JP2007143495A - Method for crushing material to be crushed in sample - Google Patents

Method for crushing material to be crushed in sample Download PDF

Info

Publication number
JP2007143495A
JP2007143495A JP2005343698A JP2005343698A JP2007143495A JP 2007143495 A JP2007143495 A JP 2007143495A JP 2005343698 A JP2005343698 A JP 2005343698A JP 2005343698 A JP2005343698 A JP 2005343698A JP 2007143495 A JP2007143495 A JP 2007143495A
Authority
JP
Japan
Prior art keywords
sample
nucleic acid
crushed
crushing
powder
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
JP2005343698A
Other languages
Japanese (ja)
Other versions
JP4797602B2 (en
Inventor
Daisuke Miki
大輔 三木
Kenji Masui
健治 増井
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tosoh Corp
Original Assignee
Tosoh Corp
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 Tosoh Corp filed Critical Tosoh Corp
Priority to JP2005343698A priority Critical patent/JP4797602B2/en
Publication of JP2007143495A publication Critical patent/JP2007143495A/en
Application granted granted Critical
Publication of JP4797602B2 publication Critical patent/JP4797602B2/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for crushing a sample which is difficult to crush and efficiently extracting a nucleic acid in a short time. <P>SOLUTION: In the method for crushing a material to be crushed in a sample by adding a powdery solid material which is amorphous to a sample containing a material to be crushed which is a microorganism or an organic tissue, and stirring the sample, major axis of the powdery solid material is ≤32 μm and the specific gravity is ≥3.5 and the hardness (Hv10) is ≥600. A method for extracting a nucleic acid from the microorganism and the organic tissue are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、粉末状の固形物を用いて試料中の微生物又は生物組織を効率よく破砕する方法に関するものである。   The present invention relates to a method for efficiently crushing microorganisms or biological tissues in a sample using a powdery solid.

細菌やウイルスによる感染症その他の病気の原因を確定診断するためには、その病気の原因となっている細菌やウイルスを検出し同定することが必要である。   In order to make a definitive diagnosis of the cause of infection or other diseases caused by bacteria or viruses, it is necessary to detect and identify the bacteria or viruses that cause the disease.

臨床診断等の分野では、試料から核酸を抽出し、標的細菌またはウイルスの遺伝子を増幅し検出することにより、病気の確定診断が行われている(特開2000−14400号公報「標的核酸の定量方法」(特許文献1);Anal.Biochem.,314,77−86(2003)(非特許文献1))。さらに、人の各組織中の細胞から抽出した核酸は、癌やその他の病気の診断マーカーとして利用可能であり(Ann.Surg.Oncol.,11,778−785(2004)(非特許文献2))、病気の早期診断、治療のモニタリング、予後判定、治療方針決定の指標に有用である(J.Clin.Microbiol.,42,4284−4292(2004)(非特許文献3);J.Clin.Microbiol.,43,2489−2491(2005)(非特許文献4))。これら遺伝子検査の場合、試料からの核酸の抽出が重要な操作となっている。核酸抽出のための細胞破砕法には、ホモジェナイザー等による機械的方法、変性剤等による化学的な方法(Molecular Cloning:A laboratory manual Appendix 7.23−7.25(New York:Cold Spring Harbor Laboratory,1989)、酵素等による生物学的方法がある(Molecular Cloning:A laboratory manual Appendix E3−E4(New York:Cold Spring Harbor Laboratory,1989)。又、凍結した試料に粒子状の固形物を添加し混在させ、試料を攪拌することにより細胞を破砕する方法がある。   In the field of clinical diagnosis and the like, a definite diagnosis of a disease is performed by extracting a nucleic acid from a sample and amplifying and detecting a target bacterial or viral gene (Japanese Patent Laid-Open No. 2000-14400, “Quantification of target nucleic acid”). Method (Patent Document 1); Anal. Biochem., 314, 77-86 (2003) (Non-Patent Document 1)). Furthermore, nucleic acids extracted from cells in human tissues can be used as diagnostic markers for cancer and other diseases (Ann. Surg. Oncol., 11, 778-785 (2004) (Non-patent Document 2)). ), Early diagnosis of disease, treatment monitoring, prognosis determination, and index of treatment policy determination (J. Clin. Microbiol., 42, 4284-4292 (2004) (Non-patent Document 3); J. Clin. Microbiol., 43, 2489-2491 (2005) (non-patent document 4)). In these genetic tests, extraction of nucleic acids from a sample is an important operation. Cell disruption methods for nucleic acid extraction include mechanical methods using homogenizers, chemical methods using denaturants, etc. (Molecular Cloning: A laboratory manual Appendix 7.23-7.25 (New York: Cold Spring Harbor). (Laboratory, 1989), and biological methods using enzymes, etc. (Molecular Cloning: A laboratory manual Appendix E3-E4 (New York: Cold Spring Harbor Laboratory, 1989). There is a method of crushing cells by mixing them and stirring the sample.

臨床診断における遺伝子検査において、これら従来法による核酸抽出法では、微生物によっては細胞壁が硬く十分に細菌を破砕することができず細菌の遺伝子を効率良く抽出することが困難なものも存在するという問題点があった。又、核酸抽出のために細胞壁を破砕するために時間がかかり、結果的に核酸の抽出に時間がかかるという問題点があった。又、凍結した試料に粒子状の固形物を添加し混在させ、試料を攪拌することにより組織や細胞を破砕する場合は、試料を予め凍結する必要があり、凍結のための時間がかかったり、試料を処理している間に試料が溶解して、効率よく細胞を破砕できないという問題点があった。又、その場合、試料を凍結したまま破砕するという特別な装置が必要であった。   In genetic testing in clinical diagnosis, these conventional nucleic acid extraction methods have problems that some microorganisms have a hard cell wall that cannot sufficiently crush bacteria, making it difficult to efficiently extract bacterial genes. There was a point. In addition, it takes time to crush the cell wall for nucleic acid extraction, and as a result, it takes time to extract nucleic acid. In addition, in the case where tissues and cells are disrupted by adding and mixing particulate solids to a frozen sample and stirring the sample, it is necessary to freeze the sample in advance, which takes time for freezing, There was a problem that the sample was dissolved while the sample was being processed, and the cells could not be efficiently disrupted. In that case, a special apparatus for crushing the sample while it was frozen was necessary.

