JPH1189567A - System for sterilizing microorganism and gene recovery system - Google Patents
System for sterilizing microorganism and gene recovery systemInfo
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- JPH1189567A JPH1189567A JP9258816A JP25881697A JPH1189567A JP H1189567 A JPH1189567 A JP H1189567A JP 9258816 A JP9258816 A JP 9258816A JP 25881697 A JP25881697 A JP 25881697A JP H1189567 A JPH1189567 A JP H1189567A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/03—Electric current
- A61L2/035—Electrolysis
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/46—Treatment of water, waste water, or sewage by electrochemical methods
- C02F1/461—Treatment of water, waste water, or sewage by electrochemical methods by electrolysis
- C02F1/467—Treatment of water, waste water, or sewage by electrochemical methods by electrolysis by electrochemical disinfection; by electrooxydation or by electroreduction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
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- Genetics & Genomics (AREA)
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- Plant Pathology (AREA)
- Hydrology & Water Resources (AREA)
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- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Biophysics (AREA)
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- Animal Behavior & Ethology (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Water Treatment By Electricity Or Magnetism (AREA)
Abstract
Description
【0001】[0001]
【発明が属する技術分野】本発明は微生物の殺菌システ
ム、及び特定微生物から遺伝子を回収するシステムに関
するものである。The present invention relates to a system for killing microorganisms and a system for recovering genes from specific microorganisms.
【0002】[0002]
【従来の技術】近年、病原性微生物による悪影響が多々
論じられている。問題の第1は、人間の生活水を汚染す
る微生物の存在である。水や食物を通じて感染し、人体
に激しい下痢や腹痛を起こす原虫「クリプトスポリジウ
ム」は、水道水の水源等を汚染する。厄介なことにこの
原虫は、塩素系殺菌は効果がない。水源には塩素系殺菌
剤の殺菌効果を越える薬剤を使用することは出来ない。2. Description of the Related Art In recent years, the adverse effects of pathogenic microorganisms have been widely discussed. The first problem is the presence of microorganisms that contaminate human water. Protozoa "Cryptosporidium", which is transmitted through water and food and causes severe diarrhea and abdominal pain in the human body, contaminates the water source of tap water. Unfortunately, this protozoa is ineffective in chlorine-based disinfection. As a water source, it is not possible to use an agent that exceeds the bactericidal effect of a chlorine-based bactericide.
【0003】第2は、院内感染や日和見感染の原因とな
る緑膿菌である。この緑膿菌はビルの貯留水の汚染減と
もなり、空調機やビル内の飲料水を介して人体に感染す
るおそれがある。緑膿菌は薬剤に対して抵抗性を持って
いる。[0003] The second is Pseudomonas aeruginosa, which causes nosocomial and opportunistic infections. The Pseudomonas aeruginosa also reduces the contamination of the stored water in the building, and may infect the human body via the air conditioner and drinking water in the building. Pseudomonas aeruginosa is resistant to drugs.
【0004】第3は、家庭用24時間風呂の汚染源とな
るレジオネラ菌である。レジオネラ菌は、肺炎の原因と
なる。この菌の繁殖を抑制するために、紫外線を利用す
ることが取りざたされているが、十分な殺菌方法とは言
えない。[0004] The third is Legionella bacteria, which is a source of pollution in home 24-hour baths. Legionella bacteria cause pneumonia. In order to suppress the growth of this bacterium, the use of ultraviolet rays has been sought, but it cannot be said to be a sufficient sterilization method.
【0005】第4は、食中毒となる病原性大腸菌、特に
O−157である。食中毒の感染源を特定することは、
その防止に不可欠である。感染ルートの特定には、O−
157の遺伝子分析が必要である。遺伝子分析について
は、例えば、特開平9−178752号が存在する。[0005] Fourth, pathogenic Escherichia coli causing food poisoning, particularly O-157. Identifying the source of food poisoning is
It is essential for its prevention. O-
157 genetic analyzes are required. Regarding gene analysis, for example, Japanese Patent Application Laid-Open No. 9-178752 exists.
【0006】[0006]
【発明が解決しようとする課題】生活水を汚染する微生
物に対しては、人体に対する悪影響を防止しなけらばな
らないことから、塩素系殺菌が中心となる。しかしなが
ら、「クリプトスポリジウム」等はこれに対して抵抗性
がある。With respect to microorganisms contaminating living water, chlorine-based disinfection is mainly used since it is necessary to prevent adverse effects on the human body. However, "Cryptosporidium" and the like are resistant to this.
