JPH04131080A - Filter membrane-attached microorganism detector and rapid detection of microorganism - Google Patents
Filter membrane-attached microorganism detector and rapid detection of microorganismInfo
- Publication number
- JPH04131080A JPH04131080A JP25309390A JP25309390A JPH04131080A JP H04131080 A JPH04131080 A JP H04131080A JP 25309390 A JP25309390 A JP 25309390A JP 25309390 A JP25309390 A JP 25309390A JP H04131080 A JPH04131080 A JP H04131080A
- Authority
- JP
- Japan
- Prior art keywords
- microorganism
- microorganisms
- atp
- detector
- hydrophilic
- 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.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 39
- 238000001514 detection method Methods 0.000 title claims abstract description 12
- 239000012528 membrane Substances 0.000 claims abstract description 25
- 239000005089 Luciferase Substances 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims description 18
- 108060001084 Luciferase Proteins 0.000 claims description 11
- 238000004020 luminiscence type Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 abstract description 4
- 239000011148 porous material Substances 0.000 abstract description 4
- 239000004809 Teflon Substances 0.000 abstract description 2
- 229920006362 Teflon® Polymers 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 15
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 15
- 238000000034 method Methods 0.000 description 9
- 238000007796 conventional method Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 3
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 3
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 3
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 101150072179 ATP1 gene Proteins 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 229960001456 adenosine triphosphate Drugs 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、沢過膜を装着した微生物検出器ならびにこれ
を使用した微生物の迅速検出方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a microorganism detector equipped with a filtration membrane and a method for rapid detection of microorganisms using the same.
（従来の技術）
飲料や飲料水中の微生物を検出するためには、従来は寒
天培地上でこれを培養し、４８時間ないし７２時間にわ
たって菌体を成育させ、コロニーをカウントする方法が
採られていたが、これには長時間を必要とし、迅速に結
果を得ることができなかった。また、培養や検査には無
菌施設や専門的な技術を必要とするので、簡易に測定を
行うことも困難であった。(Prior art) In order to detect microorganisms in drinks and drinking water, the conventional method was to culture them on an agar medium, grow the microorganisms for 48 to 72 hours, and then count the colonies. However, this required a long time and results could not be obtained quickly. Furthermore, culture and testing require sterile facilities and specialized techniques, making it difficult to perform simple measurements.
これに対し、微生物中のアデノシン５′−三リン酸（以
下ｒＡＴＰＪという）に着眼して、ＡＴＰ量を測定する
ことによって微生物を検出しようとする試みも行われて
いたが、無処理のままこれを測定するのは溶液中に多量
の微生物が存在する場合を除いては困難であった。微生
物を濾過して集菌したうえ一過膜を取り外し、これを別
の容器に入れて菌体から抽出したＡＴＰの発光量を測定
する方法も提案されている（特開平２−１６３０９８号
）′が、膜上に集菌したまま測定に供することのできる
濾過膜を有する検出器を使用するならば微生物の検出を
一層効率的に行うことができる。In response, attempts have been made to detect microorganisms by focusing on adenosine 5'-triphosphate (hereinafter referred to as rATPJ) in microorganisms and measuring the amount of ATP; It was difficult to measure this unless a large amount of microorganisms were present in the solution. A method has also been proposed in which the microorganisms are collected by filtration, the membrane is temporarily removed, and the membrane is placed in another container to measure the amount of luminescence of ATP extracted from the microbial cells (Japanese Patent Application Laid-Open No. 163098/1999). However, microorganisms can be detected more efficiently if a detector is used that has a filtration membrane that can be used for measurement while bacteria are collected on the membrane.
（発明が解決しようとする課題）
本発明は、微生物等を膜上に集菌したままそのＡＴＰ量
を測定することができる、ＡＴＰのルシフェラーゼ反応
光を透過する濾過膜を有する検出器を供給するものであ
り、また、該検出器を用いた微生物の迅速な検出方法を
供給することを目的とするものである。(Problems to be Solved by the Invention) The present invention provides a detector having a filtration membrane that transmits the luciferase reaction light of ATP, which can measure the amount of ATP while collecting microorganisms on the membrane. Another object of the present invention is to provide a method for rapidly detecting microorganisms using the detector.