以上の様に、試料に含まれる標的微生物や組織からの核酸抽出が困難であったり、時間がかかる場合は、病気の原因となっている微生物を遺伝子検査により高感度あるいは迅速に検出し特定することができない問題や、組織中の遺伝子が病気の遺伝子マーカーとして使用できない問題があった。   As described above, if extraction of nucleic acids from target microorganisms or tissues contained in a sample is difficult or takes a long time, the microorganisms causing the disease are detected and identified with high sensitivity or rapidity by genetic testing. There were problems that could not be used, and that genes in tissues could not be used as genetic markers for diseases.

特開2000−14400号公報JP 2000-14400 A

Anal.Biochem.,314,77−86(2003)Anal. Biochem. , 314, 77-86 (2003) Ann.Surg.Oncol.,11,778−785(2004)Ann. Surg. Oncol. , 11, 778-785 (2004) J.Clin.Microbiol.,42,4284−4292(2004)J. et al. Clin. Microbiol. , 42, 4284-4292 (2004) J.Clin.Microbiol.,43,2489−2491(2005)J. et al. Clin. Microbiol. , 43, 2489-2491 (2005)

本発明の目的は、破砕困難な試料を破砕し、効率よく短時間で核酸を抽出する方法を提供することにある。これにより、効率よく短時間で微生物から核酸を抽出し、遺伝子を検出同定することにより、細菌やウイルスによる感染症の確定診断を行うことができる。又、組織から短時間で核酸を抽出し、病気のマーカー遺伝子を検出することにより、手術中の治療方針決定、治療のモニタリングの指標にすることができる。   An object of the present invention is to provide a method for crushing a sample that is difficult to crush and efficiently extract nucleic acid in a short time. Thereby, a nucleic acid is efficiently extracted from a microorganism in a short time, and a gene is detected and identified, so that a definitive diagnosis of an infection caused by bacteria or viruses can be performed. Further, by extracting nucleic acid from a tissue in a short time and detecting a marker gene for a disease, it can be used as an index for determination of treatment policy during surgery and monitoring of treatment.

本発明者らは研究を重ねた結果、試料中の微生物および組織を効率よく破砕する方法を見出した。
本発明は、粉末状固形物を、被破砕物を含む試料に添加し、該試料を攪拌することにより試料中の被破砕物を破砕する方法において、該粉末状固形物の長径が32μm以下であり、その比重が3.5以上であり、その硬度(Hv10)が600以上である、被破砕物の破砕方法を提供する。
好適な態様において、該粉末状固形物は無定形である。さらに、該被破砕物は微生物又は生物組織であることができる。より好ましい態様において、該粉末状固形物はジルコニアである。
別の観点において、本発明は上記方法により破砕した被破砕物から核酸を抽出することを特徴とする核酸の抽出方法も提供する。
As a result of repeated studies, the present inventors have found a method for efficiently disrupting microorganisms and tissues in a sample.
The present invention relates to a method for crushing a material to be crushed in a sample by adding the powdered solid material to a sample containing the material to be crushed and stirring the sample, and the major axis of the powdered solid material is 32 μm or less. There is provided a method for crushing an object to be crushed, having a specific gravity of 3.5 or more and a hardness (Hv10) of 600 or more.
In a preferred embodiment, the powdered solid is amorphous. Furthermore, the material to be crushed can be a microorganism or a biological tissue. In a more preferred embodiment, the powdered solid is zirconia.
In another aspect, the present invention also provides a method for extracting nucleic acid, characterized in that nucleic acid is extracted from the material to be crushed by the above method.

本発明に従えば、試料中の微生物又は生物組織が、硬い細胞壁を持った物であっても効率よく破砕することができ、それらの核酸を抽出し遺伝子の高感度・迅速検査を行うことが可能である。また、試料を凍結する必要がないことから、装置の低価格化、小型化が可能である。これにより、効率よく短時間で微生物から核酸を抽出し、遺伝子を検出同定することにより、細菌やウイルスによる感染症の確定診断を行うことができる。又、組織から短時間で核酸を抽出し、病気のマーカー遺伝子を検出することにより、手術中の治療方針決定、治療のモニタリングの指標にすることができる。   According to the present invention, microorganisms or biological tissues in a sample can be efficiently disrupted even if they have a hard cell wall, and their nucleic acids can be extracted to perform high-sensitivity and rapid testing of genes. Is possible. In addition, since it is not necessary to freeze the sample, it is possible to reduce the cost and size of the apparatus. Thereby, a nucleic acid is efficiently extracted from a microorganism in a short time, and a gene is detected and identified, so that a definitive diagnosis of an infection caused by bacteria or viruses can be performed. Further, by extracting nucleic acid from a tissue in a short time and detecting a marker gene for a disease, it can be used as an index for determination of treatment policy during surgery and monitoring of treatment.

