WO2000008136A1 - Method for enzymatic amplification of nucleic acid - Google Patents
Method for enzymatic amplification of nucleic acid Download PDFInfo
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- 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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- 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.
Abstract
A method for amplifying a nucleic acid which is little affected by interfering components coexisting in a sample. In this method, various factors inhibiting the reaction of enzymatic amplification of a nucleic acid can be eliminated with the use of an organic solvent. Thus, even a fecal sample which should be subjected to the genome extraction and purification prior to PCR in the conventional method can be subjected to PCR merely after washing with an organic solvent.
Description
核酸の酵素的増幅方法 技術分野 Method for enzymatic amplification of nucleic acids
本発明は、 核酸の酵素的な増幅反応に関する。 特に、 酵素反応に対する阻害因 子が試料中に共存するときに、 この阻害因子の影響を受けること無く核酸の増幅 反応を実施する方法に関する。 背景技術 The present invention relates to an enzymatic amplification reaction of a nucleic acid. In particular, 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. Background art
核酸の酵素的な合成に基づく増幅反応は、 遺伝子の検出技術として広く実施さ れている。 ことに病原性微生物の検出と同定においては、 核酸の増幅反応は不可 欠な分析技術である。 核酸の増幅反応としてもっとも普及しているのがポリメラ —ゼ連鎖反応 （polymerase chain reaction; PCR) である。 PCRは、 Taqポリメラ —ゼ等の MA依存性 MAポリメラーゼが、 相補鎖にハイブリダイズしたブラィマー の 3'末端を起点として相補鎖の合成を行う酵素反応を核酸の増幅に応用したもの である。 増幅すべきセグメントのセンス鎖とアンチセンス鎖に対して各鎖の 3，末 端領域にァニールするプライマ一を用意し、 その 3，末端を起点に DNAポリメラ一ゼ による相補鎖合成反応を行う。 生成物を鎵型として同じ相補鎖増幅反応を繰り返 せば、 核酸を指数的に増幅することが可能である。 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. Prepare a primer that anneals to the 3 and 3 end regions of the sense and antisense strands of the segment to be amplified, and perform a complementary strand synthesis reaction with DNA polymerase from the 3 and end points. If the same complementary strand amplification reaction is repeated with the product as type III, nucleic acids can be amplified exponentially.
PCR反応は特異性や増幅効率の点で優れているが、 現実には共存成分の影響を逃 れられないという問題点を持っている。 つまり、 錡型となる核酸が由来する生物 試料に共存する様々な成分が PCRにおいて阻害的な影響を与えるのである。 そのた め、 一般的には PCRに先立って錶型となる核酸の抽出精製処理が必要とされている 。 核酸の抽出精製は、 たとえばプロテアーゼゃフエノールによるタンパク質の変 性、 更にそれらの処理後の水溶液から核酸を分離するためのアルコール沈でん等
により実施されている。 これらの処理は多量の試料の処理には不向きであるし、 処理を通じて長鎖 DNAが切断されてしまう可能性を伴っていた （新生化学実験講座 •核酸 I 「分離精製」 、 日本生化学会編、 P13-15)。 Although the 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. In other words, various components coexisting in the biological sample from which the type III nucleic acid is derived have an inhibitory effect on PCR. For this reason, it is generally necessary to carry out extraction and purification of nucleic acids of type I prior to 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).
このような操作の省略を目的として、 共存成分の影響を受け難いように改良さ れた特殊な DNAポリメラ一ゼ （"TaKaRa ExTaq"、 宝酒造社製、 登録商標） が市販さ れている。 この DNAポリメラ一ゼは、 通常の DNAポリメラーゼでは試料の前処理無 しでは増幅が困難な血液や培地成分共存下での PCRを可能とする。 For the purpose of omitting such an operation, 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.
他方、 共存成分の影響をブロックしょうとするアプローチも行われている。 た とえば、 AmpDirect (島津製作所社製、 登録商標） は、 血液試料に共存するタンパ ク質ゃ糖による PCRの阻害の影響をブロックしうる成分を含んでいる。 PCRに先立 つて AmpDirectで処理するだけで、 血液試料の PCRが可能とされている。 しかしこ れらの対策は、 単独では効果が不十分な場合があり、 通常は両者を組み合わせて PCRを実施している。 加えて、 AmpDirectは DNAの増幅には有効だが、 RNAポリメラ ーゼによる増幅反応においては AmpDirect処理後も酵素反応の妨害が観察されるの で利用できないという制限があった。 On the other hand, some approaches are trying to block the effects of co-existing components. For example, 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.
しかも阻害因子を含むとはいえ、 血液はどちらかというと個体間の違いの少な い比較的取り扱いやすい試料である。 糞便や尿といった生体試料では、 量的にも 質的にも成分の変動が大きいため、 より確実な妨害因子対策が望まれるが、 未だ に有効な対策は知られていない。 In addition, although containing inhibitors, blood is a relatively easy-to-handle sample with relatively few differences between individuals. For biological samples such as feces and urine, the components fluctuate greatly both quantitatively and qualitatively, so more reliable countermeasures against interfering factors are desired. However, no effective countermeasures have yet been known.