【0007】レジオネラ菌を紫外線で殺菌しようとする
のは、薬剤を使用することによって人体に悪影響が及ぶ
ことを防止するためでもある。しかしながら、既述の微
生物は、殺菌効果が高い薬剤を使用しなければ十分殺菌
できないという問題があった。[0007] The reason why Legionella bacteria are to be sterilized with ultraviolet rays is also to prevent the use of a drug from adversely affecting the human body. However, there is a problem that the microorganisms described above cannot be sufficiently sterilized unless a drug having a high sterilizing effect is used.
【0008】一方、遺伝子分析による菌の検出につい
て、既述の従来例では、酵素を用いる化学的分析法であ
るために、分析系に夾雑物が混入し分析精度が劣ること
になる。また化学的処理に時間を要することになる。On the other hand, regarding the detection of bacteria by genetic analysis, in the above-described conventional example, since the chemical analysis method using an enzyme is used, impurities are mixed in the analysis system, and the analysis accuracy is inferior. In addition, it takes time for the chemical treatment.
【0009】本願出願人は、特開平9−37763号に
おいて、酵素等の化学成分を使用することなく微生物の
菌体を破壊し細胞内の遺伝子を回収することを提案し
た。しかしながら、感染ルートを迅速に確定するため
に、素早い遺伝子の特定が求められる食中毒原因菌に対
して、迅速かつ精度が高い遺伝子分析法にまでの配慮
はない。[0009] The applicant of the present application has proposed in Japanese Patent Application Laid-Open No. 9-37763 that the cells of microorganisms are destroyed without using a chemical component such as an enzyme and the gene in the cell is recovered. However, in order to quickly determine the route of infection, fast and accurate genetic analysis methods for food poisoning bacteria that require quick identification of genes
There is no.
【0010】そこで、本発明の目的は、各種の病原性微
生物が与える悪影響を、簡単な操作によって除去するこ
とができるシステムを提供することである。詳しくは、
薬剤を使用することなく、生活水を汚染する微生物に対
して効果的な殺菌システムを提供することである。他
に、薬剤を使用することなく、緑膿菌、そしてレジオネ
ラ菌を殺菌するシステムを提供することである。さらに
他に、食中毒の原因菌に対して迅速かつ精度が高い遺伝
子分析法のための、核酸検出システムを提供することで
ある。[0010] Therefore, an object of the present invention is to provide a system capable of removing the adverse effects of various pathogenic microorganisms by a simple operation. For more information,
An object of the present invention is to provide an effective disinfection system for microorganisms contaminating living water without using chemicals. Another object is to provide a system for killing Pseudomonas aeruginosa and Legionella bacteria without using drugs. It is still another object of the present invention to provide a nucleic acid detection system for a rapid and highly accurate gene analysis method for the causative bacteria of food poisoning.
【0011】[0011]
【課題を解決するための手段】この目的を達成するため
に、本発明は、多孔質膜にコーティングされた金属膜を
有する通電手段と、この通電手段に対する電源と、前記
通電手段に微生物を供給する供給手段と、を備え、この
微生物が生活水に対する汚染源であり、前記通電手段を
電極として電源から微生物に通電して殺菌するシステム
であることを特徴とする。In order to achieve this object, the present invention provides an energizing means having a metal film coated on a porous film, a power source for the energizing means, and supplying a microorganism to the energizing means. Supply means for supplying water to the living water, wherein the microorganisms are a source of contamination of living water, and the system is a system for disinfecting the microorganisms by supplying electricity to the microorganisms from a power supply using the electricity supply means as an electrode.
【0012】この微生物は、特に、既述の「クリプトス
ポリジウム」、緑膿菌、レジオネラ菌、病原性大腸菌で
ある。This microorganism is, in particular, the aforementioned "Cryptosporidium", Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli.
【0013】多孔質膜は、市販の中空糸やそれが平膜状
になった平膜、特に、多孔質樹脂からなるもの、セラミ
ック中空管、焼結管等である。多孔質樹脂に金属をコー
ティングするためには、既述の特開平9−37763号
に記載の方法を使用すれば良い。この公報に記載されて
いるように、多孔質樹脂と金属とは化学結合しているこ
とが好ましい。こうすることにより、より多くの量の金
属を多孔樹脂にコーティングすることができる。The porous membrane is a commercially available hollow fiber or a flat membrane in which the hollow fiber is formed into a flat membrane, particularly one made of a porous resin, a hollow ceramic tube, a sintered tube, or the like. In order to coat the porous resin with a metal, the method described in JP-A-9-37763 may be used. As described in this publication, the porous resin and the metal are preferably chemically bonded. By doing so, a larger amount of metal can be coated on the porous resin.