以下、本発明を詳細に説明する。
本発明における粉末状固形物質とは、長径が32μm以下であり、その比重が3.5以上であり、その硬度(Hv10)が600以上であるという条件を満たす物質であれば何でも良く、無機材料でも有機材料であってもよい。かかる粉末状固形物質の材料の限定でない例として、例えば、ジルコニア、ダイアモンド、鉄、合金、例えばタングステンカーバイド−コバルト合金(WC−Co合金)といった無機材料等が挙げられる。
Hereinafter, the present invention will be described in detail.
The powdered solid substance in the present invention may be any inorganic substance as long as it satisfies the condition that the major axis is 32 μm or less, the specific gravity is 3.5 or more, and the hardness (Hv10) is 600 or more. However, it may be an organic material. Non-limiting examples of the material of the powdered solid substance include inorganic materials such as zirconia, diamond, iron, alloys such as tungsten carbide-cobalt alloy (WC-Co alloy).

本発明における粉末状固形物は不均一で無定形の形状の粉末であることが好ましいが、均一な形状のものであってもよい。粉末状固形物は球形、楕円形等の滑らかな角を持った形状ではなく、鋭利な角を持つ角張った形状を有することが好ましい。このような形状の具体例としては、例えば先端が角形状を有する1個所以上の縁部を保有する半球、円錐、三角錐、四角錐、円柱、立方体、三角乃至十六角柱、板状、例えば三角形、四角形、五角形等を含む多角形状の板、これらの形状が複合した形状、鋭利な突起を有する星型形状、などが挙げられる。粉末状固形物の形状は例えば位相差光学顕微鏡や走査型もしくは透過型電子顕微鏡などにより観察で確認することができる。   The powdered solid in the present invention is preferably a non-uniform and amorphous powder, but may be of a uniform shape. The powdered solid material preferably has an angular shape with sharp corners, not a shape with smooth corners such as a sphere or an ellipse. Specific examples of such a shape include, for example, a hemisphere, a cone, a triangular pyramid, a quadrangular pyramid, a cylinder, a cube, a triangular to a hexagonal prism, a plate, For example, a polygonal plate including a triangle, a quadrangle, a pentagon, and the like, a shape in which these shapes are combined, a star shape having a sharp protrusion, and the like can be given. The shape of the powdered solid can be confirmed by observation with, for example, a phase-contrast optical microscope, a scanning type, or a transmission type electron microscope.

粒子の大きさはその粉末の径の最も長い部分が32μm以下であればよいが、好ましくは20μm以下であればよい。その比重は、攪拌の運動エネルギーを利用して組織を破砕することから、大きい方が好ましく、3.5以上あればよい。粒子の比重の上限は特に限定されるものではないが、粒子の製造や入手のし易さを考慮すれば、例えば最大で約20、より好ましくは最大で約10程度であろう。試料と粉末状固形物との分離は遠心分離で行えばよく、比重が試料溶液より大きい場合は、静置により粉末状固形物が沈降し試料と分離することができる。また、粉末状固形物にあらかじめ磁性体を含有させておくことにより、磁力による分離が可能である。粉末状固形物の硬さは、硬いほど好ましく、ビッカース硬度(Hv10)にして600以上、好ましくは1000以上であることが好ましい。硬度はまた、試料への粉末状固形物の添加量は、試料の種類、攪拌方法、攪拌時間によって適宜決められる。硬度は例えば、JISハンドブック:セラミックス、財団法人日本規格協会、p315−317に従い測定することができる。   As for the size of the particles, the longest part of the diameter of the powder may be 32 μm or less, preferably 20 μm or less. The specific gravity is preferably larger, because the tissue is crushed using the kinetic energy of stirring, and may be 3.5 or more. The upper limit of the specific gravity of the particles is not particularly limited, but in consideration of the production and availability of the particles, for example, it will be about 20 at the maximum, more preferably about 10 at the maximum. The sample and the powdered solid may be separated by centrifugation. When the specific gravity is larger than the sample solution, the powdered solid can settle and be separated from the sample by standing. Moreover, separation by magnetic force is possible by incorporating a magnetic substance in the powder solid. The hardness of the powdery solid is preferably as hard as possible, and is preferably 600 or more, preferably 1000 or more in terms of Vickers hardness (Hv10). For the hardness, the amount of powdered solid added to the sample is appropriately determined according to the type of sample, the stirring method, and the stirring time. The hardness can be measured, for example, according to JIS Handbook: Ceramics, Japanese Standards Association, p315-317.

本発明における粉末状固形物は市販のものであっても、あるいは調製したものであってもよい。例えば、本発明における粉末状固形物の原材料となる物質、例えば板状ジルコニアを粉砕機により適宜破砕し、得られた粉末状を、所望する大きさの粉末を選別するのに適切な孔径を有するふるい(例えば、粒子の長径が32μm以下の粉末を所望する場合はφ32μm以下のふるい、粒子の長径が20μm以下の粉末を所望する場合はφ20μm以下のふるい)にかけ、長径が所望の大きさの粉末を回収し、好ましくは滅菌処理することで調製することができる。粉末の大きさ及び形状は例えば走査型電子顕微鏡観察により確認することができる。   The powdery solid in the present invention may be commercially available or prepared. For example, a material that is a raw material of the powdered solid material in the present invention, for example, plate-shaped zirconia is appropriately crushed by a pulverizer, and the obtained powder has an appropriate pore size for selecting a powder having a desired size. Sieve (for example, when a powder having a major axis of particles of 32 μm or less is desired, a sieve having a diameter of 32 μm or less, and when a powder having a major axis of particles of 20 μm or less is desired, a powder having a major axis having a desired size) Can be recovered, and preferably sterilized. The size and shape of the powder can be confirmed, for example, by observation with a scanning electron microscope.