PCRそのものの感度を考慮すれば理論的には糞便中の微生物の直接検出も不可能 ではない。 しかし現実には、 糞便中の多様な反応阻害因子の存在により、 糞便試 料から直接 PCRを行うことは困難である。 そのため、 リン酸緩衝液を利用した一般 的な糞便抽出液では病原性大腸菌を十分な感度で検出できなかったり（J.Clin.Mi crobiol .，33/3,pp519-524, 1995)、 あるいは DNAを吸着する特殊なガラスマトリク ス素材による糞便からの DNAの抽出によって感度の向上を試みている（J. Clin.Mic robiol . , 33/5， ρρ1054-1059, 1995) ο これらの先行技術文献においては、 糞便中に
共存する胆汁酸塩やピリルビン等が PCRに対して阻害的に作用することが推測され ている。 したがって、 通常はゲノムの抽出精製や複製によって、 PCRの実施を可能 とする核酸試料を得ているが、 多くの試料について煩雑な抽出精製処理を行うの は現実的ではない。 こうした背景があって、 糞便中の病原微生物の検出において は、 PCRではなく微生物の分離培養が第一の選択肢となつているのが現状である。 あるいは PCRを利用して培養操作を省く場合には、 糞便中の共存物質の影響を避け るために 1000倍以上の希釈が求められる。 しかしこのような希釈操作が感度の低 下につながることは言うまでもない。 たとえば病原性大腸菌 0157:H7では、 ソルビ トール ·マッコンキー寒天培地に便検体を塗沫し、 8時間の培養後に生じるコロニ —の性状によってスクリーニングしている。 病原性大腸菌であることが疑われる ものについては、 更にラテックス凝集反応により確認を行って最終的な判定をし ている。 病原性微生物の検出と同定は治療方針を左右する重要な情報なので、 時 間のかかる培養ステップを省き、 糞便試料から直接、 高い感度で PCRを行うことが 理想である。 発明の開示 Considering the sensitivity of PCR itself, it is theoretically not impossible to detect microorganisms in feces directly. However, in reality, it is difficult to perform PCR directly from stool samples due to the presence of various reaction inhibitors in stool. For this reason, pathogenic Escherichia coli cannot be detected with sufficient sensitivity using a common fecal extract using phosphate buffer (J. Clin. Microbiol., 33/3, pp519-524, 1995), or DNA (J. Clin. Microbiol., 33/5, ρ1054-1059, 1995) ο attempts to improve sensitivity by extracting DNA from feces using a special glass matrix material that adsorbs Is in the feces It is presumed that coexisting bile salts, pyrilrubin, etc. act inhibitory to PCR. Therefore, nucleic acid samples that enable PCR to be performed are usually obtained by extracting and purifying or duplicating genomes, but it is not practical to perform complicated extraction and purification processes on many samples. Against this background, isolation and culture of microorganisms, rather than PCR, are currently the primary option for detecting pathogenic microorganisms in feces. Alternatively, when culturing is omitted by using PCR, a dilution of 1000 times or more is required to avoid the effects of coexisting substances in feces. However, it goes without saying that such a dilution operation leads to a decrease in sensitivity. For example, in the case of pathogenic Escherichia coli 0157: H7, stool samples are spread on sorbitol-MacConkey agar medium and screened for the properties of colonies formed after 8 hours of culture. For those suspected to be pathogenic Escherichia coli, they are further confirmed by latex agglutination to make a final decision. Since the detection and identification of pathogenic microorganisms is important information that determines the course of treatment, it is ideal to eliminate time-consuming culturing steps and perform PCR with high sensitivity directly from stool samples. Disclosure of the invention
本発明の課題は、 糞便のような生体試料に存在する微生物を試料とする核酸の 酵素的増幅方法において、 煩雑な核酸の抽出精製操作を省略することができる、 新規な方法の提供にある。 より具体的には、 酵素的な合成反応を阻害する因子を 確実に除去することができ、 しかもより簡単な操作で実施可能な、 核酸の酵素的 増幅方法の提供が本発明の課題である。 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.
本発明者らは、 前記課題を解決するために、 酵素的な合成反応を阻害する因子 の影響を確実に取り除く方法について検討を行った。 その結果、 特定の有機溶媒 で核酸含有試料を洗浄することにより、 核酸の酵素的合成反応を阻害する因子の 影響が効果的に除去されることを見出し本発明を完成した。 すなわち、 本発明は 以下の核酸の酵素的増幅方法とその応用に関する。
(I) 有機溶媒で被検試料を洗浄して核酸の酵素的増幅反応を阻害する物質を除 去し、 該被検試料中に含まれる細胞の核酸を酵素的に増幅させることを含む、 核 酸の酵素的増幅方法。 In order to solve the above-mentioned problems, 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.
( 2 ) 有機溶媒が親水性溶媒または両親媒性の有機溶媒である、 （ 1 ) に記載の 核酸の酵素的増幅方法。 (2) The method for enzymatic amplification of a nucleic acid according to (1), wherein the organic solvent is a hydrophilic solvent or an amphiphilic organic solvent.
( 3 ) 有機溶媒の比誘電率 εが 5〜40である、 （ 2 ) に記載の核酸の酵素的増幅方 (3) The method for enzymatic amplification of nucleic acid according to (2), wherein the relative permittivity ε of the organic solvent is 5 to 40.
(4) 有機溶媒が 70%エタノール水溶液、 メタノール、 2-プロパノール、 アセトン 、 ァセトニトリル、 ジメチルスルホキシド、 ブ夕ノール、 2-ブ夕ノール、 および 酢酸ェチルで構成される群から選択される （3) に記載の核酸の酵素的増幅方法 (4) 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
(5) 細胞が微生物細胞または血液細胞である、 （1) 〜 （4) のいずれかに記 載の核酸の酵素的増幅方法。 (5) The method for enzymatic amplification of a nucleic acid according to any one of (1) to (4), wherein the cell is a microbial cell or a blood cell.
( 6 ) 微生物細胞が病原性大腸菌である、 （ 5 ) に記載の核酸の酵素的増幅方法 (6) The method for enzymatic amplification of nucleic acid according to (5), wherein the microbial cell is pathogenic Escherichia coli.
(7)被検試料が糞便である （1) 〜 （6) のいずれかに記載の核酸の酵素的増 幅方法。 (7) The method for enzymatic amplification of nucleic acids according to any one of (1) to (6), wherein the test sample is feces.
(8) 有機溶媒による洗浄工程の前に、 微生物細胞よりも大きな残滓の一部また は全部を除去する工程を含む、 (7) に記載の核酸の酵素的増幅方法。 (8) The method for enzymatically amplifying a nucleic acid according to (7), comprising a step of removing a part or all of a residue larger than the microorganism cells before the washing step with an organic solvent.
(9) 有機溶媒による洗浄を遠心分離により実施する、 （1) 〜 （8) のいずれ かに記載の核酸の酵素的増幅方法。 (9) The method for enzymatic amplification of a nucleic acid according to any one of (1) to (8), wherein washing with an organic solvent is performed by centrifugation.
(10) 酵素的増幅を PCRによって行う、 （1) 〜 （9) のいずれかに記載の核酸 の酵素的増幅方法。 (10) The method for enzymatic amplification of a nucleic acid according to any one of (1) to (9), wherein the enzymatic amplification is performed by PCR.
(I I) (1) に記載の方法により生成する増幅生成物の有無を調べることによ り、 被検試料中の微生物を検出する方法。 (II) A method for detecting microorganisms in a test sample by examining the presence or absence of an amplification product produced by the method according to (1).