【0014】本発明において通電は必須のものである。
金属板を電極としたものに比較して、殺菌効果がより高
いのは、多孔質に微生物が捕捉されながら、通電を受け
るためであると考えられる。通電を受けた場合と受けな
い場合では、殺菌率について顕著な相違を示す。In the present invention, energization is essential.
It is considered that the reason why the sterilizing effect is higher than that in the case where the metal plate is used as the electrode is that the porous plate is energized while the microorganisms are captured. There is a marked difference in the germicidal rate between when energized and when not energized.
【0015】この殺菌システムにおいて、通電の過程で
微生物の膜構造が破壊されて、細胞内容物である核酸を
得ることが可能となる。この核酸を遺伝子分析に利用す
れば、病原性大腸菌の遺伝子が特定できることになり、
その結果、感染ルートを解析することが可能となる。先
に説明した従来の特開平9−178752では細胞の膜
構造を破壊するために、酵素を使用しており、これでは
分析系に夾雑物が混入するのと、分析に時間を要する問
題がある。In this sterilization system, the membrane structure of the microorganism is destroyed in the course of energization, and it is possible to obtain a nucleic acid which is a cell content. If this nucleic acid is used for gene analysis, the pathogenic E. coli gene can be identified,
As a result, it becomes possible to analyze the infection route. In the above-mentioned conventional Japanese Patent Application Laid-Open No. Hei 9-178752, an enzyme is used in order to destroy the membrane structure of a cell, which causes a problem that contaminants are mixed in an analysis system and that analysis requires time. .
【0016】通電の形態としては、パルス通電、直流通
電、交流通電、インパルス通電がある。直流通電が有効
ではあることは後述の実施例から明らかである。また、
パルス通電が、細菌の細胞を破壊する上で有効なことは
特開平9−37763号に示されるとおりである。As a form of energization, there are pulse energization, DC energization, AC energization, and impulse energization. It is apparent from the examples described later that the direct current is effective. Also,
The fact that pulsed current is effective in destroying bacterial cells is as described in JP-A-9-37763.
【0017】[0017]
【実施例】次に、本発明の第1の実施例に係わる緑膿菌
の殺菌システムにつて説明する。Next, a sterilization system for Pseudomonas aeruginosa according to a first embodiment of the present invention will be described.
【0018】システム構成:多孔質ポリプロピレン製樹
脂からなる平膜(東燃タピルス(株)P120UA-04F)にSystem configuration: A flat membrane made of porous polypropylene resin (P120UA-04F, Tonen Tapils Co., Ltd.)
【0019】特開平9−37763のJapanese Patent Application Laid-Open No. 9-37763
【0043】の方法を応用して、銀をコーティングし
た。図1に示すように、これを4cm四方に切断し、低
融点半田とエポキシ樹脂で導線をこの導電性平膜に固定
した。次いで、蒸留水を3ml載せ、3000rpm、
10分間で膜を洗浄した。Silver was coated by applying the above method. As shown in FIG. 1, this was cut into a square of 4 cm, and a conductive wire was fixed to this conductive flat film with low melting point solder and epoxy resin. Next, 3 ml of distilled water was placed, and 3000 rpm,
The membrane was washed for 10 minutes.
【0020】この導電性平膜をエチルアルコール中で1
0分間、次いで蒸留水100ml中で10分洗浄し、乾
燥後、容器の開放側にキャップを被せて固定した。この
導電性平膜の電気抵抗は、0.3オームであった。This conductive flat film was prepared by
After washing for 0 minutes and then for 10 minutes in 100 ml of distilled water, and after drying, the open side of the container was capped and fixed. The electric resistance of this conductive flat film was 0.3 ohm.
【0021】使用微生物:緑膿菌(Pseudomonas aerugi
nosa)とレジオネラ菌(Legionella pneumophila serogr
oup 1)を使用して殺菌試験を行った。実験に使用したレ
ジオネラ菌は、ビル空調の冷却水から分離され、生化学
的性状と血清学的検査から1型レジオネラ菌であること
が確認された。Microorganism used: Pseudomonas aerugi
nosa) and Legionella pneumophila serogr
A sterilization test was performed using oup 1). The Legionella bacterium used in the experiment was separated from the cooling water of the building air conditioner, and was confirmed to be type 1 Legionella bacterium by biochemical properties and serologic tests.