本発明における試料には、喀痰、膿、コロニー、スワブ等の生体試料の試料懸濁液、食物試料のホモジェナイズ等の試料懸濁液、あるいは血液、尿、血液培養液等の試料溶液等が挙げられる。試料が喀痰の場合は、NALC(N-アセチル-L-システイン)処理等の試料の粘性を落す前処理を行うとより好ましい結果が得られる。また、環境分析等における環境水や排水、土壌の懸濁液等が挙げられる。   Examples of the sample in the present invention include sample suspensions of biological samples such as sputum, pus, colonies and swabs, sample suspensions such as homogenization of food samples, and sample solutions such as blood, urine and blood culture fluid. It is done. When the sample is sputum, a more preferable result can be obtained by performing a pretreatment such as NALC (N-acetyl-L-cysteine) treatment to reduce the viscosity of the sample. In addition, environmental water and wastewater in environmental analysis and the like, soil suspension, and the like can be mentioned.

試料中の微生物は、細菌では例えば大腸菌、サルモネラ、肺炎桿菌、セラチア菌、腸炎ビブリオ、インフルエンザ桿菌等のグラム陰性菌、黄色ぶどう球菌、レンサ球菌、破傷風菌、ボツリヌス菌、炭疽菌等のグラム陽性菌、および結核菌が挙げられる。ウイルスでは例えばノロウイルス、A型肝炎ウイルス、B型肝炎ウイルス、C型肝炎ウイルス、エイズウイルス、SARSウイルス、鳥インフルエンザウイルス等が挙げられる。試料中の生物組織は、人の病理組織、例えば乳癌の組織や肝臓癌の組織等、人の臓器の組織、動物の臓器、植物の組織が挙げられる。   Microorganisms in the sample are gram-positive bacteria such as Escherichia coli, Salmonella, Klebsiella pneumoniae, Serratia bacteria, Vibrio parahaemolyticus, H. influenzae, etc., Staphylococcus aureus, Streptococcus, Tetanus, Clostridium botulinum, Bacillus anthracis, etc. , And Mycobacterium tuberculosis. Examples of viruses include norovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, AIDS virus, SARS virus, avian influenza virus and the like. Examples of biological tissues in the sample include human pathological tissues, such as breast cancer tissues and liver cancer tissues, human organ tissues, animal organs, and plant tissues.

試料の攪拌方法は、試料を入れた容器を手で往復振動させれば良く、振幅の方向は縦でも横でもよいが、好ましくは縦方向に1秒当たり約2〜10往復程度の速さで往復振動させるのがよい。あるいは、ボルテックスミキサーで攪拌してもよい。攪拌時間は、微生物および組織の細胞膜が破壊されるまででよく、具体的には2分間以上でよい。組織が植物の組織の様に、丈夫な細胞壁を持ち、硬い場合には、それ以上長くても良い。   The sample can be stirred by reciprocating the container containing the sample by hand, and the direction of the amplitude may be vertical or horizontal, but is preferably about 2 to 10 reciprocations per second in the vertical direction. It is better to reciprocate. Or you may stir with a vortex mixer. The agitation time may be until the cell membranes of microorganisms and tissues are destroyed, specifically, 2 minutes or more. If the tissue has a strong cell wall and is hard like a plant tissue, it may be longer.

本発明に従い標的細菌および組織を破砕することで、これらから様々な生理活性物質、例えば核酸、タンパク質、糖質、脂質、ホルモン等が容易に抽出される。好ましくは、破砕した細菌又は組織の残骸は、例えば遠心分離や濾過などの周知の手段により除去する。本発明の方法は特に核酸の抽出に極めて有用である。本発明の破砕方法を通じて抽出された核酸は様々な処理にかけられることができ、その典型例は核酸増幅検査である。核酸増幅検査には、PCR(Polymerase Chain Reaction)、RT−PCR(Reverse−Transcriptase PCR)、LCR(Ligase Chain Reaction)、LAMP(Loop−mediated Isothermal Amplification of DNA)等のDNA増幅法、NASBA(Nucleic Acid Sequence Based Amplification)、TMA(Transcription−Mediated Amplification)あるいはTRC(Transcription Reverse Transcription Concerted Reaction、医学のあゆみ、Vol.206.No8、2003)等のRNA増幅法が挙げられる。   By disrupting the target bacteria and tissues according to the present invention, various physiologically active substances such as nucleic acids, proteins, carbohydrates, lipids, hormones and the like are easily extracted from them. Preferably, disrupted bacteria or tissue debris is removed by well-known means such as centrifugation or filtration. The method of the present invention is particularly useful for nucleic acid extraction. The nucleic acid extracted through the disruption method of the present invention can be subjected to various treatments, and a typical example is a nucleic acid amplification test. Nucleic acid amplification tests include PCR (Polymerase Chain Reaction), RT-PCR (Reverse-Transscript PCR), LCR (Ligase Chain Reaction), LAMP (Loop-Mediated Amplification of DNA Amplification DNA) Sequence Based Amplification), TMA (Transscription-Mediated Amplification), or TRC (Transcribation Reverse Transaction Reaction, Ayumi of Medicine, Vol. 206, No. 200, No. 200, etc.). RNA amplification methods.