(12) 比誘電率 £が 5〜40である有機溶媒を含む、 酵素的増幅用核酸基質の洗浄
剤。 (12) Washing of nucleic acid substrates for enzymatic amplification, including organic solvents with relative permittivity of £ 5 to 40 Agent.
本発明の対象となる被検試料とは、 糞便、 尿、 もしくは汗のような***物、 血 液、 ***、 唾液、 胃液、 もしくは胆汁のような体液等である。 あるいは、 外科的 に生体から取り出した組織、 または毛髪のように生体から脱落した組織であって も良い。 またこれらの生体試料を更に分画してその一部を取り出したものであつ ても良い。 たとえば糞便では、 未消化の固形分のように比較的大きな残滓が多く 含まれている。 このような固形分は正確なピぺヅティング操作の妨げになるので 、 予めろ過や軽度の遠心分離によって取り除いておくと良い。 また組織のような 固形試料にあっては、 洗浄を助けるためにホモジェナイズしておくことが望まし い。 これら生体試料の中でも、 糞便や尿のように、 成分が食事や体調によって大 きく変動するものでは、 除去すべき反応阻害因子の質的、 そして量的な変動も大 きいことから、 本発明による効果が大きい。 すなわち、 このような試料に本発明 を応用すれば、 単に測定感度の向上が期待できるのみならず、 試料によって変動 する阻害の影響を小さくすることができ、 結果として測定精度が高まる。 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. Alternatively, it may be a tissue that has been surgically removed from a living body or a tissue that has fallen from a living body such as hair. Further, these biological samples may be further fractionated and a part thereof may be taken out. For example, 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. In addition, it is desirable to homogenize solid samples such as tissues to facilitate washing. Among these biological samples, those whose components fluctuate greatly depending on the diet and physical condition, such as feces and urine, have a large qualitative and quantitative fluctuation of the reaction inhibitory factors to be removed. Great effect. That is, if the present invention is applied to such a sample, not only the improvement of the measurement sensitivity can be expected, but also the influence of the inhibition that varies depending on the sample can be reduced, and as a result, the measurement accuracy increases.
本発明において、 洗浄によってその影響を除去すべき核酸の酵素的合成反応を 阻害する因子とは、 酵素反応系に混入することにより後に述べる酵素的な合成反 応を阻害する不特定の成分を意味する。 不特定ではあるが、 阻害因子として作用 する物質の除去作用は、 酵素的な増幅反応の結果を観察すれば容易に確認するこ とができる。 たとえば糞便を試料とする場合には、 胆汁酸塩等ポルフィリン化合 物の PCRに対する阻害作用が推測されていることは既に述べた。 しかし糞便の構成 成分は多様性に富んでおり、 これらの化合物だけで糞便試料の PCRに対する阻害作 用を説明することは難しい。 In the present invention, 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.
本発明において、 有機溶媒を使った生体試料の洗浄は、 様々な操作により実施 することができる。 たとえば、 遠心分離による洗浄は代表的な洗浄操作である。 具体的な操作について、 糞便試料に対し本発明を適用する場合を例に説明する。 まず糞便試料を適当な緩衝液に懸濁し、 大きな固形分を取り除くために 2000rpmで
5分程度の遠心分離を行い上清を分取する。 得られた上清を 15000rpmで 15分間遠心 分離して上清を捨て、 残った沈でんに親水性有機溶媒を加えて再度 15000rpmで 10 分の遠心分離を行い、 洗浄を実施する。 この結果沈でんとして回収できる分画は 、 そのまま PCRに適用することができる。 もしも良い結果を得られない場合には、 再度有機溶媒による洗浄を繰り返すと、 反応阻害因子をより確実に取り除くこと ができる。 In the present invention, 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.
本発明による洗浄操作はフィル夕一を組み合わせて実施することもできる。 た とえば大腸菌の菌体を基質として利用したい場合には、 大腸菌菌体をトラップし それよりも小さな分子を通過させるフィルターを利用し、 本発明に基づく洗浄操 作を実施する。 最終的にフィルター上に捕捉した大腸菌菌体を回収し、 PCRのため の基質とすれば良い。 糞便のような不溶性固形分を多く含む試料でフィルターの 目詰まりが心配される場合には、 大きな固形分を除去するためのプレフィル夕一 を通した後に洗浄用のフィル夕一にトラップするように構成すれば有利である。 フィルターによる本発明の洗浄操作に必要な複数のフィル夕一を送液用のシリン ジゃ洗浄用の有機溶媒と組み合わせてキット化することができる。 フィル夕一に よる洗浄には、 遠心分離と比較すると特別な装置が無い環境でも簡単に実施でき るという利点がある。 あるいは、 限外ろ過フィルターを装着したフィルタープレ —トを利用して、 一連の洗浄工程を自動化することも可能である。 微生物のゲノ ムゃプラスミ ドの抽出工程を自動化したシステムは公知である。 この種のシステ ムの動作原理は、 本発明による洗浄方法にも適用することができる。 The washing operation according to the invention can also be carried out in combination with the filling process. For example, when it is desired to use Escherichia coli cells as a substrate, 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. Finally, 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.
本発明において、 種々の有機溶媒を利用することが可能である。 中でも好まし いのは親水性あるいは両親媒性の有機溶媒である。 親水性有機溶媒としては、 た とえば、 エタノール、 メタノール、 2-プロパノ一ル、 プロパノン （アセトン） 、 ェ夕ン二トリル （ァセトニトリル）、 ジメチルスルホキシド（DMS0)等を利用する ことができる。 また、 両親媒性の有機溶媒としては、 ブ夕ノ一ル、 2-ブ夕ノール 、 酢酸ェチル等を利用することができる。
溶媒の親水性は、 比誘電率 (permittivity)を指標として定量的に比較すること ができる。 本発明において、 好ましい有機溶媒としては洗浄を実施する温度 （20 〜25°C) における比誘電率 εが 5〜40の範囲にあるものを示すことができる。 中で も特に比誘電率 eが 10〜25の範囲にある有機溶媒が効果的である。 誘電率とは、 物質内部の巨視的電界を Eとし、 電束密度を Dとしたときに、 D= £ Eの関係を与える εとして表される。 比誘電率とは真空の誘電率を 1とした場合の比で、 一般に誘 電率と言う場合には比誘電率を意味することが多い。 物質の比誘電率は、 あるコ ンデンサー （蓄電器） に試料を入れたときと空のときの電気容量 （それぞれ Cと C ») を比較し、 £ =C/C«の関係を使えば容易に測定することができる。 £の大きさは 溶液中のイオンの間の相互作用の強さに重要な影響を与える。 たとえば水 （25°C ) の比誘電率は 78で、 真空中に比べて水の中では静電的な力が 1/78になることを 意味している。 In the present invention, various organic solvents can be used. Of these, hydrophilic or amphiphilic organic solvents are preferred. As the hydrophilic organic solvent, for example, ethanol, methanol, 2-propanol, propanone (acetone), ether nitrile (acetonitrile), dimethyl sulfoxide (DMS0) and the like can be used. Further, as the 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. In the present invention, preferred organic solvents include those having a relative dielectric constant ε in the range of 5 to 40 at the temperature at which washing is performed (20 to 25 ° C). Among them, an organic solvent having a relative dielectric constant e in the range of 10 to 25 is particularly effective. The dielectric constant is expressed as ε, which gives the relationship D = £ E, where E is the macroscopic electric field inside the material and D is the electric flux density. 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 relative permittivity of a substance can be easily determined by comparing the capacitance (C and C ») between a sample placed in a certain capacitor (capacitor) and an empty sample (C and C», respectively), and using the relationship £ = C / C «. Can be measured. The magnitude of £ has a significant effect on the strength of the interaction between ions in solution. For example, the relative permittivity of water (25 ° C) is 78, which means that the electrostatic force in water is 1/78 of that in vacuum.