【0022】殺菌方法:導電性平膜に一定菌数に調整し
た菌液1mlを載せ、遠心により膜に菌を捕捉させた。
その後、乾燥を防ぐ目的で生理食塩水をさらに滴下し1
Aの電流を10分通電した。通電終了後、膜を反転し、
裏側に生理食塩水を1ml載せ遠心して膜に捕捉された
菌を強制的に膜から離脱させて回収した。対照には、通
電のみを行わない平膜を用意し、この膜から回収された
菌数を計測した。Sterilization method: 1 ml of a bacterial solution adjusted to a certain number of bacteria was placed on a conductive flat membrane, and the bacteria were captured on the membrane by centrifugation.
Then, in order to prevent drying, physiological saline was further added dropwise.
A current was applied for 10 minutes. After energization is completed, the membrane is inverted,
1 ml of physiological saline was placed on the back side and centrifuged, and the bacteria captured on the membrane were forcibly removed from the membrane and collected. As a control, a flat membrane in which only current was not applied was prepared, and the number of bacteria recovered from this membrane was counted.
【0023】実験結果:図2に実験結果を示す。また、
図3には実験手順の工程を示す。図2の各表は、それぞ
れ印加した電流値での殺菌効果を示している。また、no
currentは膜に菌液を捕捉させた後、通電処理のみを行
わないものを意味し、通電処理を行ったものに関しては
それぞれの通電値を表中に示してある。菌数は、初期菌
数が膜に載せた菌液の中の菌数、濾液中菌数が膜を通過
した菌数、これらの初期菌数から濾液中菌数を減じた値
が捕捉菌数である。通電の効果は、表中の回収菌数によ
り計算することができる。回収菌数は菌を膜に捕捉した
後膜を反転し裏側に生理食塩水を載せて遠心を行い、膜
中の菌を強制的に離脱させた液中の菌数である。Experimental Results: FIG. 2 shows the experimental results. Also,
FIG. 3 shows the steps of the experimental procedure. Each table in FIG. 2 shows the bactericidal effect at the applied current value. Also no
“current” means that only the energization process is not performed after the bacterial solution is captured on the membrane, and the respective energization values are shown in the table for those subjected to the energization process. The number of bacteria is the number of bacteria in the bacterial solution on the membrane, the number of bacteria in the filtrate is the number of bacteria that passed through the membrane, and the value obtained by subtracting the number of bacteria in the filtrate from the initial number of bacteria is the number of captured bacteria. It is. The effect of energization can be calculated from the number of recovered bacteria in the table. The number of recovered bacteria is the number of bacteria in a liquid obtained by capturing bacteria on the membrane, inverting the membrane, placing a physiological saline solution on the back side, performing centrifugation, and forcibly removing bacteria in the membrane.
【0024】緑膿菌の調整は次のようにして行った。ア
ンプル(3ml)中で一晩前培養する。遠心分離器を用
いて2回洗浄する。生理食塩水3mlで再浮遊して、緑
膿菌懸濁液(108−109CFU/ml)を調整した。Preparation of Pseudomonas aeruginosa was performed as follows. Preculture overnight in ampoules (3 ml). Wash twice using a centrifuge. The suspension was resuspended in 3 ml of physiological saline to prepare a Pseudomonas aeruginosa suspension (10 8 -10 9 CFU / ml).
【0025】図2に示す結果から、緑膿菌に対する通電
の効果は、印加する直流電流値が750mA間では通電
しない群に比較して25%程度の減少に留まっている
が、電流値を1Aとした場合には、未通電群に比較して
その菌数が0.03%迄減少することが分かった。これ
らの結果は、従来抗生物質や各種の化学療法剤等の薬剤
で容易に死滅することが無かった緑膿菌を、これらの薬
剤を使用することなく短時間で殺菌できることが明らか
となった。よって、通電量は750mA以上であることが
好ましい。From the results shown in FIG. 2, it can be seen that the effect of energizing Pseudomonas aeruginosa is only about 25% lower than that of the group not energizing when the applied DC current value is between 750 mA, but the current value is 1 A. It was found that the number of bacteria decreased to 0.03% as compared with the non-energized group. These results revealed that Pseudomonas aeruginosa, which had not been easily killed by drugs such as antibiotics and various chemotherapeutic agents, can be sterilized in a short time without using these drugs. Therefore, the amount of current is preferably 750 mA or more.