核酸の抽出は、酵素や界面活性剤で細胞膜や細胞壁、または外皮蛋白質を破壊し、複合体の蛋白質を分解して核酸を遊離させた後、フェノール/クロロホルムを添加して遊離した核酸を抽出する、いわゆるフェノール/クロロホルム法(Molecular Cloning:A laboratory manual Appendix E3−E4(New York:Cold Spring Harbor Laboratory,1989年))、塩酸グアニジンまたはチオシアン酸グアニジンで処理して細胞膜や細胞壁、または外皮蛋白質を破壊し、核酸との複合体を形成している蛋白質を変性して核酸を遊離させた後、エタノール等を添加して遊離した核酸を抽出する、いわゆるグアニジン法(Molecular Cloning:A laboratory manual Appendix 7.23〜7.25(New York:Cold Spring Harbor Laboratory,1989年))等がある。しかしこれらの方法は、処理工程数が多く、煩雑でかつ時間を要する。また、酸性溶液であるフェノールや、有機溶媒であるクロロホルムや、強力な変性剤である塩酸グアニジンあるいはチオシアン酸グアニジンは刺激性、毒性がある。そのため人体に有害であり取り扱いに注意が必要で、廃液が自然環境へ及ぼす悪影響も大きい。本発明では、短時間で、これらの酸性溶液、有機溶媒、および変性剤を使用することなく、細菌の細胞や組織を破砕し、それらの持つ核酸を抽出することが可能である。   Nucleic acid extraction is performed by destroying cell membranes, cell walls, or coat proteins with enzymes and surfactants, decomposing complex proteins to release nucleic acids, and then adding phenol / chloroform to extract the released nucleic acids. The so-called phenol / chloroform method (Molecular Cloning: A laboratory manual Appendix E3-E4 (New York: Cold Spring Harbor Laboratory, 1989)), disrupting cell membranes or protein walls by treating guanidine hydrochloride or cell wall, Then, the protein forming a complex with the nucleic acid is denatured to release the nucleic acid, and then the so-called guanidine method (Molecular Cloning) in which the nucleic acid is extracted by adding ethanol or the like. A laboratory manual Appendix 7.23~7.25 (New York: Cold Spring Harbor Laboratory, 1989 years)), and the like. However, these methods have many processing steps, are complicated, and require time. In addition, phenol as an acidic solution, chloroform as an organic solvent, and guanidine hydrochloride or guanidine thiocyanate as strong modifiers are irritating and toxic. Therefore, it is harmful to the human body and needs to be handled with care, and the waste liquid has a great negative effect on the natural environment. In the present invention, bacterial cells and tissues can be crushed and their nucleic acids can be extracted in a short time without using these acidic solutions, organic solvents, and denaturing agents.

また核酸増幅検査のための核酸の抽出を行う場合、試料によっては細菌の細胞壁が硬いあるいは組織が硬く、核酸の抽出が困難な場合がある。喀痰中の好酸菌などは硬い細胞壁を持っており、通常の核酸抽出方では全ての好酸菌を効率よく破砕し、核酸を抽出することは困難である。しかしながら本発明により、硬い細胞壁を持つ細菌および組織を効率よく破砕し核酸を抽出することが可能である。   When nucleic acid is extracted for nucleic acid amplification test, depending on the sample, the bacterial cell wall is hard or the tissue is hard, and it may be difficult to extract the nucleic acid. Eosinophils and the like in the cocoon have a hard cell wall, and it is difficult to extract nucleic acids by efficiently crushing all the eosinophilic bacteria with a normal nucleic acid extraction method. However, according to the present invention, it is possible to efficiently disrupt bacteria and tissues having hard cell walls and extract nucleic acids.

これまでにも、試料にビーズ等を添加し攪拌することにより細胞や組織を破砕する方法はあったが、試料を凍結しなければならず、あるいは攪拌中の試料の溶解を防ぐため、冷凍した状態で攪拌しなければならなかった。そのため試料を凍結する時間が長時間必要であり、迅速検査を行うことは不可能であった。また、冷凍機能を持った装置でなければならず、価格も高価であり装置の小型化も困難であった。また、試料を凍結しない場合は、破砕効率が低いため長時間攪拌しなければならず、試料の温度が上昇してしまい、細胞中のたんぱく質が変性してしまう場合があった。本発明では、試料を凍結する必要がないため凍結にかかる時間を省くことができ、攪拌時間を短時間にすることが可能でることから、抽出した核酸による迅速検査を行うことが可能である。また、冷凍機能も必要でないため、装置の低価格化、小型化が可能である。   Until now, there was a method of crushing cells and tissues by adding beads etc. to the sample and stirring, but the sample must be frozen or frozen to prevent dissolution of the sample during stirring The state had to be stirred. For this reason, it takes a long time to freeze the sample, and it was impossible to perform a rapid inspection. Moreover, the apparatus must have a refrigeration function, is expensive in price, and it is difficult to downsize the apparatus. In addition, when the sample is not frozen, the crushing efficiency is low, so it must be stirred for a long time, the temperature of the sample rises, and the protein in the cell may be denatured. In the present invention, since it is not necessary to freeze the sample, the time required for freezing can be omitted, and the stirring time can be shortened. Therefore, it is possible to perform a rapid test using the extracted nucleic acid. Further, since no refrigeration function is required, the apparatus can be reduced in price and size.

以下に、発明を更に詳細に説明するために実施例を示すが、本発明はこれら実施例に限定されるものではない。   Examples are shown below to describe the invention in more detail, but the invention is not limited to these Examples.