（課題を解決するための手段）
本発明は、ＡＴＰのルシフェラーゼ反応光を透過する親
水性、耐薬品性濾過Ｍ（例えば親水性テフロン、ポリカ
ーボネート等を材質とする）をモニター部に装着したこ
とを特徴とする微生物検出器の発明であり、この濾過膜
は湿潤状態にすることによって透過性を具備するように
なるものでもよい。また、一過膜を装着したモニター部
は脱着自在とできれば一層便宣である。(Means for Solving the Problems) The present invention includes a monitor unit equipped with a hydrophilic, chemical-resistant filtration M (made of hydrophilic Teflon, polycarbonate, etc.) that transmits ATP luciferase reaction light. The invention is characterized by a microorganism detector, and the filtration membrane may become permeable by keeping it in a wet state. Furthermore, it would be even more convenient if the monitor section equipped with the transient membrane could be made detachable.
さらに、本発明はＡＴＰのルシフェラーゼ反応光を透過
する親水性、耐薬品性濾過膜を装着した微生物検出器を
使用し、該−過膜において検体を沢過したうえ、−過捕
捉した微生物のＡＴＰ発光量を測定することによって微
生物を迅速に検出する方法の発明である。Furthermore, the present invention uses a microorganism detector equipped with a hydrophilic, chemical-resistant filtration membrane that transmits ATP luciferase reaction light, and allows the sample to pass through the filtration membrane. This invention is a method for rapidly detecting microorganisms by measuring the amount of luminescence.
本発明で使用する一過膜は、検査目的や微生物により異
なるが通常は０．１〜１．０μｍ程度の孔径のものが好
適である。The transient membrane used in the present invention preferably has a pore diameter of about 0.1 to 1.0 μm, although it varies depending on the purpose of the test and microorganisms.
微生物の検出は、ＡＴＰが酵素ルシフェラーゼの存在下
においてルシフェリンと反応することによって発生する
微弱な光を測定することによって行うことができる。Detection of microorganisms can be performed by measuring the weak light produced by the reaction of ATP with luciferin in the presence of the enzyme luciferase.
微生物の検出に際しては濾過膜上に捕捉した微生物を予
め滅菌水等で洗浄し、菌体外にある遊離状態のＡＴＰを
排出除去しておくことが必要であり、これによって過大
な測定値が誤検出される虞れを除くことができる。When detecting microorganisms, it is necessary to wash the microorganisms captured on the filtration membrane with sterile water or the like in advance to discharge and remove free ATP outside the microbial cells. The risk of being detected can be eliminated.
（実施例）
以下、本発明の詳細な説明するために実施例を述べるが
、もとより本発明の範囲がこれら実施例のみに限定され
るのではない。(Examples) Examples will be described below to explain the present invention in detail, but the scope of the present invention is not limited to these Examples.
実施例１ 次のようにして本発明の微生物検出器を製造した。Example 1 The microorganism detector of the present invention was manufactured as follows.
底部に内径４０■のモニター部（３）を有する円筒状の
ファネル（２） （容量５００ｍ１）をプラスチックに
より各々成形し、嵌合した。ファネル上部に！（１）、
モニター部下部にｒ過膜捜入部を設け、これに５６０〜
５７０ｒｉｎの光線を透過する孔径０．２μｍで親水性
、耐薬品性の濾過膜（４）を挟入した。Cylindrical funnels (2) (capacity: 500 ml) each having a monitor portion (3) with an inner diameter of 40 mm at the bottom were molded from plastic and fitted together. At the top of the funnel! (1),
A membrane detection section is provided at the bottom of the monitor section, and 560~
A hydrophilic, chemical-resistant filtration membrane (4) with a pore diameter of 0.2 μm that transmits 570 rin of light was inserted.
また、−過膜の下部に底蓋（５）を脱着自在に設置した
。底蓋は、透過集菌後にとりつけ、これによりＡＴＰ反
応液の漏出を防止することができ、底蓋もＡＴＰのルシ
フェラーゼ反応光を透過する材質を用いた。Further, a bottom cover (5) was detachably installed at the bottom of the membrane. The bottom lid was attached after the permeation and bacterial collection to prevent leakage of the ATP reaction solution, and the bottom lid was also made of a material that transmits the ATP luciferase reaction light.
実施例２
ファネル部（１００ｍｌ）、モニター部（内径２５ ｉ
ｎ ）から構成された検出器を使用し、モニター部には
孔径０．２μｍの一過膜を装着した。この濾過膜は湿潤
するとＡＴＰのルシフェラーゼ反応光５６０〜５７０ｎ
ｍ発光波長の光を透過する性買を有する。Example 2 Funnel part (100 ml), monitor part (inner diameter 25 i
A detector was used, and a transient membrane with a pore size of 0.2 μm was attached to the monitor section. When this filtration membrane gets wet, the luciferase reaction light of ATP is 560 to 570n.
It has a structure that transmits light of m emission wavelength.
５ｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ（ＩＦ
Ｏ１３２７６）をｍ−ＴＧＥ Ｂｒｏｔｈで３７℃、
１夜培養した後、生理食塩水で約４Ｘ１０’ｃｆｕ／ｌ
、になるように希釈し、そのＩＬを微生物の検出に供し
た。5taphylococcus aureus (IF
O13276) in m-TGE Broth at 37°C.
After overnight incubation, approximately 4X10'cfu/l in physiological saline.
, and the resulting IL was used to detect microorganisms.
検出液ＩＪｌを微生物検出器で濾過し、菌体を捕捉した
後、モニター部に底ｉ （ＡＴＰのルシフェラーゼ反応
光を透過する）をとりつけた。After filtering the detection solution IJl with a microorganism detector and capturing the bacterial cells, a bottom i (which transmits light from the luciferase reaction of ATP) was attached to the monitor section.
これにフルーツジューステスｌヘキット（ＬｕｍａＣ社
製）のｌｕｍｉｔ ｂｕｆｆｅｒｌｏｏｕｌとＬ−Ｎ
ＢＳ１００μｌを加えて３０秒間ＡＴＰの抽出を行った
。その後モニター部を取り外して、ルミカウンター（ア
ロカ社製ルミネッセンスリーダＢＬＲ４０１）にセット
し、これにＬｕｍｉｔ−ＰＭ １００μｌを加え３０
秒間の発光量を積算した。To this, fruit juice Tessl Hekit (manufactured by LumaC) lumit bufferloool and L-N
ATP was extracted for 30 seconds by adding 100 μl of BS. After that, remove the monitor unit and set it on a Lumi Counter (Luminescence Reader BLR401 manufactured by Aloka), add 100 μl of Lumit-PM to it, and add 30 μl of Lumit-PM.
The amount of light emitted per second was integrated.
発光は次の反応によって行われる。Luminescence is produced by the following reaction.
ルシフェラーゼ
ルシフ・リン＋ＡＴＰ−−−−−−−１ルシフェリン
ルシフェラーゼ−ＡＭＰ＋Ｐ＋Ｐ
ルシフェリン
ルシフェラーゼ−ＡＭＰ
千ＣＯ２
＋光
ＡＴＰにもとづく発光は、例えば、波長５６０〜５７０
ｎｍの微弱な発光であり、この発光反応を利用した微生
物の検出限界は、一般的に言えば、細菌で１０’ｃｆｕ
／ｍ１以上、酵母で１０３ｃｆｕ／ ｍｌであるとされ
ている。Luciferase Lin+ATP-------1 Luciferin luciferase-AMP+P+P Luciferin luciferase-AMP 1,000 CO2 + light ATP-based light emission has a wavelength of 560 to 570, for example.
It is a weak luminescence of nanometers, and the detection limit for microorganisms using this luminescent reaction is generally 10'cfu for bacteria.
/ml or more, and for yeast it is said to be 103 cfu/ml.
本発明によってＡＴＰｊＬを測定し、これにもとづいて
換算した菌体数と、従来の寒天培地培養法（プレート法
）によって測定した菌体数とを比較したのが表−１であ
る。Table 1 shows a comparison between the number of bacterial cells calculated based on the measurement of ATPjL according to the present invention and the number of bacterial cells measured by the conventional agar culture method (plate method).
これによれば、本発明の方法による微生物検出の結果は
、従来法による測定結果とよく合致するものであった。According to this, the results of microorganism detection by the method of the present invention were in good agreement with the measurement results by the conventional method.
実施例３
Ｓｔｒｅｐｔｏｃｏｃｃｕｓ １ａｃｔｉｓ（ＩＦＯ
１２５４６）をｍ−ＴＧＥ Ｂｒｏｔｈで３７℃、１
夜培養した後、生理食塩水で約４ｘｌｏ’ｃｆｕ／Ａに
なるように希釈し、その１！を実施例２と同様に濾過し
て測定した。その結果を表−２に示すが、検出の結果は
、従来法による測定結果とよく合致するものであった。Example 3 Streptococcus 1actis (IFO
12546) with m-TGE Broth at 37°C for 1
After culturing overnight, dilute with physiological saline to approximately 4xlo'cfu/A, and prepare Part 1! was filtered and measured in the same manner as in Example 2. The results are shown in Table 2, and the detection results were in good agreement with the measurement results by the conventional method.
