 of that in vacuum.
  • hydrophilic organic solvents or amphiphilic solvents may be used alone or as a mixture of a plurality of organic solvents. When mixing, the combination can be determined in consideration of the washing effect, operational safety, influence on the enzymatic amplification reaction, or economics. In addition, when an inhibitory effect on Taq polymerase was confirmed for a wide range of organic solvents including the organic solvent used in the examples, no inhibitory effect on the enzyme activity was found. In the case of a hydrophilic organic solvent, a solution appropriately diluted with water can be used in consideration of the washing effect of the inhibitor and the effect on DNA.
  • ethanol when ethanol is used for washing a nucleic acid sample for PCR derived from pathogenic Escherichia coli in feces, the best results can be obtained when water is mixed so that the ethanol concentration becomes 70 to 90%.
  • Various conditions at the time of washing are appropriately selected depending on the combination of the kind of the hydrophilic organic solvent used and the sample. For example, in the case of ethanol, treatment at room temperature is possible.
  • lipophilic organic solvents can be used in the present invention. That is, for example, ethyl acetate, trichloromethane (chloroform), benzene, Methylbenzene (xylene) and the like can be used in the present invention. With these lipophilic organic solvents, it was confirmed that although the sensitivity was not as high as that of the hydrophilic organic solvent depending on the combination with the sample, the lipophilic organic solvent clearly showed the inhibitory effect of the inhibitor. These lipophilic organic solvents may be used alone or in combination of two or more.
  • the nucleic acid to be amplified is not particularly limited, such as a gene of a pathogenic microorganism and a gene derived from a living body.
  • Pathogenic microorganisms are particularly important analytes in stool samples having a large effect of removing the reaction inhibitor according to the present invention.
  • Examples of the pathogenic microorganism include bacteria, fungi, and rickettsia. All of these pathogenic microorganisms may appear in human feces, and amplifying and detecting their nucleic acids has clinical significance.
  • pathogenic Escherichia coli, cholera bacteria, dysentery amoeba, etc. represented by 0157: H7, etc. are simple and easy to use, because once infected, suspected infections, their families, and even the route of infection and a wide range of tests are required. Quick analysis operation is required. Therefore, it is very effective to apply the present invention to such inspection targets.
  • the enzymatic nucleic acid amplification method of the present invention means an amplification reaction with various enzymes using a nucleic acid as a substrate.
  • DNA-dependent DNA polymerases such as Taq polymerase, RNA-dependent DNA polymerases such as reverse transcriptase, and DNA-dependent RNA polymerases such as T7 RNA polymerase
  • This is an amplification reaction using an enzyme such as DNA ligase or DNA ligase.
  • PCR which is a DNA amplification reaction using a thermostable DNA polymerase, is highly evaluated in terms of both sensitivity and specificity, and is a typical amplification method that is currently widely used in many research and testing facilities. It is a reaction.
  • the amplification reaction of nucleic acids based on these enzymes is inhibited by various components coexisting in the sample, but according to the present invention, these inhibitors are reliably removed by a washing step using an organic solvent. can do.
  • RT-PCR also has no effect on reverse transcriptase activity
  • RT-PCR is a method of amplifying a gene by first synthesizing complementary MA using RNA as a type II using reverse transcriptase (RT), and then using this as a type II for PCR to detect mRNA.
  • RT reverse transcriptase
  • AmpD irect a known measure against inhibitors, inhibited the reverse transcriptase reaction that synthesizes DNA from RNA.
  • the effect on reverse transcriptase is negligible, and the RNA to be analyzed is stably maintained during the treatment, so that it can be easily applied to RT-PCR.