1.無定形ジルコニア粉末の調整
板状ジルコニアTZ−8YS(東ソー社製、110mmX110mm、厚さ0.3mm、比重6.05)をニットー電動スタンプミルANS143(愛知電機株式会社製)により、120rpmで5分間破砕した。得られた粉末状のジルコニアをφ32μmのふるいにかけ、粒子の長径が32μm以下のジルコニア粉末を回収し、121℃で20分間滅菌処理し、無定形ジルコニア粉末(φ32μm以下)とした。該無定形ジルコニア粉末(φ32μm以下)の走査型電子顕微鏡写真を図1に示した。粒子の長径が32μm以下で、鋭利な角を持った不均一な形状の無定形粉末であることが示される。φ32μmのふるいにかけた残りのジルコニア粉末を同様にφ75μmのふるいにかけ、φ32μm以上φ75μm以下の粉末とφ75μm以上の粉末をそれぞれ回収し、121℃で20分間滅菌処理し、それぞれ無定形ジルコニア粉末(φ32μm以上φ75μm以下)、無定形ジルコニア粉末(φ75μm以上)とした。
1. Preparation of amorphous zirconia powder Plate-shaped zirconia TZ-8YS (manufactured by Tosoh Corporation, 110 mm x 110 mm, thickness 0.3 mm, specific gravity 6.05) was crushed at 120 rpm for 5 minutes with a knit-electric stamp mill ANS143 (manufactured by Aichi Electric Co., Ltd.) did. The obtained powdery zirconia was passed through a sieve having a diameter of 32 μm, and a zirconia powder having a major particle diameter of 32 μm or less was collected and sterilized at 121 ° C. for 20 minutes to obtain an amorphous zirconia powder (φ32 μm or less). A scanning electron micrograph of the amorphous zirconia powder (φ32 μm or less) is shown in FIG. It is shown that the powder is a non-uniform shaped amorphous powder having a long axis of 32 μm or less and a sharp angle. The remaining zirconia powder applied to a sieve of φ32 μm is similarly applied to a sieve of φ75 μm, and a powder of φ32 μm or more and φ75 μm or less and a powder of φ75 μm or more are respectively collected and sterilized at 121 ° C. for 20 minutes. φ75 μm or less) and amorphous zirconia powder (φ75 μm or more).

2.細菌を含んだ試料の調整
MRSA(メシチリン耐性黄色ブドウ球菌)のPBP−2’をコードするmecA遺伝子(FEBS Lett. 221(1)、167−171,1987)を組み込んだpUC19により形質転換を行ったJM109を、100μg/mlのアンピシリンを添加したLB培地により37℃で一晩培養した。該培養液を、波長600nmにおける吸光度が0.5となるようにPBSで希釈し、試料とした。
2. Preparation of samples containing bacteria Transformation was performed with pUC19 incorporating the mecA gene (FEBS Lett. 221 (1), 167-171, 1987) encoding PBP-2 ′ of MRSA (mesitillin-resistant Staphylococcus aureus). JM109 was cultured overnight at 37 ° C. in LB medium supplemented with 100 μg / ml ampicillin. The culture solution was diluted with PBS so that the absorbance at a wavelength of 600 nm was 0.5 to prepare a sample.

3.試料中の細菌の破砕による核酸抽出
2.により調整した試料1mlを2ml容量のエッペンドルフチューブ(エッペンドルフ社製)に入れ、これに1.により調整した無定形ジルコニア粉末(φ32μm以下)あるいは無定形ジルコニア粉末(φ32μm以上φ75μm以下)、あるいは無定形ジルコニア粉末(φ75μm以上)を3.0g添加した。該試料と無定形ジルコニア粉末を含んだチューブを室温で2分間激しく攪拌した。その後、遠心分離により上清を回収した。一方、2ml容量のエッペンドルフチューブに入れた該試料1mlに上記無定形ジルコニア粉末と同容積の球形ジルコニア(φ30μm)あるいはアルミナ粉末(比重3.5、φ50μm以下)を添加し、上記操作と同様に試料を攪拌し、上清を回収した。
3. Nucleic acid extraction by disruption of bacteria in the sample 1 ml of the sample prepared by the above procedure is put into a 2 ml Eppendorf tube (manufactured by Eppendorf). 3.0 g of amorphous zirconia powder (φ32 μm or less), amorphous zirconia powder (φ32 μm or more and φ75 μm or less) or amorphous zirconia powder (φ75 μm or more) prepared by the above was added. The tube containing the sample and amorphous zirconia powder was vigorously stirred at room temperature for 2 minutes. Thereafter, the supernatant was collected by centrifugation. On the other hand, spherical zirconia (φ30 μm) or alumina powder (specific gravity 3.5, φ50 μm or less) having the same volume as the amorphous zirconia powder is added to 1 ml of the sample placed in a 2 ml Eppendorf tube, and the sample is subjected to the same procedure as above. The supernatant was recovered.

4.遺伝子の検出
3.で調整した上清を滅菌水で2倍希釈し、そのうち2μlを用いて、プライマー(MecP4:5’−TGCTATCCACCCTCAAACAGG−3’(配列番号1)とプライマー(MecP2:5’−AACGTTGTAACCACCCCAAGA−3’(配列番号2)を用いたライトサイクラー(ロッシュ・ダイアグノスティックス社製)による定量PCRを行うことにより上清中のmecA遺伝子を定量した。PCR反応は、94℃で20秒、43℃で30秒、72℃で2分を1サイクルとして40サイクル行った。対照として、2.により調整した該試料1mlを13000回転で10分間遠心した沈殿からQIA prep Spin Miniprep Kit(QIAGEN社製)によりプラスミドを抽出し、定量PCRを行なった。
4). 2. Gene detection The supernatant prepared in (2) was diluted 2-fold with sterilized water, 2 μl of which was used as a primer (MecP4: 5′-TGCTATCCACCCTCAAACAGG-3 ′ (SEQ ID NO: 1) and primer (MecP2: 5′-AACGTTGTATAACCACCCCCAAGA-3 ′ (sequence The mecA gene in the supernatant was quantified by quantitative PCR using a light cycler (manufactured by Roche Diagnostics) using No. 2) The PCR reaction was performed at 94 ° C. for 20 seconds and at 43 ° C. for 30 seconds. And 40 cycles of 2 minutes at 72 ° C. As a control, a plasmid was extracted from the precipitate prepared by centrifuging at 13,000 rpm for 10 minutes using QIA prep Spin Miniprep Kit (manufactured by QIAGEN). Quantitative PCR was performed.