【0026】(第2実施例)レジオネラ菌に対する殺
菌。次に第1実施例と同様な方法によりレジオネラ菌に
対する殺菌試験を行った。(Second embodiment) Sterilization against Legionella bacteria. Next, a bactericidal test against Legionella was performed in the same manner as in the first example.
【0027】図4に殺菌試験の結果を示す。この試験結
果から、1012個の菌を捕捉させた後、遠心を用いて菌
を強制的に回収しても109個程度の菌数しか回収でき
ないことから、導電性平膜は細菌に対して高い捕捉性を
有する。この高い捕捉性を導電性平膜が有するが故に、
細菌に対する通電が効果的に行われる。そして、回収さ
れる菌数が通電処理によりさらに1/100程度に減少
されていることから、導電処理でレジオネラ菌が殺菌で
きたことが分かる。従来、緑膿菌及びレジオネラ菌は殺
菌処理が困難であったが、本システムによれば効果的に
これらの細菌が殺菌されたことになる。FIG. 4 shows the results of the sterilization test. From this test result, after capturing 10 12 bacteria, only about 10 9 bacteria can be recovered even if the bacteria are forcibly recovered using centrifugation. And high capture performance. Because the conductive flat membrane has this high trapping property,
Energization of bacteria is effectively performed. And since the number of collect | recovered bacteria is further reduced to about 1/100 by an electricity supply process, it turns out that Legionella bacteria could be sterilized by the electroconductive treatment. Conventionally, it has been difficult to sterilize Pseudomonas aeruginosa and Legionella bacteria, but according to the present system, these bacteria are effectively sterilized.
【0028】(第3実施例)(Third Embodiment)
【0029】特開平9−37763号のJapanese Patent Application Laid-Open No. 9-37763 discloses
【0043】 −−
【0058】までにしたがって、病原性大腸菌(Escher
ichia coli serotype O−157)から、遺伝子の抽出
を行った。The pathogenic Escherichia coli (Escher
From ichia coli serotype O-157), a gene was extracted.
【0030】次いで、入手した遺伝子について、テクネ
社製プロジーンを用いたPCR法によって遺伝子の増幅
を行った。得られた遺伝子について、電気泳動を行い電
気泳動のパターンを得た。図5は、そのパターンを示す
ものである。Next, the obtained gene was amplified by a PCR method using Progene manufactured by Techne. The obtained gene was subjected to electrophoresis to obtain an electrophoresis pattern. FIG. 5 shows the pattern.
【0031】図5において、は遺伝子マーカー(ベロ
毒素を見付けるための遺伝子)である。は標的遺伝子
(ベロ毒素生成遺伝子)である。は108Cell/mlである
化学的抽出法(BIO-RAD社製DNA精製・回収試薬「インス
タジーン」を使用。)によって得た遺伝子のパターンで
ある。は、1/10化学的抽出法(107cell/ml)
である。は未通電による抽出法である。、、、
はそれぞれ、通電100mA、100mA、300m
A、500mA(但し、未通電、通電ともに108ce
lls/mL対象)の条件での通電法により抽出された
遺伝子の結果である。白線中に白く見えるバンドが、ベ
ロ毒素遺伝子である。In FIG. 5, is a gene marker (gene for finding verotoxin). Is a target gene (verotoxin producing gene). Shows a gene pattern obtained by a chemical extraction method using BIO-RAD DNA purification / recovery reagent “Instagene” at 10 8 Cell / ml. Is the 1/10 chemical extraction method (10 7 cell / ml)
It is. Is an extraction method using no current. ,,,
Are 100 mA, 100 mA, and 300 m, respectively.
A, 500mA (however, 10 8 ce for both non-energized and energized)
It is the result of the gene extracted by the energization method under the condition of (11s / mL target). The band that looks white in the white line is the verotoxin gene.
【0032】化学的抽出法の内容は次の通りである。
(1)試料をリン酸緩衝食塩水で洗浄・濃縮する。
(2)試料を蒸留水で再浮遊する。(3)インスタジー
ンを試料に加えて56℃30分間インキュベーションす
る。この段階で、細胞の酵素破壊と遺伝子の凝集が行わ
れる。(4)100℃8分間の加熱処理。酵素や他の蛋
白質の熱変性と遺伝子の凝集が行われる。(5)良く混
合してPCR試料とする。The contents of the chemical extraction method are as follows.
(1) Wash and concentrate the sample with phosphate buffered saline.