これら親水性有機溶媒あるいは両親媒性溶媒は、 単独で用いても良いし、 複数 の有機溶媒を混合して用いることもできる。 混合する場合には、 洗浄効果、 作業 上の安全性、 酵素的増幅反応に与える影響、 あるいは経済性等を考慮して組み合 わせを決定することができる。 なお実施例で用いた有機溶媒をはじめとする幅広 い有機溶媒について、 Taqポリメラ一ゼに対する阻害作用を確認したところ、 酵素 活性に重大な阻害作用を与えるものは確認できなかった。 また親水性有機溶媒の 場合には、 阻害因子の洗浄効果と DNAに与える影響などを考慮して適宜水で希釈し たものを利用することができる。 たとえばエタノールを糞便中の病原性大腸菌に 由来する PCR用核酸試料の洗浄に利用する場合、 エタノール濃度 70〜90%となるよ うに水を混和したときにもっとも良い結果を得ることができる。 洗浄時の各種条 件は、 用いる親水性有機溶媒の種類と試料との組み合わせによって適宜選択する 。 たとえば、 エタノールの場合には、 室温での処理が可能である。 These 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. For example, 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.
一方、 親油性有機溶媒であっても本発明に利用可能なものがある。 すなわち、 たとえば酢酸ェチル、 トリクロロメタン （クロ口ホルム） 、 ベンゼン、 およびジ
メチルベンゼン （キシレン） 等は本発明に利用することができる。 これらの親油 性有機溶媒については、 試料との組み合わせによっては親水性有機溶媒ほどの感 度は期待できないものの、 明らかに阻害因子の洗浄効果を示すことが確認された 。 これら親油性有機溶媒についても、 単独で用いてもよいし、 複数種を混合する ことも可能である。 On the other hand, some 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.
本発明において、 増幅の対象となる核酸は、 病原性微生物の遺伝子、 生体由来 の遺伝子等、 特に制限されない。 本発明による反応阻害因子の除去効果が大きい 糞便試料中においては、 病原性微生物は特に重要な分析対象である。 病原性微生 物とは、 細菌、 真菌、 およびリケッチア等を例示することができる。 これらの病 原性微生物は、 いずれもヒト糞便中に出現する可能性のあるもので、 その核酸を 増幅し検出することは臨床的にも大きな意味を持つ。 特に 0157:H7に代表される病 原性大腸菌、 コレラ菌、 赤痢アメーバ等は、 いったん流行すると感染を疑われる 患者、 その家族、 更には感染ルートと、 幅広い検査が必要となることから、 簡便 で迅速な分析操作が求められる。 したがって、 このような検査対象について本発 明を適用する場合には、 非常に効果的である。 In the present invention, 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. In particular, 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.
本発明の酵素的な核酸の増幅方法とは、 核酸を基質とする各種酵素による増幅 反応を意味する。 具体的には、 Taqポリメラ一ゼのような DNA依存性 DNAポリメラ一 ゼ、 逆転写酵素等の RNA依存性の DNAポリメラ一ゼ、 T7RNAポリメラ一ゼのような D NA依存性の RNAポリメラ一ゼ、 あるいは DNAリガ一ゼといった酵素を利用した、 増 幅反応である。 特に耐熱性 DNAポリメラーゼを利用した DNAの増幅反応である PCRは 、 感度の面でも、 特異性の面でも高く評価され、 現在多くの研究施設、 あるいは 検査施設において広く実施されている代表的な増幅反応である。 これらの酵素に 基づく核酸の増幅反応は、 試料中に共存する多様な成分により阻害的な影響を受 けるが、 本発明によれば有機溶媒を使った洗浄工程によって、 これら阻害因子を 確実に除去することができる。 The enzymatic nucleic acid amplification method of the present invention means an amplification reaction with various enzymes using a nucleic acid as a substrate. Specifically, 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. In particular, 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.
また本発明は、 逆転写酵素活性に対しても影響を与えないことから、 RNAを錡型
として反応を開始する RT- PCRへの応用も可能である。 RT- PCRは、 まず逆転写酵素 (RT; reverse transcriptase)により RNAを錄型として相補的 MAを合成し、 更にこ れを錡型として PCRを行う遺伝子の増幅方法で、 mRNAを検出対象とすれば実際に発 現している遺伝子の分析を可能とする。 しかし、 公知の阻害因子対策である AmpD irectを使うと、 RNAから DNAを合成する逆転写酵素反応が阻害されていた。 一方、 本発明では、 逆転写酵素への影響は無視できるうえ、 処理中には分析対象である RNAが安定に維持されることから、 RT- PCRへも容易に適用することができる。 更に、 本発明は、 Nucleic Acid Sequence-based Amp 1 i f i cat i on ( NASB A ) ( Tr ans cription Mediated Amplification(TMA)法とも呼ばれる）などの Aポリメラーゼ を利用した増幅反応への応用も可能である。 NASBAは、 標的 RNAを錶型として T7プ ロモ—夕—を付加したプライマーで DNAポリメラーゼによる DNA合成を行い、 これ を更に第 2のプライマーで 2本鎖とし、 生成する 2本鎖 DNAを鎵型として T7RNAポ リメラ一ゼによる転写を行わせて多量の RNAを増幅する反応系である（Nature, 350 , 91-92, 1991)。 本発明では、 Aポリメラ一ゼゃ DNAポリメラ一ゼに対する影響が 小さいので、 このような複雑な酵素反応も確実に実施することができる。 The present invention also has no effect on reverse transcriptase activity, It is also possible to apply to RT-PCR to start the reaction as 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. For example, it would be possible to analyze the genes that actually appear. However, the use of AmpD irect, a known measure against inhibitors, inhibited the reverse transcriptase reaction that synthesizes DNA from RNA. On the other hand, in the present invention, 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. Furthermore, 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). . NASBA conducts DNA synthesis using DNA polymerase with a primer to which the T7 promoter is added using the target RNA as type II, then converts it into double-stranded with the second primer, and converts the resulting double-stranded DNA into type II. This is a reaction system that amplifies a large amount of RNA by performing transcription using T7 RNA polymerase (Nature, 350, 91-92, 1991). In the present invention, since the influence on the A-polymerase and the DNA polymerase is small, such a complicated enzyme reaction can be surely performed.