結果を図2に示した。図2に示すグラフは、各種ジルコニア粉末又はアルミナ粉末を用いて、大腸菌を含んだ試料から大腸菌を破砕し核酸を抽出した場合の、核酸抽出率を見たものである。波長600nmにおける吸光度が0.5の大腸菌溶液1mlから抽出したプラスミドの量を100%とし、それに対する各試料から抽出されたプラスミドの割合を核酸抽出率とした。
(i)は長径が32μm以下の無定形ジルコニア粉末により、大腸菌を破砕し核酸を回収した場合、(ii)は長径が32μm以上75μm以下の無定形ジルコニア粉末により、大腸菌を破砕し核酸を回収した場合、(iii)は長径が75μm以上の無定形ジルコニア粉末により、大腸菌を破砕し核酸を回収した場合、(iv)は粒子径が30μm以下の球形ジルコニアにより、大腸菌を破砕し核酸を回収した場合、(v)は長径が50μm以下の無定形アルミナ粉末により、大腸菌を破砕し核酸を回収した場合を示す。
The results are shown in FIG. The graph shown in FIG. 2 shows the nucleic acid extraction rate when various kinds of zirconia powder or alumina powder are used to crush E. coli from a sample containing E. coli and extract the nucleic acid. The amount of plasmid extracted from 1 ml of an E. coli solution having an absorbance at a wavelength of 600 nm of 0.5 was defined as 100%, and the ratio of the plasmid extracted from each sample relative thereto was defined as the nucleic acid extraction rate.
When (i) crushes E. coli with amorphous zirconia powder having a major axis of 32 μm or less and collects nucleic acid, (ii) crushes Escherichia coli with amorphous zirconia powder with a major axis of 32 μm or more and 75 μm or less to collect nucleic acid In the case of (iii), when the nucleic acid is recovered by crushing E. coli with amorphous zirconia powder having a major axis of 75 μm or more, and (iv) in the case of recovering the nucleic acid by crushing E. coli with spherical zirconia having a particle size of 30 μm or less (V) shows the case where the nucleic acid was recovered by crushing Escherichia coli with amorphous alumina powder having a major axis of 50 μm or less.

その結果、対照の結果を100%とすると、3.で無定形ジルコニア粉末(φ32μm以下)により細菌を破砕し抽出した遺伝子の回収率は、約100%であった。一方、無定形ジルコニア粉末(φ32μm以上φ75μm以下)、あるいは無定形ジルコニア粉末(φ75μm以上)、球形ジルコニア(φ30μm)、アルミナ粉末(比重3.5、粒径50μm以下)により細菌を破砕し抽出した遺伝子の回収率は、全て約50%以下であった。このことは、本法により試料中の細菌を効率よく破砕し、100%の効率で核酸を回収することが可能であることを示す。   As a result, assuming that the control result is 100%, 3. The recovery rate of the gene obtained by crushing and extracting bacteria with amorphous zirconia powder (φ32 μm or less) was about 100%. On the other hand, genes obtained by crushing and extracting bacteria with amorphous zirconia powder (φ32 μm or more and φ75 μm or less), amorphous zirconia powder (φ75 μm or more), spherical zirconia (φ30 μm), alumina powder (specific gravity 3.5, particle size 50 μm or less) The recovery rates of all were about 50% or less. This shows that it is possible to efficiently crush bacteria in the sample and recover nucleic acid with 100% efficiency by this method.

実施例1.で示した無定形ジルコニア粉末(φ32μm以下)の走査型電子顕微鏡写真である。写真中右下の傍線は30μmを示す。Example 1. 2 is a scanning electron micrograph of amorphous zirconia powder (φ32 μm or less) shown in FIG. The side line in the lower right of the photograph indicates 30 μm.

各種ジルコニア粉末又はアルミナ粉末を用いて、大腸菌を含んだ試料から大腸菌を破砕し核酸を抽出した場合の、核酸抽出率を示す。The nucleic acid extraction rate is shown when nucleic acids are extracted by crushing E. coli from a sample containing E. coli using various zirconia powders or alumina powders.

Claims (5)

粉末状固形物を、被破砕物を含む試料に添加し、該試料を攪拌することにより試料中の被破砕物を破砕する方法において、該粉末状固形物の長径が32μm以下であり、その比重が3.5以上であり、その硬度(Hv10)が600以上である、被破砕物の破砕方法。   In the method of crushing the material to be crushed by adding the powdered solid material to the sample containing the material to be crushed and stirring the sample, the major axis of the powdered solid material is 32 μm or less, and its specific gravity Is a method of crushing an object to be crushed, having a hardness (Hv10) of 600 or more. 該該粉末状固形物が無定形である、請求項1記載の方法。   The method of claim 1, wherein the powdered solid is amorphous. 被破砕物が微生物又は生物組織である、請求項1又は2に記載の方法。   The method according to claim 1 or 2, wherein the material to be crushed is a microorganism or a biological tissue. 該粉末状固形物がジルコニアである、請求項1から3のいずれかに記載の方法。   The method according to claim 1, wherein the powdered solid is zirconia. 請求項1から4のいずれかに記載の方法により被破砕物を破砕することで該被破砕物から核酸を抽出することを特徴とする、核酸の抽出方法。   A nucleic acid extraction method, wherein nucleic acids are extracted from the crushed material by crushing the crushed material by the method according to claim 1.
JP2005343698A 2005-11-29 2005-11-29 Method for crushing objects to be crushed in a sample Expired - Fee Related JP4797602B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2005343698A JP4797602B2 (en) 2005-11-29 2005-11-29 Method for crushing objects to be crushed in a sample