(2) Resuspend the sample in distilled water. (3) Add Instagene to the sample and incubate at 56 ° C for 30 minutes. At this stage, cell enzymatic destruction and gene aggregation occur. (4) Heat treatment at 100 ° C. for 8 minutes. Thermal denaturation of enzymes and other proteins and gene aggregation occur. (5) Mix well to obtain a PCR sample.
【0033】図5から明らかなように、未通電のもので
は、バンドが現れていないのに対して、通電のもので
は、バンドが出現して、確かにO−157の遺伝子が分
離されたのが確認できる。化学的抽出法では、バンド
(標的遺伝子)以外の上方に夾雑物のパターンが出現し
ている。通電のものにこれは出現していない。したがっ
て、通電のものでは、化学的抽出法に比較して、より純
粋な状態で遺伝子を抽出することが可能となる。As is clear from FIG. 5, no band appeared in the non-energized one, whereas the band appeared in the energized one, and the O-157 gene was certainly isolated. Can be confirmed. In the chemical extraction method, a pattern of contaminants appears above the band (target gene). This does not appear in energized ones. Therefore, it is possible to extract genes in a more pure state in the case of energization as compared with the chemical extraction method.
【0034】図6は、通電法による遺伝子抽出迄の時間
と化学的抽出法による遺伝子回収迄の時間とを比較した
特性図である。前者のものは、後者のものに比較してほ
ぼ1/2の時間で遺伝子の抽出が完了する。すなわち、
両者は、菌の前培養と菌体の収集については同程度の時
間を要するが、菌体の破壊について、前者が12分程度
で終了するのに対して、後者は、1時間以上を有する。FIG. 6 is a characteristic diagram comparing the time until the gene extraction by the energization method and the time until the gene recovery by the chemical extraction method. In the former case, the gene extraction is completed in approximately half the time in the latter case. That is,
In both cases, the same time is required for pre-culture of bacteria and collection of cells, but for destruction of cells, the former takes about 12 minutes, whereas the latter takes 1 hour or more.
【0035】また、遺伝子抽出のための前培養に要する
時間についても両者において顕著な差が存在する。すな
わち、前者においては、純度や収率が高い遺伝子の回
収、抽出法であるために、必要とする菌体の数が後者の
1/10以下で良い。後者のものでは、16時間の前培
養で菌体数が(108cells/mL)程度のサンプルが必要
である。これに対して、前者のものはこの1/10以下
で良いので、前培養としては、1時間以内で良い。すな
わち、後者の方法では、菌の前培養から遺伝子の抽出ま
で17時間近くを要するのに対して、前者の方法では、
2時間以内で済む。There is also a remarkable difference between the two in the time required for preculture for gene extraction. That is, in the former method, the number of required bacterial cells may be 1/10 or less of the latter, because the method is a method for collecting and extracting genes having high purity and high yield. In the latter case, a sample in which the number of cells is about (10 8 cells / mL) in the pre-culture for 16 hours is required. On the other hand, since the former is less than 1/10 of this, the pre-culture may be less than 1 hour. That is, in the latter method, it takes about 17 hours from the preculture of the bacterium to the extraction of the gene, whereas in the former method,
It takes less than 2 hours.
【0036】要するに、前者の方法は、後者の化学的抽
出法と異なり、操作が簡単かつ素早く、また、純度や収
率が高い遺伝子抽出法である。また、用いる化学的成分
が後者に比べて少ないために、その分他物質の混入が少
なくて済み、より純度が高い遺伝子を得ることが可能と
なる。In short, the former method, unlike the latter chemical extraction method, is a gene extraction method that is simple and quick in operation, and has high purity and high yield. In addition, since less chemical components are used than in the latter case, the amount of other substances to be mixed is small, and a gene with higher purity can be obtained.
【0037】なお、既述の実施例1及び2では、平膜を
利用したが、金属を被覆した中空糸膜を利用しても良
い。In the first and second embodiments, the flat membrane is used. However, a hollow fiber membrane coated with metal may be used.
【0038】[0038]
【発明の効果】以上説明したように本発明によれば、各
種の病原性微生物が与える悪影響を、簡単な操作によっ
て除去することができるシステムを提供することができ
る。すなわち、薬剤を使用することなく、生活水を汚染
する微生物に対して効果的な殺菌システムを提供する。
他に、薬剤を使用することなく、緑膿菌、そしてレジオ
ネラ菌を殺菌するシステムを提供する。さらに他に、食
中毒の原因菌に対して迅速かつ精度が高い遺伝子分析法
のための、核酸検出システムを提供する。As described above, according to the present invention, it is possible to provide a system capable of removing the adverse effects of various pathogenic microorganisms by a simple operation. That is, the present invention provides an effective disinfection system for microorganisms contaminating living water without using a chemical.