この他、 相補的な配列上で隣接するプロ一ブが DNAリガーゼによってライゲーシ ヨンされる Ugase Chain Reaction (LCR) (Laff ler, T. G. et al. , Ann. Biol. Clin. (Paris) , 1993， 51 :9, 821-6) において本発明を応用することができる。 また、 鎖置換を行う特殊な DNAポリメラーゼとプライマーにニックを入れる制限酵 素とを組み合わせ、 複雑な温度制御を不要とした Strand Displacement Amplific ation(SDA)法（Walker GT et.al. ; Nucleic Acids Res, 1992 Apr 11, 20:7, 169 1-6) に対しても適用することができる。 このように各種ポリメラ一ゼを用い、 標 的核酸の存在によって何らかの増幅生成物を与える酵素反応であればいずれも本 発明を応用することができる。 In addition, Ugase Chain Reaction (LCR) in which adjacent probes on complementary sequences are ligated by DNA ligase (Laffler, TG et al., Ann. Biol. Clin. (Paris), 1993, 51 : 9, 821-6), the present invention can be applied. The Strand Displacement Amplification (SDA) method (Walker GT et.al .; Nucleic Acids Res.) That eliminates the need for complicated temperature control by combining a special DNA polymerase that performs strand displacement and a restriction enzyme that nicks primers. , 1992 Apr 11, 20: 7, 169 1-6). As described above, the present invention can be applied to any enzymatic reaction that provides various amplification products in the presence of a target nucleic acid using various polymerases.
以下実施例に基づいて本発明を具体的に説明する。
図面の簡単な説明 Hereinafter, the present invention will be specifically described based on examples. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明による糞便中に含まれる核酸の酵素的増幅方法のフローチヤ一 トである。 発明を実施するための最良の形態 FIG. 1 is a flowchart of the method for enzymatically amplifying nucleic acids contained in feces according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
( 1 ) 試料の調製 (1) Sample preparation
糞便等の生体試料中に存在する病原性大腸菌 0157の検出を試みた。 被検試料と しては、 次の 4種類を用意した。 一連の操作は図 1に示した。 An attempt was made to detect pathogenic Escherichia coli 0157 present in a biological sample such as feces. The following four types of test samples were prepared. The sequence of operations is shown in FIG.
1 ) 病原性大腸菌 0157 :H7感染患者の糞便検体： 7例 （下痢便） 1) Fecal specimens from patients with pathogenic E. coli 0157: H7 infection: 7 cases (diarrheal stool)
2 ) 病原性大腸菌 0157:H7健康保菌者の糞便検体： 1例 （冷凍便） 2) Fecal specimen of pathogenic Escherichia coli 0157: H7 healthy carrier: 1 case (frozen stool)
3 ) 培養した病原性大腸菌 0157:H7を健常糞便検体に添加： 1例 （固体便） 3) Cultured pathogenic Escherichia coli 0157: H7 added to healthy feces specimen: 1 case (solid stool)
4 ) 培養した病原性大腸菌 0157:H7を A型血液に添加： 1例 4) Cultured pathogenic E. coli 0157: H7 added to blood type A: 1 case
以下の操作に基づいて、 本発明による核酸の増幅反応のための上記試料を調製 した。 The above-mentioned sample for the nucleic acid amplification reaction according to the present invention was prepared based on the following operations.
まず、 O. nLの糞便 （または血液） を採取し、 リン酸緩衝液 (ρΗ7· 0)に懸濁させ た。 これを 2000rpmで 5分間遠心分離して上清を回収し、 菌体よりも大きな残滓を 除いた。 回収した上清は、 15000rpmで 15分間遠心分離し、 沈でんとして菌体を濃 縮した。 上清を捨て、 沈でんに 0.9mLの各種有機溶媒を加え、 良く混和して再び遠 心分離（15000rpm、 10分） した。 この沈でんを以降の酵素的増幅反応の試料とした 。 実験に用いた有機溶媒は表 2に示したとおり、 メタノール、 エタノール （70〜 90%) 、 2-プロパノール （イソプロパノール） 、 プロパノン （アセトン） 、 ェ夕ン 二トリル （ァセトニトリル） 、 ジメチルスルホキシド（DMS0)、 ブ夕ノール、 2-ブ 夕ノール、 酢酸ェチル、 トリクロロメタン （クロ口ホルム） 、 ；[-へキサノール、 ジェチルエーテル、 n-へキサン、 ベンゼン、 メチルベンゼン （トルエン） 、 およ びジメチルベンゼン （キシレン） である。 この他に、 対照として有機溶媒に代え てリン酸緩衝液を洗浄に用いた試料を用意した。
( 2 ) PCR洗浄後の沈でん 1 L分を採取し、 ExTaq用緩衝液 （宝酒造製） 1 /Lに錡 型として加えた。 99°Cで 20分間、 次いで 95°C3分間インキュベートして DNAを 1本鎖 とし、 プライマ一、 dNTP、 および 2倍量の TaKaRa ExTaqポリメラーゼ （宝酒造製） を添加して PCRを開始した。 PCRは、 94°C/1分間； 61°C/0.5分間； 72°C/1分間を 40 サイクルとし、 40サイクルの後に 72°Cで 7分間ィンキュベートし、 次いで 4°Cに冷 却した。 反応後、 反応液の一部を電気泳動してェチジゥムプロマイ ド染色により 増幅生成物を確認した。 プライマーには配列番号： 1 ~ 3に示したセンスプライ マ一に、 配列番号： 4で示したアンチセンスプライマ一を組み合わせて用いた。 これらのプライマーと増幅生成物の関係は以下に示すとおりである。 各試料と確 認された感度については表 1に結果を示した。 また、 健常者の糞便に培養した大 腸菌 0157を添加したものを基質とし、 各溶媒で遠心洗浄 （1回） して PCRを行った 結果を表 2にまとめた。 First, feces (or blood) of O. nL were collected and suspended in a phosphate buffer (ρΗ7.0). 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). , Butanol, 2-butanol, ethyl acetate, trichloromethane (chloroform), [-hexanol, getyl ether, n-hexane, benzene, methylbenzene (toluene), and dimethylbenzene (Xylene). In addition, a sample using phosphate buffer instead of the organic solvent for washing was prepared as a control. (2) 1 L of the precipitate after PCR washing was collected and added to 1 / L of ExTaq buffer solution (Takara Shuzo) as a 錡 type. The DNA was made single-stranded by incubating at 99 ° C for 20 minutes and then at 95 ° C for 3 minutes, and PCR was started by adding a primer, dNTP, and 2 times the amount of TaKaRa ExTaq polymerase (Takara Shuzo). The PCR was performed at 40 cycles of 94 ° C / 1 minute; 61 ° C / 0.5 minute; 72 ° C / 1 minute. After 40 cycles, the mixture was incubated at 72 ° C for 7 minutes, and then cooled to 4 ° C. After the reaction, a part of the reaction solution was subjected to electrophoresis, and the amplification product was confirmed by ethidium bromide staining. 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.