  • the present invention can be applied to an amplification reaction using A polymerase such as Nucleic Acid Sequence-based Amp 1 ifi cation (NASB A) (also called Transduction Mediated Amplification (TMA) method).
  • a polymerase such as Nucleic Acid Sequence-based Amp 1 ifi cation (NASB A) (also called Transduction Mediated Amplification (TMA) method).
  • T7 promoter also called Transduction Mediated Amplification
  • T7 RNA polymerase T7 RNA polymerase
  • FIG. 1 is a flowchart of the method for enzymatically amplifying nucleic acids contained in feces according to the present invention.
  • the above-mentioned sample for the nucleic acid amplification reaction according to the present invention was prepared based on the following operations.
  • feces (or blood) of O. nL were collected and suspended in a phosphate buffer ( ⁇ 7.0). This was centrifuged at 2000 rpm for 5 minutes to recover the supernatant, and the residue larger than the cells was removed. The collected supernatant was centrifuged at 15000 rpm for 15 minutes, and the cells were concentrated as sediment. The supernatant was discarded, 0.9 mL of various organic solvents were added to the precipitate, mixed well, and centrifuged again (15000 rpm, 10 minutes). This precipitate was used as a sample for subsequent enzymatic amplification reactions.
  • the organic solvents used in the experiment are as shown in Table 2, methanol, ethanol (70-90%), 2-propanol (isopropanol), propanone (acetone), nitrile nitrile (acetonitrile), and dimethyl sulfoxide (DMS0).
  • a sample using phosphate buffer instead of the organic solvent for washing was prepared as a control.
  • the primer used was a combination of the sense primer shown in SEQ ID NOS: 1 to 3 and the antisense primer shown in SEQ ID NO: 4. The relationship between these primers and the amplification product is as shown below. Table 1 shows the results for each sample and the confirmed sensitivity. Table 2 summarizes the results of PCR after centrifugal washing (once) with each solvent using a substrate to which Escherichia coli 0157 cultured on stool of a healthy person was added as a substrate.
  • NASBA was performed on the blood sample containing pathogenic E. coli (washed once with 90% ethanol) prepared in (1).
  • an enzymatic reaction was carried out using a kit supplied commercially from Toyobo Co., Ltd. according to the attached instructions. The operation is as follows.
  • the 50 / L portion of the sediment after washing obtained in (1) was collected, ethanol was dried, and then sterilized water was added thereto and stirred well.
  • the sample was collected and used as a sample for NASBA.
  • a 10 / L primer solution (as described below) was added, and the mixture was incubated at 61 ° C for 5 minutes.
  • the enzyme mix required for NASBA layer-RT, RNaseH, 5 L
  • T7 RNA polymerase, BSA, etc. was added, and reacted at 41 ° C for 95 minutes.
  • DNA is synthesized starting from the first primer that has been annealed to RNA, and then the MA, which has become type II, is enzymatically removed, and the second primer anneals.
  • the DNA becomes a double strand containing the T7 promoter, and this is used as a type II to transcribe RNA having the antisense sequence of the target sequence.
  • the same set as the primer used in the PCR of (2) was used.
  • a T7 promoter sequence of 25 bp was added to the 5th side, and used as the first primer (SEQ ID NO: 5). After the reaction, a part of the reaction solution was subjected to electrophoresis, and the amplified product was confirmed by ethidium die opening staining.
  • Primer solution One NASBE freeze-dried reagent
  • a sample for performing an enzymatic synthesis reaction of a nucleic acid can be easily prepared.
  • even for complex coexisting systems such as feces it is possible to reliably remove the reaction inhibitory factor.
  • the effect of the present invention on various enzyme reactions is small, so that the present invention can be applied to various reactions.
  • AmpDirect cannot be applied to RT-PCR or NASBA, but the present invention can be easily applied.