In addition, there is provided a system for killing Pseudomonas aeruginosa and Legionella bacteria without using a drug. Still another object of the present invention is to provide a nucleic acid detection system for a rapid and highly accurate gene analysis method for the causative bacteria of food poisoning.
【図1】本発明に使用する、金属を被覆した平膜の製造
工程図。FIG. 1 is a manufacturing process diagram of a metal-coated flat membrane used in the present invention.
【図2】緑膿菌に対する殺菌結果を示す特性図。FIG. 2 is a characteristic diagram showing the results of sterilization of Pseudomonas aeruginosa.
【図3】実施例1の工程図。FIG. 3 is a process chart of Example 1.
【図4】レジオネラ菌の試験結果を示す特性図。FIG. 4 is a characteristic diagram showing test results of Legionella bacteria.
【図5】病原性大腸菌からの遺伝子回収試験結果に依
る、電気泳動法パターンの特性図である。FIG. 5 is a characteristic diagram of an electrophoresis pattern based on the results of a gene recovery test from pathogenic Escherichia coli.
【図6】遺伝子の化学抽出法と本発明システムによる抽
出法がそれぞれ要する所用時間の比較をするための工程
図である。FIG. 6 is a process chart for comparing required times required for the chemical extraction method of the gene and the extraction method by the system of the present invention.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 勝浦 信夫 神奈川県相模原市西橋本2丁目23番3号 日幸工業株式会社R&Dセンター内 (72)発明者 五十嵐 治 神奈川県相模原市西橋本2丁目23番3号 日幸工業株式会社R&Dセンター内 (72)発明者 中山 敦 神奈川県相模原市西橋本2丁目23番3号 日幸工業株式会社R&Dセンター内 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Nobuo Katsuura 2-323 Nishihashimoto, Sagamihara City, Kanagawa Prefecture Inside the R & D Center (Nikko Industries Co., Ltd.) (72) Osamu Igarashi 2-23, Nishihashimoto, Sagamihara City, Kanagawa Prefecture No. 3 in the R & D center of Nippon Kogyo Co., Ltd. (72) Inventor Atsushi Nakayama 2-3-3 Nishihashimoto, Sagamihara-shi, Kanagawa Pref.
Claims (11)
有する通電手段と、この通電手段に対する電源と、前記
通電手段に微生物を供給する供給手段と、を備え、この
微生物が生活水に対する汚染源であり、前記通電手段を
電極として前記電源から微生物に通電して微生物を殺菌
するシステム。1. An electric power supply having a metal film coated on a porous film, a power supply for the electric power supply, and a supply means for supplying microorganisms to the electric power supply. A system for disinfecting microorganisms by supplying electricity to the microorganisms from the power supply using the current supply means as an electrode.
緑膿菌、レジオネラ菌、病原性大腸菌の少なくとも一つ
である請求項1記載のシステム。2. The method according to claim 1, wherein the microorganism is Cryptosporidium,
The system according to claim 1, wherein the system is at least one of Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli.
有する通電手段と、この通電手段に対する電源と、前記
通電手段に微生物を供給する供給手段と、を備え、この
微生物が緑膿菌、レジオネラ菌、病原性大腸菌の少なく
とも1種の菌であり、前記通電手段を電極として前記電
源から微生物に通電して微生物を殺菌するシステム3. An electricity supply means having a metal film coated on a porous film, a power supply for the electricity supply means, and a supply means for supplying microorganisms to the electricity supply means, wherein the microorganisms are Pseudomonas aeruginosa, Legionella A system for sterilizing microorganisms by supplying electricity to the microorganisms from the power source using the current supply means as an electrode, wherein the microorganisms are at least one kind of bacteria or pathogenic Escherichia coli.
通電する、請求項1又は3記載のシステム。4. The system according to claim 1, wherein said energizing means applies a direct current to said microorganism.
捕捉して通電する手段である請求項1又は3記載のシス
テム。5. The system according to claim 1, wherein the energizing means is means for capturing the microorganism on a porous membrane and energizing the porous membrane.
脂に化学的に結合している請求項1又は3記載のシステ
ム。6. The system according to claim 1, wherein the metal is chemically bonded to a porous resin forming the porous membrane.