配列番号： 1 配列番号： 2 配列番号： 3 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3
配列番号： 4 143bp 256bp 391bp SEQ ID NO: 4 143bp 256bp 391bp
表 1 table 1
90%エタノールで洗浄した各試料における病原性大腸菌の検出感度 試料 病原性大腸菌を添加した Sensitivity of detection of pathogenic E. coli in each sample washed with 90% ethanol Sample Pathogenic E. coli was added
健常者の糞便 5 x l03CFU/0. 1g便 Normal person's feces 5 x l0 3 CFU / 0.1g stool
血液 5 x l02CFU/0. 1mL血液 表 1に示したとおり、 エタノールを用いた洗浄の結果、 糞便、 ならびに血液の いずれの試料においても、 病原性大腸菌の高感度な検出が可能であった。 なおェ 夕ノールの濃度と検出結果の間に特別な傾向は見られず、 70〜90%の間では同程度
の検出感度を得ることができた。 表 1にまとめた結果に対して、 病原性大腸菌 01 57:H7感染患者の糞便検体： 5例についても同様の結果が得られた。 Blood 5 x 10 2 CFU / 0.1 mL blood As shown in Table 1, washing with ethanol enabled highly sensitive detection of pathogenic Escherichia coli in both fecal and blood samples. . No special tendency was observed between the concentration of ethanol and the detection result, and the same level was observed between 70% and 90%. Could be obtained. In contrast to the results summarized in Table 1, similar results were obtained for stool specimens of 5 patients infected with pathogenic Escherichia coli 01 57: H7.
表 2 各種溶媒によって洗浄した 0157添加試料の PCRによる病原性大腸菌の検出結果 溶媒 構造 PCRの結果 誘電率 e 対照 （リン酸緩衝液） 親水性有機溶媒 Table 2 Detection results of pathogenic Escherichia coli by PCR of 0157 spiked sample washed with various solvents Solvent Structure PCR result Dielectric constant e Control (phosphate buffer) Hydrophilic organic solvent
メタノール CHaOH + 32.68 Methanol CHaOH + 32.68
エタノール CH3CH20H + 24.5 Ethanol CH 3 CH 2 0H + 24.5
(70-90%) (70-90%)
2-プロパノール CH3CHCH30H + 19.52 2-propanol CH 3 CHCH 3 0H + 19.52
(イソプロパノール） (Isopropanol)
プロパノン 0 + 20.7 Propanone 0 + 20.7
(ァセ卜ン） CH3-C-CH3 (Acetone) CH 3 -C-CH 3
エタンニトリル CH,C≡N + 37.5 Ethanitrile CH, C≡N + 37.5
(ァセトニトリル） (Acetonitrile)
ジメチルスルホキシド 0 Dimethyl sulfoxide 0
(DMS0) CH3-S-CH3 + 48.9 (DMS0) CH 3 -S-CH 3 + 48.9
両親媒性溶媒 Amphiphilic solvent
ブタノ一ル
+ 17.5 Butanol + 17.5
0H 0H
2 -ブタノール CiyJHCH^ + 16.56 2-butanol CiyJHCH ^ + 16.56
0 0
ft酸ェチル CH,-C-0-CH,CH, + 6.02 ft Ethyl CH, -C-0-CH, CH, + 6.02
親油性有機溶媒 Lipophilic organic solvent
トリクロロメタン CHCI, + 4.86 Trichloromethane CHCI, + 4.86
(クロ口ホル厶） (Kuroguchi Holm)
1-へキサノール
13.3 1-hexanol 13.3
ジェチルエーテル CH3CH2-0-CH2CH3 4.335 Getyl ether CH 3 CH 2 -0-CH 2 CH3 4.335
n-へキサン
2 n-hexane Two
ベンゼン + 2.274 Benzene + 2.274
メチルベンゼン CH3 2.379 Methylbenzene CH3 2.379
(トルエン） (Toluene)
ジメチルベンゼン CH3 + 2.37 Dimethylbenzene CH 3 + 2.37
(キシレン） ©-OH,
一方、 溶媒間の比較においては、 親水性有機溶媒によって洗浄した場合には PC Rの結果はすべて陽性となっているのに対して、 親油性有機溶媒で洗浄した場合に 陰性の結果を与えるものが見られた （表 2 ) 。 なお、 リン酸緩衝液で洗浄した場 合には PCiUこよる検出は不可能であった。 また、 陰性の結果を与えた有機溶媒につ いて別に PCRへの影響を確認したところ、 特に阻害的な影響は観察されなかった。 したがって、 以上の結果は洗浄に用いる溶媒の選択が PCRの結果に影響しているこ とを示すものと考えられた。 (Xylene) © -OH, On the other hand, in the comparison between solvents, all the PCR results were positive when washed with a hydrophilic organic solvent, whereas the results were negative when washed with a lipophilic organic solvent. (Table 2). Note that detection with PCiU was not possible when washing with phosphate buffer. In addition, when the effect on PCR of the organic solvent that gave a negative result was separately confirmed, no particularly inhibitory effect was observed. Therefore, the above results were considered to indicate that the choice of solvent used for washing had an effect on the PCR results.