実施例４
Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ（ＩＦＯ１３８９
８）をＭ−Ｃｏｌｉｆｏｒｍ Ｂｒｏｔｈで３７℃、
１夜培養した後、生理食塩水で約３Ｘ１０’ｃｆｕ／Ａ
になるように希釈し、その１！を実施例２と同様に処理
して測定した。その結果を表−３に示すが、従来法によ
る測定結果とよく合致するものであった。Example 4 Escherichia coli (IFO1389
8) in M-Coliform Broth at 37°C.
After overnight incubation, approximately 3X10'cfu/A in physiological saline.
Dilute it so that it becomes, Part 1! was treated and measured in the same manner as in Example 2. The results are shown in Table 3, and were in good agreement with the results measured by the conventional method.
実施例５
酵母Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ（ＩＦｏ
１９７４＞をＷＬ Ｎｕｔｒｉｅｎｔ Ｂｒｏｔｈ
で３０℃、１夜培養した後、生理食塩水で約４Ｘ１０３
ｃｆｕ／Ａとなるように希釈し、その１！を実施例２と
同様に処理して測定した。その結果を表−４に示す力釈
従来法による測定結果をよく合致するのであった。Example 5 Yeast Candida albicans (IFo
1974> WL Nutrient Broth
After culturing overnight at 30°C, incubate approximately 4x103 with physiological saline.
Dilute to cfu/A, Part 1! was treated and measured in the same manner as in Example 2. The results are shown in Table 4, and were in good agreement with the results measured by the conventional method.
（発明の効果）
本発明の装置または方法を使用することによって、検出
液中の微生物を培養することなく迅速に検出することが
でき、特別の無菌施設や培養技術を必要としない。また
、検出中の微生物が少量であっても、これを迅速に検出
することができる。(Effects of the Invention) By using the device or method of the present invention, microorganisms in a detection liquid can be detected rapidly without culturing, and special sterile facilities and culture techniques are not required. Furthermore, even if the number of microorganisms being detected is small, it can be detected quickly.
第１図は、本発明の微生物迅速検出器の実施例。
１は蓋、２はファネル、３はモニター部、４は濾過膜、
５は底蓋。第２図はモニター部の拡大図。FIG. 1 shows an embodiment of the microorganism rapid detector of the present invention. 1 is the lid, 2 is the funnel, 3 is the monitor part, 4 is the filtration membrane,
5 is the bottom cover. Figure 2 is an enlarged view of the monitor section.
Claims (4)
、耐薬品性ろ過膜をモニター部に装着したことを特徴と
する微生物検出器。(1) A microorganism detector characterized in that a monitor unit is equipped with a hydrophilic, chemical-resistant filtration membrane that transmits ATP luciferase reaction light.
過する親水性、耐薬品性ろ過膜を装着した請求項第１項
記載の微生物検出器。(2) The microorganism detector according to claim 1, further comprising a hydrophilic and chemical-resistant filtration membrane that transmits ATP luciferase reaction light under humid conditions.
求項第１項または第２項記載の微生物検出器。(3) The microorganism detector according to claim 1 or 2, wherein the monitor section to which the filtration membrane is attached is detachably attached.
、耐薬品性ろ過膜を装着した微生物検出器を使用し、ろ
過捕捉した微生物ＡＴＰの発光量を測定することを特徴
とする微生物迅速検出方法。(4) A rapid microorganism detection method characterized by using a microorganism detector equipped with a hydrophilic, chemical-resistant filtration membrane that transmits luciferase reaction light of ATP, and measuring the amount of luminescence of microorganism ATP captured by filtration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25309390A JPH04131080A (en) | 1990-09-21 | 1990-09-21 | Filter membrane-attached microorganism detector and rapid detection of microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25309390A JPH04131080A (en) | 1990-09-21 | 1990-09-21 | Filter membrane-attached microorganism detector and rapid detection of microorganism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04131080A true JPH04131080A (en) | 1992-05-01 |
Family
ID=17246397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP25309390A Pending JPH04131080A (en) | 1990-09-21 | 1990-09-21 | Filter membrane-attached microorganism detector and rapid detection of microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04131080A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5506629A (en) * | 1993-07-02 | 1996-04-09 | Mitsubishi Denki Kabushiki Kaisha | Projecting-type display apparatus |
| WO2007116687A1 (en) * | 2006-03-27 | 2007-10-18 | The University Of Electro-Communications | Heterocyclic compound and luminescence method |
-
1990
- 1990-09-21 JP JP25309390A patent/JPH04131080A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5506629A (en) * | 1993-07-02 | 1996-04-09 | Mitsubishi Denki Kabushiki Kaisha | Projecting-type display apparatus |
| WO2007116687A1 (en) * | 2006-03-27 | 2007-10-18 | The University Of Electro-Communications | Heterocyclic compound and luminescence method |
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