有する通電手段と、この通電手段に対する電源と、前記
通電手段に病原性大腸菌を供給する供給手段と、を備
え、前記通電手段を電極として電源から微生物に通電し
て前記大腸菌の菌体が破壊されることによって生じる菌
内容物から遺伝子を選択的に抽出するようにした、遺伝
子回収システム。7. An electricity supply means having a metal film coated on a porous film, a power supply for the electricity supply means, and a supply means for supplying pathogenic Escherichia coli to the electricity supply means, wherein the electricity supply means is used as an electrode. A gene recovery system, wherein a gene is selectively extracted from microbial contents caused by the destruction of the E. coli cells by energizing the microorganism from a power supply.
生活水に対する汚染源となる微生物を適用し、次いで、
電源から当該金属膜に通電することにより、前記微生物
を殺菌する方法。8. A porous membrane coated with a metal,
Apply microorganisms that are a source of pollution to domestic water,
A method for disinfecting the microorganisms by energizing the metal film from a power supply.
緑膿菌、レジオネラ菌、病原性大腸菌の少なくとも一つ
である請求項8記載の方法。9. The method according to claim 8, wherein the microorganism is Cryptosporidium,
The method according to claim 8, which is at least one of Pseudomonas aeruginosa, Legionella, and pathogenic Escherichia coli.
求項7項記載のシステム。10. The system according to claim 7, wherein said porous film is a porous resin film.
求項8記載の方法。11. The method according to claim 8, wherein said porous film is a porous resin film.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9258816A JPH1189567A (en) | 1997-09-24 | 1997-09-24 | System for sterilizing microorganism and gene recovery system |
GB9820822A GB2329633A (en) | 1997-09-24 | 1998-09-24 | Sterilizing system and system for recovering genes |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9258816A JPH1189567A (en) | 1997-09-24 | 1997-09-24 | System for sterilizing microorganism and gene recovery system |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH1189567A true JPH1189567A (en) | 1999-04-06 |
Family
ID=17325448
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP9258816A Pending JPH1189567A (en) | 1997-09-24 | 1997-09-24 | System for sterilizing microorganism and gene recovery system |
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JP (1) | JPH1189567A (en) |
GB (1) | GB2329633A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005261365A (en) * | 2004-03-19 | 2005-09-29 | Yukie Iwamoto | Plate for recovering biomaterial |
CN103951118A (en) * | 2014-04-12 | 2014-07-30 | 大连双迪科技股份有限公司 | Business water machine |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10115633A1 (en) * | 2001-03-23 | 2002-09-26 | Fuma Tech Gmbh | Shower water filter cartridge incorporates numerous porous hollow fibres |
WO2005083391A1 (en) | 2004-02-26 | 2005-09-09 | Thomsen Bioscience A/S | Method, chip, device and system for collection of biological particles |
CN100535112C (en) | 2004-02-26 | 2009-09-02 | 汤姆森生物公司 | Method, chip, device and system for extraction of biological materials |
ES2330347T3 (en) | 2004-02-26 | 2009-12-09 | DELTA, DANSK ELEKTRONIK, LYS & AKUSTIK | METHOD, CHIP, DEVICE AND INTEGRATED SYSTEM FOR THE DETECTION OF BIOLOGICAL PARTICLES. |
DE102004032375A1 (en) * | 2004-06-30 | 2006-01-26 | Klaus Dr. Rennebeck | Fiber, in particular hollow fiber and its use |
AU2020101945B4 (en) * | 2020-04-05 | 2020-12-24 | Williams, Andrew C MR | A covering for attenuating microorganisms. |
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US5510195A (en) * | 1991-03-01 | 1996-04-23 | Nikko Kogyo Kabushiki Kaisha | Resin membrane having metallic layer and method of producing the same |
EP0577026A2 (en) * | 1992-06-29 | 1994-01-05 | Yoshiaki Nagaura | Filtration method and filter device |
-
1997
- 1997-09-24 JP JP9258816A patent/JPH1189567A/en active Pending
-
1998
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005261365A (en) * | 2004-03-19 | 2005-09-29 | Yukie Iwamoto | Plate for recovering biomaterial |
JP4549084B2 (en) * | 2004-03-19 | 2010-09-22 | 幸英 岩本 | Biomaterial recovery plate |
CN103951118A (en) * | 2014-04-12 | 2014-07-30 | 大连双迪科技股份有限公司 | Business water machine |
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GB9820822D0 (en) | 1998-11-18 |
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