( 3 ) NASBA (3) NASBA
( 1 ) で調製した病原性大腸菌添加血液試料 （90%エタノールで 1回洗浄したも の） について、 NASBAを試みた。 NASBAには東洋紡績株式会社から商業的に供給さ れているキットを用い、 添付の指示書にしたがって酵素反応を実施した。 操作は 以下のとおりである。 NASBA was performed on the blood sample containing pathogenic E. coli (washed once with 90% ethanol) prepared in (1). For NASBA, 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.
( 1 ) で得られた洗浄後の沈でん 50 /L分を採取し、 エタノールを乾燥させてか ら の滅菌水を加えてよく攪袢した。 その を分取して NASBA用の試料とし 、 10 /Lのプライマ一溶液 （下記のとおり） を加えて 61°Cで 5分間インキュベート 後、 更に NASBAに必要な酵素ミックス （層- RT、 RNaseH, T7RNAポリメラ一ゼ、 BS Aなどを含有、 5 L)を加えて 41°Cで 95分間反応させた。 この反応により RNAにァニ —ルした第 1プライマーを起点として DNAが合成され、 次いで錶型となった MAが 酵素的に除去されて第 2ブラィマ一がァニールする。 第 2プライマーが合成起点 となって、 DNAは T7プロモー夕一を含む 2本鎖となり、 これを錶型として標的配列 のアンチセンス配列を持つ RNAが転写される。 プライマ一には、 （2 ) の PCRで用 いたものと同じセッ トを用いた。 ただし、 アンチセンス側のプライマ一には、 そ の 5，側に T7プロモーター配列 25bpを付加して第 1プライマーとした （配列番号： 5 ) 。 反応後、 反応液の一部を電気泳動してェチジゥムブ口マイ ド染色により増 幅生成物を確認した。 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. Then, the enzyme mix required for NASBA (layer-RT, RNaseH, 5 L) containing T7 RNA polymerase, BSA, etc. was added, and reacted at 41 ° C for 95 minutes. By this reaction, 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. Using the second primer as a starting point for synthesis, 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. However, to the primer on the antisense side, 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.
プライマー溶液：
NASBE凍結乾燥試薬 1本 Primer solution: One NASBE freeze-dried reagent
NASBE溶解液 50//L NASBE solution 50 // L
KC1溶液 KC1 solution
10 /M第 1ブラィマ一溶液 5 L 10 / M 1st Braima Solution 5 L
10〃M第 2プライマ一溶液 5〃L 10〃M second primer solution 5 一 L
NASBATK Sl . Gjul NASBATK Sl. Gjul
合計 120 /L Total 120 / L
その結果、 いずれのプライマ一セットを利用した場合にも、 3.2 x l03CFU/0. 1m L血液の感度で病原性大腸菌の検出が可能であった。 この実験により前処理技術を 利用した市販の AmpDirectとは異なり、 本発明によれば血液を試料として逆転写酵 素反応を伴う酵素的な増幅反応を実施できることが確認された。 産業上の利用の可能性 As a result, it was possible to detect pathogenic Escherichia coli with sensitivity of 3.2 × 10 3 CFU / 0.1 mL of blood using any set of primers. This experiment confirmed that unlike the commercially available AmpDirect using the pretreatment technique, according to the present invention, an enzymatic amplification reaction involving a reverse transcriptase reaction can be performed using blood as a sample. Industrial applicability
本発明によれば、 核酸の酵素的な合成反応を行うための試料を、 容易に調製す ることができる。 特に糞便のような複雑な共存系に対しても、 確実に反応阻害因 子を除去することができる。 加えて本発明は、 各種の酵素反応に対する影響が小 さいので、 さまざまな反応に対して応用が可能である。 たとえば、 AmpDirectは R T-PCRや NASBAに応用できないが、 本発明は容易に適用することができる。 According to the present invention, a sample for performing an enzymatic synthesis reaction of a nucleic acid can be easily prepared. In particular, even for complex coexisting systems such as feces, it is possible to reliably remove the reaction inhibitory factor. In addition, the effect of the present invention on various enzyme reactions is small, so that the present invention can be applied to various reactions. For example, AmpDirect cannot be applied to RT-PCR or NASBA, but the present invention can be easily applied.
本発明により、 たとえば糞便中の病原性大腸菌の迅速な検出が実現する。 糞便 の培養を行うこと無く、 糞便中に存在する病原性大腸菌の遺伝子を高い感度で直 接検出可能なことから、 長い間望まれていた培養時間を必要としない微生物検査 が現実のものとなる。
According to the present invention, for example, rapid detection of pathogenic E. coli in feces is realized. The ability to directly detect the pathogenic Escherichia coli gene present in stool with high sensitivity without stool cultivation makes microbial testing that does not require long culturing time a reality .
Claims
1 . 有機溶媒で被検試料を洗浄して核酸の酵素的増幅反応を阻害する物質を除去 し、 該被検試料中に含まれる細胞の核酸を酵素的に増幅させることを含む、 核酸 の酵素的増幅方法。 1. A nucleic acid enzyme comprising washing a test sample with an organic solvent to remove a substance that inhibits an enzymatic amplification reaction of nucleic acids, and enzymatically amplifying nucleic acids of cells contained in the test sample. Amplification method.
2 . 有機溶媒が親水性溶媒、 または両親媒性の有機溶媒である、 請求項 1に記載 の核酸の酵素的増幅方法。 2. The method according to claim 1, wherein the organic solvent is a hydrophilic solvent or an amphiphilic organic solvent.
3 . 有機溶媒の比誘電率 £が 5〜40である、 請求項 2に記載の核酸の酵素的増幅方 法。 3. The method for enzymatic amplification of nucleic acids according to claim 2, wherein the relative permittivity of the organic solvent is from 5 to 40.