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2005343698A JP4797602B2 (en) 2005-11-29 2005-11-29 Method for crushing objects to be crushed in a sample

Publications (2)

Publication Number Publication Date
JP2007143495A true JP2007143495A (en) 2007-06-14
JP4797602B2 JP4797602B2 (en) 2011-10-19

Family

ID=38205657

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2005343698A Expired - Fee Related JP4797602B2 (en) 2005-11-29 2005-11-29 Method for crushing objects to be crushed in a sample

Country Status (1)

Country Link
JP (1) JP4797602B2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019074091A1 (en) * 2017-10-12 2019-04-18 三井化学株式会社 mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01317112A (en) * 1988-06-15 1989-12-21 Sumitomo Electric Ind Ltd Polycrystalline diamond having high strength and production thereof
JP2001178444A (en) * 1999-10-13 2001-07-03 Yasui Kikai Kk Method for crushing and apparatus therefor
JP2004209322A (en) * 2002-12-27 2004-07-29 Sysmex Corp Crushing method, crushing device and crushing kit

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01317112A (en) * 1988-06-15 1989-12-21 Sumitomo Electric Ind Ltd Polycrystalline diamond having high strength and production thereof
JP2001178444A (en) * 1999-10-13 2001-07-03 Yasui Kikai Kk Method for crushing and apparatus therefor
JP2004209322A (en) * 2002-12-27 2004-07-29 Sysmex Corp Crushing method, crushing device and crushing kit

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019074091A1 (en) * 2017-10-12 2019-04-18 三井化学株式会社 mecA GENE AMPLIFICATION PRIMER PAIR, mecA GENE DETECTION KIT AND mecA GENE DETECTION METHOD
JPWO2019074091A1 (en) * 2017-10-12 2020-04-16 三井化学株式会社 Primer pair for mecA gene amplification, mecA gene detection kit, and mecA gene detection method
CN111183223A (en) * 2017-10-12 2020-05-19 三井化学株式会社 Primer pair for mecA gene amplification, mecA gene detection kit and mecA gene detection method
JP2022025117A (en) * 2017-10-12 2022-02-09 三井化学株式会社 PRIMER PAIR FOR mecA GENE AMPLIFICATION, mecA GENE DETECTION KIT, AND mecA GENE DETECTION METHOD
AU2018350052B2 (en) * 2017-10-12 2022-06-02 Mitsui Chemicals, Inc. Meca gene amplification primer pair, meca gene detection kit and meca gene detection method
JP7199367B2 (en) 2017-10-12 2023-01-05 三井化学株式会社 mecA gene amplification primer pair, mecA gene detection kit, and mecA gene detection method

Also Published As

Publication number Publication date
JP4797602B2 (en) 2011-10-19

Similar Documents

Publication Publication Date Title
US10087439B1 (en) Rapid methods for the extraction of nucleic acids from biological samples
Pearlman et al. Low-resource nucleic acid extraction method enabled by high-gradient magnetic separation
JP5253451B2 (en) Materials and methods for capturing pathogens from samples and removing aurintricarboxylic acid
Hammarstrom et al. Acoustic trapping for bacteria identification in positive blood cultures with MALDI-TOF MS
EP1992689B1 (en) Method for extraction of nucleic acid from biological material
US20080171337A1 (en) Nucleic acid isolation method by heating on magnetic support
EP3067112A1 (en) Magnetic lysis method and device
JP2012511318A (en) Automated system for lysis of microorganisms present in samples intended for analysis and extraction and purification of nucleic acids of these microorganisms
Basha et al. Towards multiplex molecular diagnosis—A review of microfluidic genomics technologies
JP7071473B2 (en) A method for separating nucleic acid from a sample containing nucleic acid and a device for that purpose.
CN107667168A (en) Composition and method for disrupting tissue material
CN108410951A (en) A kind of new nucleic acid extracting reagent and its application
Powell et al. Pressure cycling technology in systems biology
JP4797602B2 (en) Method for crushing objects to be crushed in a sample
WO2008075501A1 (en) Rotary extraction container, method of identifying cell species and method of detecting gene using the same, and automatic nucleic acid extractor
JP2019520825A (en) High-throughput microbiologically applied high resolution system, kit, apparatus and method using magnetic particles
CN108603221A (en) Comprehensive sample processing system
JP5543694B2 (en) Separation and collection method of biological materials
US10640745B2 (en) Method for deforming and/or fragmenting a cell, spore or virus with a vibrating plate
JP2024501497A (en) Systems, methods and devices for enrichment and identification of microorganisms from blood
US20170081656A1 (en) Method and reagent for extracting nucleic acid
Sawant et al. Detection of Mycobacterium tuberculosis from pulmonary sputum sample using SPION mediated DNA extraction method
JP2013005820A (en) Material and method for capture of pathogen and removal of aurintricarboxylic acid from sample
Chang et al. An integrated microfluidic system for rapid isolation and detection of live bacteria in periprosthetic joint infections
JP2018143184A (en) Microbe rna detection method

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20080922

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20110426

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20110616

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20110705

A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20110718

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20140812

Year of fee payment: 3

R151 Written notification of patent or utility model registration

Ref document number: 4797602

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R151

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20140812

Year of fee payment: 3

LAPS Cancellation because of no payment of annual fees