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Abstract

La présente invention concerne un procédé d'amplification d'acide nucléique peu sensible aux composants parasites coexistant dans le prélèvement. En l'occurrence, l'utilisation d'un solvant organique permet d'éliminer de nombreux facteurs inhibiteurs de la réaction d'amplification enzymatique d'un acide nucléique. Ainsi, un lavage avec un solvant organique suffit, même dans le cas d'un prélèvement de matières fécales devant subir une extraction et purification génomique avant la PCR selon le procédé conventionnel.
PCT/JP1999/004189 1998-08-04 1999-08-03 Amplification enzymatique d'acide nucleique WO2000008136A1 (fr)

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Application Number Priority Date Filing Date Title
AU49340/99A AU4934099A (en) 1998-08-04 1999-08-03 Method for enzymatic amplification of nucleic acid

Applications Claiming Priority (4)

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JP10/220446 1998-08-04
JP22044698 1998-08-04
JP9560099 1999-04-01
JP11/95600 1999-04-01

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006101818A (ja) * 2004-10-08 2006-04-20 Eiken Chem Co Ltd 核酸増幅反応における阻害を回避する方法
US7498407B2 (en) 2001-11-09 2009-03-03 Georgetown University Vascular endothelial cell growth inhibitor, VEGI-192a
WO2009139317A1 (fr) * 2008-05-12 2009-11-19 オリンパス株式会社 Procédé de traitement d’excréments et contenant pour le traitement d’excréments
WO2010010914A1 (fr) * 2008-07-23 2010-01-28 オリンパス株式会社 Procédé de collecte d'acide nucléique à partir d'un échantillon fécal, procédé d'analyse d'acide nucléique et appareil pour le traitement d'échantillon fécal
WO2010134246A1 (fr) * 2009-05-20 2010-11-25 オリンパス株式会社 Procédé de préparation d'un échantillon contenant un acide nucléique
EP2392670A1 (fr) 2010-06-03 2011-12-07 Olympus Corporation Procédé pour la détection d'acide nucléique dans un échantillon biologique
US8465924B2 (en) 2004-12-07 2013-06-18 Sysmex Corporation Treatment solution for preparing sample solution for nucleic acid amplification reaction and method for detecting nucleic acid by using treatment solution
JP2017525383A (ja) * 2014-09-04 2017-09-07 テックラブ，インコーポレーテッド 阻害剤を除去するために有機溶媒を用いる核酸抽出
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum
JP2023522054A (ja) * 2020-04-15 2023-05-26 ヴォスビオ，インコーポレーテッド 保存および分析のための核酸試料を調製する方法、装置、およびキット
PL444578A1 (pl) * 2023-04-25 2024-10-28 Uniwersytet Kazimierza Wielkiego Sposób detekcji komponentów wieprzowych w produkcie żywnościowym

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JPH05211898A (ja) * 1990-01-19 1993-08-24 F Hoffmann La Roche Ag 水試料中の水系微生物病原体およびヒト糞便汚染の指示微生物の検出方法とそのためのキット
JPH06165676A (ja) * 1992-06-16 1994-06-14 Wako Pure Chem Ind Ltd マイコバクテリウム属の菌体からの核酸鎖抽出方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05211898A (ja) * 1990-01-19 1993-08-24 F Hoffmann La Roche Ag 水試料中の水系微生物病原体およびヒト糞便汚染の指示微生物の検出方法とそのためのキット
JPH06165676A (ja) * 1992-06-16 1994-06-14 Wako Pure Chem Ind Ltd マイコバクテリウム属の菌体からの核酸鎖抽出方法

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7498407B2 (en) 2001-11-09 2009-03-03 Georgetown University Vascular endothelial cell growth inhibitor, VEGI-192a
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum
JP2006101818A (ja) * 2004-10-08 2006-04-20 Eiken Chem Co Ltd 核酸増幅反応における阻害を回避する方法
US8465924B2 (en) 2004-12-07 2013-06-18 Sysmex Corporation Treatment solution for preparing sample solution for nucleic acid amplification reaction and method for detecting nucleic acid by using treatment solution
US8597954B2 (en) 2008-05-12 2013-12-03 Olympus Corporation Stool sample processing method and stool sample processing container
WO2009139317A1 (fr) * 2008-05-12 2009-11-19 オリンパス株式会社 Procédé de traitement d’excréments et contenant pour le traitement d’excréments
CN102027132A (zh) * 2008-05-12 2011-04-20 奥林巴斯株式会社 粪便处理方法及粪便处理容器
JP5616786B2 (ja) * 2008-05-12 2014-10-29 オリンパス株式会社 糞便処理容器
JP5710969B2 (ja) * 2008-07-23 2015-04-30 オリンパス株式会社 糞便試料からの核酸回収方法
WO2010010914A1 (fr) * 2008-07-23 2010-01-28 オリンパス株式会社 Procédé de collecte d'acide nucléique à partir d'un échantillon fécal, procédé d'analyse d'acide nucléique et appareil pour le traitement d'échantillon fécal
WO2010134246A1 (fr) * 2009-05-20 2010-11-25 オリンパス株式会社 Procédé de préparation d'un échantillon contenant un acide nucléique
EP2392670A1 (fr) 2010-06-03 2011-12-07 Olympus Corporation Procédé pour la détection d'acide nucléique dans un échantillon biologique
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11536632B2 (en) 2011-06-19 2022-12-27 DNA Genotek, Inc. Biological collection system
US11549870B2 (en) 2011-06-19 2023-01-10 DNA Genotek, Inc. Cell preserving solution
US11592368B2 (en) 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11198899B2 (en) 2014-03-07 2021-12-14 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
JP2017525383A (ja) * 2014-09-04 2017-09-07 テックラブ，インコーポレーテッド 阻害剤を除去するために有機溶媒を用いる核酸抽出
JP2023522054A (ja) * 2020-04-15 2023-05-26 ヴォスビオ，インコーポレーテッド 保存および分析のための核酸試料を調製する方法、装置、およびキット
PL444578A1 (pl) * 2023-04-25 2024-10-28 Uniwersytet Kazimierza Wielkiego Sposób detekcji komponentów wieprzowych w produkcie żywnościowym

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