4 . 有機溶媒が 70%エタノール水溶液、 メタノール、 2-プロパノール、 アセトン、 ァセトニトリル、 ジメチルスルホキシド、 ブ夕ノール、 2-ブ夕ノール、 および酢 酸ェチルで構成される群から選択される、 請求項 3に記載の核酸の酵素的増幅方 法。 4. The organic solvent is selected from the group consisting of 70% aqueous ethanol, methanol, 2-propanol, acetone, acetonitrile, dimethyl sulfoxide, butanol, 2-butanol, and ethyl acetate. The method for enzymatic amplification of a nucleic acid according to 1.
5 . 細胞が微生物細胞または血液細胞である、 請求項 1〜4のいずれかに記載の 核酸の酵素的増幅方法。 5. The method for enzymatic amplification of a nucleic acid according to any one of claims 1 to 4, wherein the cell is a microbial cell or a blood cell.
6 . 微生物細胞が病原性大腸菌である、 請求項 5に記載の核酸の酵素的増幅方法 ο 6. The method for enzymatic amplification of nucleic acid according to claim 5, wherein the microbial cell is pathogenic Escherichia coli.
7 . 被検試料が糞便である、 請求項 1〜 6のいずれかに記載の核酸の酵素的増幅 方法。 7. The nucleic acid enzymatic amplification method according to any one of claims 1 to 6, wherein the test sample is feces.
8 . 有機溶媒による洗浄工程の前に、 微生物細胞よりも大きな残滓の一部または 全部を除去する工程を含む、 請求項 7に記載の核酸の酵素的増幅方法。 8. The method for enzymatic amplification of a nucleic acid according to claim 7, comprising a step of removing a part or all of a residue larger than the microorganism cells before the washing step with an organic solvent.
9 . 有機溶媒による洗浄を遠心分離により実施する、 請求項 1〜8のいずれかに 記載の核酸の酵素的増幅方法。 9. The method for enzymatic amplification of a nucleic acid according to any one of claims 1 to 8, wherein washing with an organic solvent is performed by centrifugation.
1 0 . 酵素的増幅を PCRによって行う、 請求項 1〜9のいずれかに記載の核酸の酵 素的増幅方法。 10. The method for enzymatic amplification of a nucleic acid according to any one of claims 1 to 9, wherein the enzymatic amplification is performed by PCR.
1 1 . 請求項 1に記載の方法によって生成する増幅生成物の有無を調べることに
より、 被検試料中の微生物を検出する方法。 11. Examining the presence or absence of amplification products produced by the method of claim 1. A method for detecting microorganisms in a test sample.
1 2. 比誘電率 が 5〜40である有機溶媒を含む、 酵素的増幅用核酸基質の洗浄剤
1 2. Detergent for nucleic acid substrate for enzymatic amplification, containing organic solvent with relative dielectric constant of 5 to 40
Priority Applications (1)
| 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)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10/220446 | 1998-08-04 | ||
| JP22044698 | 1998-08-04 | ||
| JP11/95600 | 1999-04-01 | ||
| JP9560099 | 1999-04-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2000008136A1 true WO2000008136A1 (en) | 2000-02-17 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP1999/004189 WO2000008136A1 (en) | 1998-08-04 | 1999-08-03 | Method for enzymatic amplification of nucleic acid |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU4934099A (en) |
| WO (1) | WO2000008136A1 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006101818A (en) * | 2004-10-08 | 2006-04-20 | Eiken Chem Co Ltd | Method for avoiding inhibition in nucleic acid amplification reaction |
| US7498407B2 (en) | 2001-11-09 | 2009-03-03 | Georgetown University | Vascular endothelial cell growth inhibitor, VEGI-192a |
| WO2009139317A1 (en) * | 2008-05-12 | 2009-11-19 | オリンパス株式会社 | Method of processing excrement and container for processing excrement |
| WO2010010914A1 (en) * | 2008-07-23 | 2010-01-28 | オリンパス株式会社 | Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample |
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
| EP2392670A1 (en) | 2010-06-03 | 2011-12-07 | Olympus Corporation | Method for detecting nucleic acid in biological sample |
| 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 (en) * | 2014-09-04 | 2017-09-07 | テックラブ，インコーポレーテッド | Nucleic acid extraction using organic solvents to remove inhibitors |
| 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 |
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| JPH05211898A (en) * | 1990-01-19 | 1993-08-24 | F Hoffmann La Roche Ag | Method and kit for detecting water system pathogenic microorganism in water test sample and indicator microorganism contaminated with human excrements |
| JPH06165676A (en) * | 1992-06-16 | 1994-06-14 | Wako Pure Chem Ind Ltd | Method for extracting nucleic acid chain from cell of mycobacterium bacterium |
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|---|---|---|---|---|
| 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 |
| JP4674073B2 (en) * | 2004-10-08 | 2011-04-20 | 栄研化学株式会社 | Method for avoiding inhibition in nucleic acid amplification reaction |
| JP2006101818A (en) * | 2004-10-08 | 2006-04-20 | Eiken Chem Co Ltd | Method for avoiding inhibition in nucleic acid amplification reaction |
| 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 |
| JP5616786B2 (en) * | 2008-05-12 | 2014-10-29 | オリンパス株式会社 | Fecal treatment container |
| US8597954B2 (en) | 2008-05-12 | 2013-12-03 | Olympus Corporation | Stool sample processing method and stool sample processing container |
| WO2009139317A1 (en) * | 2008-05-12 | 2009-11-19 | オリンパス株式会社 | Method of processing excrement and container for processing excrement |
| CN102027132A (en) * | 2008-05-12 | 2011-04-20 | 奥林巴斯株式会社 | Method of processing excrement and container for processing excrement |
| WO2010010914A1 (en) * | 2008-07-23 | 2010-01-28 | オリンパス株式会社 | Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample |
| JP5710969B2 (en) * | 2008-07-23 | 2015-04-30 | オリンパス株式会社 | Nucleic acid recovery method from stool samples |
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
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| 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 |
| 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 |
| US11198899B2 (en) | 2014-03-07 | 2021-12-14 | Dna Genotek Inc. | Composition and method for stabilizing nucleic acids in biological samples |
| US10435735B2 (en) | 2014-03-07 | 2019-10-08 | Dna Genotek Inc. | Composition and method for stabilizing nucleic acids in biological samples |
| JP2017525383A (en) * | 2014-09-04 | 2017-09-07 | テックラブ，インコーポレーテッド | Nucleic acid extraction using organic solvents to remove inhibitors |
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|---|---|
| AU4934099A (en) | 2000-02-28 |
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